Mediator-Dependent Transcriptional Activation by Estrogen Receptor Bound to Distal Enhancers.

摘要

Dysregulated estrogen receptor (ER) function underlies many forms of breast cancer. This proposal is aimed at understanding how ER activates its target genes from distal enhancers in a Mediator-dependent fashion. We have hypothesized that ER-Mediator interactions would be critical for signal transduction at ER target genes through establishment of chromatin loops that facilitate long-range enhancerpromoter communication. The main aims of the proposal are thus to establish both cell based and cell-free (in vitro) transcription systems to recapitulate and mechanistically dissect Mediator-dependent ER function from a distal enhancer and further to develop peptidomimetic inhibitors of the ER -MED1 interaction to disrupt enhancer-promoter communication. In the current phase of the project we have completed reconstitution (via baculovirus expression of constituent subunits) of a recombinant Mediator complex that in addition to basal activity also shows p53 activator function. This sets the stage for incorporating ER -interacting MED1 and its mutant variants into this core Mediator for further ER -based functional studies. In view of the emerging importance of topologically associating domains (TADs) in enhancer-promoter communication, we also report the development of new functional assays in which linker histone H1-compacted higher order chromatin can be transcribed in vitro. These studies have revealed new factor dependencies that will inform our studies on ER dependent activation from distal enhancers. Towards pharmacological targeting of the ER -MED1 interaction we have begun implementing a computationally driven approach for design and testing of peptidomimetics.