Undergraduate Honors Theses

Thesis Defended

Spring 2015

Document Type

Thesis

Type of Thesis

Departmental Honors

Department

Biochemistry

First Advisor

Dylan Taatjes

Abstract

Mediator is a multi-subunit protein complex that plays an essential role in transcription by integrating DNA-binding transcription factors (TFs) bound to the enhancer and general transcription factors (GTFs) bound to the promoter of a gene. Mediator is similar to other general transcription factors in that it associates with the promoter region of DNA and interacts with RNA polymerase II (RNAPII), but its large size and many subunits allows it to interact with both GTFs at the promoter and other TFs bound to enhancer DNA. Mediator may regulate transcription by interacting with chromatin. Mediator can exist in different structural states and varying subunit composition. To date, Mediator has been isolated and analyzed from soluble nuclear extract many times to yield a well characterized subunit composition and structure. However, the subunit composition of chromatin-bound Mediator is unknown. Using basic biochemical techniques, it is possible to separate the soluble nuclear extract from the insoluble nuclear pellet, which contains genomic DNA (a.k.a. chromatin) and all factors associated with it. Mediator has not been purified from the nuclear pellet and therefore it is unknown whether its subunit composition is identical when bound to genomic DNA/chromatin. A common way to purify Mediator from the soluble nuclear extract is to use an affinity chromatography step involving the activation domains of the VP16 or the SREBP protein. The subunit composition of these “VP16-Mediator” or “SREBP-Mediator” complexes has been determined previously using mass spectrometry. For my thesis project, both SREBP-Mediator and VP16-Mediator were purified from a chromatin-bound state (i.e. the nuclear pellet) using affinity chromatography and glycerol gradient sedimentation. Analysis with mass spectrometry revealed that VP16-Mediator had similar subunit composition in both chromatin-bound and unbound “nuclear extract” states while the chromatin bound SREBP-Mediator lacked the Mediator subunits CDK8, CCNC, MED12, and MED13. These four subunits comprise a “CDK8 module” that is known to reversibly associate with the Mediator complex and control its interactions with RNAPII. This finding has implications for Mediator function on chromatin and suggests that other endogenous TFs (in addition to SREBP) may similarly associate exclusively with Mediator complexes lacking the CDK8 module on chromatin.

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