Cukier, C.D.; (2010) Transcription-responsive regulation of c-myc proto-oncogene – structural and biophysical studies. Doctoral thesis , UCL (University College London).

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Abstract

The Far-UpStream Element (FUSE) regulatory system tightly controls the expression of c-myc proto-oncogene – a master regulator of cellular proliferation and differentiation. The FUSE mechanism relies on the inter-molecular interactions between a DNA regulatory sequence – the FUSE, a transcriptional activator – FUSEBinding Protein (FBP) and a transcriptional repressor – FBP-Interacting Repressor (FIR). The FUSE DNA element serves as a sensor of the level of ongoing c-myc transcription. The FBP and FIR proteins bind sequentially to the FUSE, first to rapidly increase the expression (FBP) and then to cease it (FIR). Crucial for the regulation is a FBP – FIR interaction that acts as a transcriptional on/off switch. In my thesis, I used Nuclear Magnetic Resonance (NMR) Spectroscopy to determine a high resolution structure of the first two RNA Recognition Motifs (RRMs) of FIR (FIR RRM1-RRM2) alone and in the complex with an Nbox peptide derived from the FBP protein. The structural details provided explain how a low affinity but specific interaction between FBP and FIR is achieved. Further studies, using NMR and BioLayer Interferometry (BLI), explored the interplay between the three components in the FUSE regulatory system. The results show that FIR RRM1-RRM2 provides independent binding sites for the FUSE and FBP protein and that both FBP and FIR proteins are able to bind simultaneously to the DNA. Furthermore, FBP protein tethers FIR to the DNA, thereby contributing to the control of the transcriptional switch. In summary, these data extend our understanding of the c-myc proto-oncogene regulation and suggest a strategy for controlling c-myc expression in cancerous cells.

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