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

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 this phase of the project we have established an assay system that supports rudimentary ER function from distal binding sites. Using this as a baseline, future years will see further refinement of the in vitro systems so that contributions of Mediator and cohesin can be assessed. We have also begun generating the reagents we will require for our studies. Key reagents that we have obtained thus far include an active core of the Mediator complex that we have reconstituted through overexpression of its subunits using a baculovirus expression system. ER -interacting MED1 and its mutant variants will be incorporated into this core Mediator for further ER -based functional studies. We also have obtained GFP-derivatives of Mediator and TFIID complexes for proposed FRET analyses for looped chromatin. Peptidomimetic synthesis and analyses are also projected to commence shortly.
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