Measuring Ionic Strength Changes using Fluorescence Lifetime and Time-Resolved Anisotropy

Abstract

melanocyte development and differentiation. MITF has also been identified as a lineage-specific oncoprotein in melanoma, and knockdown of MITF function results in the activation of cellular senescence. MITF contains N-terminal and C-terminal activation domains as well as a central basic helix-loop-helix DNA binding motif. Both the N-terminal and C-terminal activation domains directly interact with the homologous histone acetyltransferases CBP/p300 and are required for MITF-dependent transcriptional activation. We have used a combination of pull-down experiments, NMR spectroscopy, biophysical studies, and functional transactivation assays to show that the N-terminal activation domain of MITF is intrinsically disordered in solution and directly interacts with the TAZ2 domain of CBP/p300 with high affinity. NMR-based titrations indicate that MITF also interacts with the KIX and TAZ1 domains of CBP/p300, raising the possibility of co-operative interactions between these domains. Competition experiments indicate that E1A is able to displace MITF from TAZ2. Experiments aimed at determining the functional effect of blocking the MITF:TAZ2 interaction are ongoing. These results provide insight as to how MITF may control gene expression in melanoma and support a model in which MITF interacts with multiple domains of CBP/p300 to activate transcription of MITF-target genes.