Harkin, S; (2007) Investigation of Smad-transcription factor interactions on the c-Jun promoter. Masters thesis , UCL (University College London).

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Abstract

Members of the TGF-J3 superfamily are secreted growth and differentiation factors that regulate diverse events in embryonic development and adult tissue homeostasis. Signalling by mammalian TGF-p involves phosphorylation of Smad2 and Smad3 by TGF-p receptor complexes, association with Smad4, nuclear accumulation of Smad complexes, DNA binding and regulation of target gene expression. Smad complexes rely on associated transcription factors and coregulators for gene regulation and availability of different partners may underlie the cell-type specificity of TGF-(5 responses. Several Smad-interacting transcription factors have been well characterised in Xenopus embryos but, despite numerous studies, the identities and functions of Smad-interacting transcription factors in adult mammalian tissues remain poorly understood. Furthermore, the best-characterised Smad-interacting transcription factors bind Smad2-Smad4 complexes rather than Smad3-Smad4 complexes. Previous work has demonstrated that a TGF-P-inducible complex forming on the 55-basepair c-Jun Smad-binding region (SBR) contains a Smad3- Smad4 heterodimer plus two unidentified factors. My objectives were to further characterise this complex in vitro and develop a purification strategy for identification of its components by mass spectrometry. In this thesis I show that TGF-p-induced complex formation on the SBR is dependent on Smad4. I have also found that the minimal SBR is 32 basepairs long and contains a (GCCA)2 site to which one of the unidentified factors binds. Similarly, sequential pull-downs confirm the presence of at least one additional factor that is essential for association of the Smad3-Smad4 dimer with the SBR and which only associates with the Smads on DNA. The thesis also describes a two-step affinity purification strategy that I have developed to isolate the components of the complex for identification by mass spectrometry. Finally, I have shown that Smad4 and the corepressor SnoN associate with the SBR in the absence of TGF-p, suggesting the possibility of a repressive complex. These studies extend the understanding of Smad-partner interactions and could lead to identification of transcriptional partners of Smad3-Smad4 complexes in adult cells.

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