Browsing by Subject "Mediator complex"

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Open Access
POLR2A/RPB1 subunit of RNA polymerase II interacts with NTDMED14 containing core mediator complex to facilitate basal and activator driven transcription
(2020-06) Jabbar, Javaid
The Metazoan Mediator is a 2-MDa protein complex that consist of 30 subunits, most of which are evolutionarily conserved from yeast to humans8. The maintenance and regulation of the cell is dependent on spatiotemporal control of RNA polymerase II (Pol II) mediated transcription as a result of intrinsic and extrinsic signals. Perturbations caused by the environment and genetics can alter the fate of the cells and can lead to many diseases such as cancer. The role of Mediator is critical in maintaining the cellular environment as it relays signal to RNA polymerase II to regulate homeostasis, cell growth, cell differentiation and development. Thus, it is essential to understand the mechanism by which Mediator regulates the expression of Pol II genes. We have utilized Multibac expression system to synthesize recombinant protein subcomplexes of Mediator and Pol II subunits to elucidate the interaction surface between core Mediator complex and RNA Polymerase II. Our data indicates that POLR2A (RPB1) subunit of Pol II interacts with ~84 kDa N terminal region of Med14 (NTD-Med14) containing core Mediator complex. Furthermore, we also show that other subunits of Pol II including POLR2C (RPB3), POLR2D (RPB4), POLR2E (RPB5), POLR2F (RPB6), POLR2G (RPB7), POLR2H (RPB8), POLR2I (RPB9) POLR2L (RPB10), POLR2J (RPB11) and POLR2K (RPB12) does not interact with core Mediator complex. The binding assay also demonstrates that the recombinant RPB1 subunit competes with endogenous Pol II for the interaction with core Mediator, forming a stable RPB1-core Mediator protein complex. The interaction between RPB1 subunit and NTD-Med14 containing core Mediator complex is independent of Med26. We propose a model for Pol II recruitment to the promoter by core Mediator complex which demonstrates that NTD-Med14 of Core Mediator complex interacts with RPB1 subunit of RNA polymerase II and recruits it to the promoter to facilitate basal and activated transcription.
Open Access
Reconstitution of the partial tail module of the human mediator complex
(2021-09) Çığırgan, Ege
RNA Polymerase II (Pol II) regulates and maintains every aspect of the cell through the act of transcription of protein coding genes. Transcription of protein coding genes depends on extrinsic and intrinsic signals to the Pol II through which it generates a calibrated response. Disturbance of the calibrated response through environmental and genetic factors are the source of many disease phenotypes including cancer. These intrinsic and extrinsic signals are relayed to the Pol II through the Mediator Complex. Mediator Complex is a 30 subunit 2-MDa protein complex that transduces signals from activators and repressors to mediate Pol II generated transcriptional response through which all processes of the cell are regulated. The Mediator Complex consist of 4 modules head middle kinase and tail. Tail module of the mediator complex is a 7 protein module recruits the mediator to the upstream activating sequences through its direct interactions with activators. Therefore reconstitution of the Mediator’s tail subunit is essential to mechanistically understand the Mediator’s regulation of Pol II and how activators cause transcriptional responses in the cell. Through MultiBac expression system we have generated each tail subunit and a partial tail complex of the mediator. Our data indicates that Med16-Med23-Med24-Med25 forms a 4 protein subcomplex through direct interactions of each protein with the tail subunit Med16 in the absence of Med14, the architectural scaffold of the Mediator. Presence of Med14 causes Med25 to be absent from this subcomplex and a subcomplex of Med14-Med16-Med23-Med24 is generated again through the direct interaction of each subunit with Med16. The remaining 3 subunits of the tail module Med15 Med27 and Med29 found to be separate from the remaining 4 subunits as they have failed to be elucidated in any of the immunoprecipitations performed. The results overall suggest the elucidation of a partial tail module. Purification of a full tail module may require additional Mediator Complex proteins.
Open Access
The role of mediator complex in tamoxifen resistance of ER-positive breast cancer
(2022-01) Ersan, Pelin Gülizar
Breast cancer is the most prevalent cancer type and the leading cause of cancer mortality among women worldwide. Estrogen receptor-positive (ER+) breast cancer is the most common clinical subtype with an incidence rate of approximately 80% of all breast cancers. Tamoxifen is a highly effective hormonal therapy for ER-positive breast cancer patients. However, its remarkable success is hampered by de novo or acquired resistance. Despite several advances in therapy options for relapsing patients, tamoxifen resistance is still an urgent clinical problem that needs to be addressed. Therefore, there is a dire need for novel targeted therapies to confer tamoxifen resistance in ER-positive breast cancer. The architecture of Mediator complex links DNA-bound transcription factors to the general transcription machinery RNA polymerase II. Mediator kinase module is dissociable part of the Mediator complex and broadly involved in human cancers. However, the role of kinase module in tamoxifen resistance has not been investigated. In this dissertation, I deciphered the association of Mediator kinase module in tamoxifen resistance both in vitro and in vivo settings. Initially, our gene expression profiling and survival analyses revealed that Mediator subunit 13 (MED13) and cyclin-dependent kinase 8 (CDK8) were significantly higher in tamoxifen-treated patients, and this outcome strongly correlated with worsened patient survival. In vitro inhibition of either MED13 via genetic modulation or CDK8 by highly selective inhibitor, SNX631, significantly reversed tamoxifen resistance. Notably, targeting MED13 or CDK8 resulted in inhibition of HER2/mTOR signaling and triggered apoptosis. Mechanistically, we identified that inhibition of either MED13 or CDK8 combined with tamoxifen treatment reduced ErbB2 mRNA level. We further demonstrated that CDK8 post-transcriptionally controls ErbB2 level via regulating mRNA stability. Moreover, inducible silencing of MED13 in combination with tamoxifen impaired the tumor growth. Similarly, in vivo treatment of SNX631 together with tamoxifen reduced tumor growth in xenografts and prolonged the lifespan in an aggressive transgenic mouse model. These results provided insight into how transcriptional programmers MED13 and CDK8, could contribute to mediating tamoxifen resistance and added new dimension to treatment strategies for ER-positive breast cancer.