Research title: Functional and Structural Characterization of the Reconstituted Mediator-NR Interaction as a Critical Therapeutic Approach
Research summary: The thirty-subunit human Mediator co-activator complex interacts with different transcription machinery as well as activators through its distinct subunits stabilizing the PIC (pre-initiation complex) formation. Our proposed work here is to 1 reconstitute the entire human Mediator complex, 2 to introduce deletion mutants, and understand which subunits of the Mediator are critical for these interactions, 3 understand the roles of the observed mutations on the isubunits in the development of certain diseases and later help in the design of certain therapeutics.
Transcriptional activation entails a series of factors, including the class designated as coactivators. Of these, the 30-subunit Mediator complex appears to be involved in the regulation of all RNA Polymerase II (Pol II) target genes and has emerged as the key integrative componant for transcription. Mediator action entails interactions of distinct subunits (for example, MED1 has been identified as one of the critical targets of nuclear receptors (NR), including ER whereas MED17 is a target of p53 and MED15 is a target of multiple activators, including SREBP1, that are involved in fatty acid metabolism). Due to the massive size of the Mediator and the resulting technical limitations in dissecting its mechanisms of action, detailed activation pathways entailing this coactivator have not yet been elaborated. However, because the subunits are organized into discrete head, middle, tail, and kinase modules, a systematic structure-function approach is nonetheless feasible. Recently, we were able to reconstitute a functional 15-subunit human Mediator complex and define it as the “core” through over expression of the individual subunits of the head and middle modules in insect cells via the state-of-the-art Multibac baculovirus expression system (Cevher et al., 2014). Importantly, this reconstituted core displays significant basal and activator (p53) driven transcription activity, as well as strong interactions with some of the (but not all) componants of the transcriptional machinery. Through collaboration, using the newly developed cross-linking/mass-spectrometry (XLMS) method, we further show the 3D architecture of this core-human Mediator complex. Our near-future plan is to build the human Mediator complex and fully understand the recruitment mechanisms of the entire transcription machinery that is dependent on the Mediator. We will additionally concentrate on revealing mechanisms underlying Mediator-NR dependent transcriptional activation concentrating mostly on ER. In the long run, this approach will be particularly helpful in investigating individual subunit mutations that have been correlated with various diseases focusing mostly on breast cancer.
Research Keywords: Transcription, transcription factors, Mediator complex, Multibac baculovirus, nuclear receptors and breast cancer.
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