Browsing by Subject "Mediator Complex"

Now showing 1 - 3 of 3

Open Access
Identification of general transcription Factor-Two-E and mediator complex interactions in the context of pre-initiation complex formation
(2018-06) Karasu, Onur Rojhat
Transcription is a multistep process which requires the presence of many different proteins and protein complexes. The minimal protein components enabling transcription to happen are identified to execute the event in vitro. However, formation of the transcriptional machinery and the interactions of the proteins while forming the machinery are still not fully understood. General Transcription Factors (GTFs) are identified as the essential elements required for the transcription yet, it is still not clearly known how they are being recruited to the promoter site. Mediator Complex is known to relay the signal received from enhancer sequences to the transcription machinery which is called the Pre-Initiation Complex (PIC) right before the transcription. Here, we are aimed to characterize how Transcription Factor-Two-E is being recruited to TATA promoter by showing this recruitment is strongly corelated with Mediator complex. Besides, this recruitment of Transcription Factor-Two-E to TATA promoter may require more than one subunit of Mediator complex in addition to functional core. Also, here we confirm that Transcription Factor-Two-E co-precipitates with RNA Polymerase II (RNAP II) and TFIIH which are consistent with the existing data in literature. However, the details and the nature of these interactions are yet to be clarified. Identification of these interaction will hopefully result in insights about the sequential formation of PIC or pre-formed holoenzyme multi proteins.
Open Access
Investigating the role of the mediator complex in estrogen receptor alpha-mediated transcription
(2018-07) Canavar, Tuğçe
Mediator is the most important coregulator in eukaryotic transcription. It conveys the signals received from transcriptional activators on enhancer sequences to transcription machinery on promoter regions. Although Mediator itself and MED1 subunit have been implicated in the activation of estrogen receptor alpha (ERα)-mediated gene expressions and breast cancer cell proliferations, the mechanism by which Mediator interacts and brings ligand-bound ERα on its cognate element site to promoter and translates their interactions to gene activation is still unknown. We aimed to identify the interaction between ERα and Mediator to clarify this mechanism. We recombinantly generated active human core Mediator complex along with other subunits of Mediator. We interestingly saw that MEDX showed a potential interaction. In addition to characterizing Mediator- ERα interaction, we also checked the role of the Mediator Complex in acquired tamoxifen resistance. We performed immobilized template recruitment assay to characterize the alterations in recruitment of Mediator subunits and RNA Pol II to 2XERE-TATA promoter in wild type and tamoxifen resistant MCF-7 cell lines. We tried to clarify how changes in pre-initiation complex formation causes resistance to tamoxifen in ERα-positive breast cancer patients. The increase in MEDZ subunit recruitment in resistant cells was striking and promising for further experiments. We hope that our findings will help to elucidate the detailed mechanism of ERα-dependent transcription and design alternative therapeutics.
Open Access
Reconstitution of RNA Polymerase II subunits and analysis of their interaction with PIC components
(2018-08) Erden, Merve
Mediator Complex is a global coactivator of RNA Pol II transcription machinery. It transmits the regulatory signals from enhancer to promoter region. In the absence of Mediator Complex, Pol II transcription is defected. It has been shown that Mediator Complex binds to the CTD domain of Pol II as well as other components of PIC (pre-initiation complex), and thus it forms the transcription loop that enables communication between enhancer region and the promoter. Our lab has found the Mediator Complex subunit (which will be mentioned as MEDX since the data is not published yet) which interacts with the purified endogenous Pol II. After these findings, we aimed to find the subunit of Pol II that interacts with MEDX. For that purpose, we cloned 8 out of 12 subunits of Pol II and so far only reconstituted 5 subunits of it (RPB4, RPB5, RPB6, RPB9 and RPB12) and did biochemical analysis with these proteins. Immunoprecipitation of MEDX with 5 subunits of Pol II showed that 4 of these 5 subunits of Pol II seemed to have pulled down MEDX eve after stringent wash conditions. However, we cannot conclude that MEDX interacts with these 4 subunits, since we are lacking other controls. Also, we will continue characterizing the other subunits of Pol II to find the subunit that interacts with the Mediator subunit MEDX. As the transcription loop is mediated by activators/Mediator/Pol II-PIC we also started cloning two model activators to observe activator mediated polymerase interactions. For this purpose we started cloning and expressing two nuclear hormone receptors: Androgen Receptor and Mineralocorticoid Receptors. Nuclear Receptors are a huge family of activators which transmits signals from the environment of the cell by the help of their ligands, hormones to the transcription machinery. Upon ligand binding, these activators enter the nucleus and bind to the enhancer sequences upstream of the promoter region. Then they recruit Mediator Complex which will start the formation of PIC at the promoter region. We therefore obtained AR from Dr. Nathan Lack and cloned MR. We expressed these proteins in insect cells and characterize PIC formation.