Browsing by Subject "RNA polymerase II"

Now showing 1 - 4 of 4

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Intronic elements in the Na+/I-symporter gene (NIS) interact with retinoic acid receptors and mediate initiation of transcription
(Oxford University Press, 2010) Alotaibi, H.; Yaman, E.; Salvatore, D.; di Dato, V.; Telkoparan, P.; di Lauro, R.; Tazebay, U. H.
Activity of the sodium/iodide symporter (NIS) in lactating breast is essential for iodide (I-) accumulation in milk. Significant NIS upregulation was also reported in breast cancer, indicating a potential use of radioiodide treatment. All-trans-retinoic acid (tRA) is a potent ligand that enhances NIS expression in a subset of breast cancer cell lines and in experimental breast cancer models. Indirect tRA stimulation of NIS in breast cancer cells is very well documented; however, direct upregulation by tRA-activated nuclear receptors has not been identified yet. Aiming to uncover cis-acting elements directly regulating NIS expression, we screened evolutionary-conserved non-coding genomic sequences for responsiveness to tRA in MCF-7. Here, we report that a potent enhancer in the first intron of NIS mediates direct regulation by tRA-stimulated nuclear receptors. In vitro as well as in vivo DNA-protein interaction assays revealed direct association between retinoic acid receptor-α (RARα) and retinoid-X-receptor (RXR) with this enhancer. Moreover, using chromatin immunoprecipitation (ChIP) we uncovered early events of NIS transcription in response to tRA, which require the interaction of several novel intronic tRA responsive elements. These findings indicate a complex interplay between nuclear receptors, RNA Pol-II and multiple intronic RAREs in NIS gene, and they establish a novel mechanistic model for tRA-induced gene transcription. © The Author(s) 2010. Published by Oxford University Press.
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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.
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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.
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Revealing the role of Med14 in Pol-II transcription regulation
(2017-08) Barış, Yasemin
Transcription of protein coding genes by RNA polymerase II (Pol II) is a multi-step process each of which requires a series of factors including coactivators. 30-subunit (the subunits are organized into discrete modules – head, middle, tail, and kinase) -2mDa human Mediator complex is the key coactivator in transcription not only for facilitating the establishment of pre initiation complex (PIC) by acting as a bridge between gene-specific activators and Pol II but also and more importantly for regulating the Pol II activity at all pre-initiation, elongation and re initiation steps. Although the diversity in the function of Mediator lies in its flexible conformation and variable subunit organization (for instance, four subunit CDK8 module can both bind to and disassociate from Mediator), detailed mechanistic studies regarding how Mediator interacts with different activators/repressors to regulate transcription, how Mediator facilitates basal transcription through interactions with Pol II and general transcription factors (GTFs) to establish a proper PIC on promoter DNA and how the regulation of subunit exchanges/rearrangements on the Mediator results in architectural and functional outcome are not well characterized. By using Multibac baculovirus expression system, the previous studies have shown that the reconstitution of a functional 15- subunit human core Mediator complex (composed of head and middle modules together with Med14) is sufficient to support basal transcription as well as selective activator-dependent transcription in vitro both with purified factors and with nuclear extracts (in the presence of Med26) as the source of GTFs. This study has uncovered a mechanism by which human core Mediator facilitates transcription by directly interacting with Pol II via its Med14 subunit and recruiting Pol II to target gene promoters. This is the first time that i unraveled the mechanism of how Pol II binds to Mediator and how it is being recruited to target gene promoters for proper PIC assembly and transcription. This way, i shed light into how protein coding genes are universally regulated via the Mediator complex and in the future , I will be in a better position to specifically target selected genes by reconstituting the entire Mediator complex and characterizing activator-Mediator-Pol II crosstalk.