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Santolin, Lisa (2006): Functional characterization of the Mediator subunit MED25. Dissertation, LMU München: Faculty of Chemistry and Pharmacy
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Abstract

In this study a structure–function analysis has been employed to analyze transcriptional regulation through the Mediator subunit MED25. A relationship could be established between predicted structural domains and functional characteristics of this protein. Most critically the region responsible for interaction of MED25 with the Mediator was identified. Immunoprecipitation experiments demonstrated that the so–called VWA domain (von–Willebrand A domain, amino acids 1–290) is both sufficient and required for this contact. Site–directed mutagenesis indicates that this binding reaction involves the non–conserved loop SR2, which is protruding from this domain. Based on the results of this analysis a model was proposed, in which the primary contact is established by ionic forces and is further stabilized by hydrophobic interactions. The previously identified ACID domain was reported to bind to VP16. Targeted mutagenesis of four different motifs in this region impaired not only transcriptional activation through MED25 but also led to reduced binding to VP16. In particluar a lysine–rich motif is also present in two domains of PTOV1, a close homolog of MED25. Noteworthy, K518 is not conserved in the PTOV1_B domain, which in contrast to PTOV1_A and the ACID domain of MED25 does not bind to VP16. This led to the hypothesis that K518 is critically involved in the binding of VP16 to MED25. Furthermore it could be demonstrated that MED25 contains an intrinsic transcriptional activation capacity, which is localized in the region 290–715. This indicates additional recruitment of other factors to promoters through this region. Together with the Mediator binding VWA–domain and the VP16–interaction domain this region might facilitate transcriptional activation. A genome–wide screen showed downregulation of c–Jun and FosB following overexpression of MED25. Interestingly, expression of GSK3β, a downstream target of which is cyclin D1, seems to be stimulated by MED25. Together with the finding that overexpression of MED25 leads to activation of a p21 reporter, this raises the possibility that MED25 is involved in cell cycle control. An overlap has been discovered by comparison of MED25 target genes and genes identified previously as target for the viral activator EBNA2. The close homology between the activation domains of EBNA2 and VP16 implies a common mechanism of transcriptional activation by these two viral proteins through MED25. The involvement of MED25 in gene activation by viral activators might indicate a role for this Mediator subunit in viral transcription.