Browsing by Subject "Transcription"
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Item Open Access Enhancer cooperativity as a novel mechanism underlying the transcriptional regulation of E-cadherin during mesenchymal to epithelial transition(Elsevier, 2015) Alotaibi, H.; Basilicata, M. F.; Shehwana, H.; Kosowan, T.; Schreck, I.; Braeutigam, C.; Konu, O.; Brabletz, T.; Stemmler, M. P.Epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) highlight crucial steps during embryogenesis and tumorigenesis. Induction of dramatic changes in gene expression and cell features is reflected by modulation of Cdh1 (E-cadherin) expression. We show that Cdh1 activity during MET is governed by two enhancers at +. 7.8. kb and at +. 11.5. kb within intron 2 that are activated by binding of Grhl3 and Hnf4α, respectively. Recruitment of Grhl3 and Hnf4α to the enhancers is crucial for activating Cdh1 and accomplishing MET in non-tumorigenic mouse mammary gland cells (NMuMG). Moreover, the two enhancers cooperate via Grhl3 and Hnf4α binding, induction of DNA-looping and clustering at the promoter to orchestrate E-cadherin re-expression. Our results provide novel insights into the cellular mechanisms whereby cells respond to MET signals and re-establish an epithelial phenotype by enhancer cooperativity. A general importance of our findings including MET-mediated colonization of metastasizing tumor cells is suggested.Item Open Access Histone H3.3 regulates mitotic progression in mouse embryonic fibroblasts(Canadian Science Publishing, 2017) Ors, A.; Papin, C.; Favier, B.; Roulland, Y.; Dalkara, D.; Ozturk, M.; Hamiche, A.; Dimitrov, S.; Padmanabhan, K.H3.3 is a histone variant that marks transcription start sites as well as telomeres and heterochromatic sites on the genome. The presence of H3.3 is thought to positively correlate with the transcriptional status of its target genes. Using a conditional genetic strategy against H3.3B, combined with short hairpin RNAs against H3.3A, we essentially depleted all H3.3 gene expression in mouse embryonic fibroblasts. Following nearly complete loss of H3.3 in the cells, our transcriptomic analyses show very little impact on global gene expression or on the localization of histone variant H2A.Z. Instead, fibroblasts displayed slower cell growth and an increase in cell death, coincident with large-scale chromosome misalignment in mitosis and large polylobed or micronuclei in interphase cells. Thus, we conclude that H3.3 may have an important under-explored additional role in chromosome segregation, nuclear structure, and the maintenance of genome integrity. © 2017 Published by NRC Research Press.Item 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 RojhatTranscription 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.Item Open Access Investigating the role of the mediator complex in estrogen receptor alpha-mediated transcription(2018-07) Canavar, TuğçeMediator 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.Item Open Access Osmanlıca kelimeleri eşleme(IEEE, 2007-06) Ataer, Esra; Duygulu, PınarOsmanlı arşivleri dünyanın pek çok yerinden araştırmacının ilgi alanına girmektedir. Fakat bu belgelerin elle çevirisi zor bir iş olduğu için, bu arşivler kullanılamaz durumdadır. Otomatik çeviri gerekmektedir, fakat Osmanlıca’nın yazma özelliklerinden dolayı karakter tabanlı tanıma sistemleri istenen başarıyı gösterememektedir. Ayrıca, belgeler minyatür ve tuğra gibi önemli kısımlar içerdiği için, imge formatında saklanmaları gerekmektedir. Bu nedenle, bu çalışmada Osmanlıca kelimeleri imge olarak görerek probleme imge erişim problemi olarak yaklaşıldı ve kelime eşleme tekniği üzerine bir çözüm önerisinde bulunuldu. Nesne tanımada başarılı olan görsel öğeler kümesi (bag-of-visterms) tekniği kelime eşleme işlemine uyarlandı ve böylece her kelime imgesi taç noktalarından çıkarılan SIFT özelliklerinin ¨ vektor¨ nicemlemesiyle sembolize edildi. Benzer kelimeler görsel ögelerin dağılımına göre eşlendi. Deneyler 10,000 kelimenin üzerindeki matbu ve elyazması belge üzerinde yapıldı. Sonuçlar sistemin benzer kelimeleri yüksek doğrulukla eşlediğini ve anlamsal benzerlikleri bulduğunu gösteriyor Large archives of Ottoman documents are challenging to many historians all over the world. However, these archives remain inaccessible since manual transcription of such a huge volume is difficult. Automatic transcription is required, but due to the characteristics of Ottoman documents, character recognition based systems may not yield satisfactory results. It is also desirable to store the documents in image form since the documents may contain important drawings, especially the signatures. Due to these reasons, in this study we treat the problem as an image retrieval problem with the view that Ottoman words are images, and we propose a solution based on image matching techniques. The bag-of-visterms approach, which is shown to be successful to classify objects and scenes, is adapted for matching word images. Each word image is represented by a set of visual terms which are obtained by vector quantization of SIFT descriptors extracted from salient points. Similar words are then matched based on the similarity of the distributions of the visual terms. The experiments are carried out on printed and handwritten documents which included over 10,000 words. The results show that, the proposed system is able to retrieve words with high accuracies, and capture the semantic similarities between words.Item 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, JavaidThe 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.Item Open Access Reconstitution of Pol II (G) responsive form of the human Mediator complex(Scientific and Technical Research Council of Turkey - TUBITAK,Turkiye Bilimsel ve Teknik Arastirma Kurumu, 2021-06-23) Cevher, Murat AlperRNA polymerase II (Pol II) is a 12 subunit protein complex from yeast to human that is required for gene expression. Gdown1 containing Pol II [Pol II (G)] is a special form of Pol II that is catalytically inactive and heavily depends on the 30-subunit Mediator complex for its activator and basal dependent function in vitro. Here we report for the first time, the identification and the generation of a 15-subunit human Mediator complex via the novel multibac baculovirus expression system that is fully responsive to Pol II (G). Our results show complete recovery of Pol II (G) dependent transcription both with full 30-subunit Mediator and also with 15-subunit recombinant Mediator that we synthesized. Moreover, we also show that the recombinant Mediator interacts with Pol II (G) as well. These results enlighten us towards understanding how a certain population of Pol II that is involved in selected gene regulation is activated by Mediator complex.Item Open Access Reconstitution of RNA Polymerase II subunits and analysis of their interaction with PIC components(2018-08) Erden, MerveMediator 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.Item Open Access Revealing the role of Med14 in Pol-II transcription regulation(2017-08) Barış, YaseminTranscription 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.Item Open Access Role of histone variant H3.3 in transcription and mitotic progression(2017-04) Örs, AyşegulChromatin structure needs to be dynamic and flexible in order for the eukaryotic cellular processes to function correctly. Incorporation of histone variants into chromatin serves to increase epigenetic plasticity by conferring new structural and functional properties to chromatin. Histone variants are implicated in many cellular processes such as transcription or cell division and their deregulation is involved in tumorigenesis. H3.3 is an evolutionarily well conserved histone variant that differs by only a few amino-acids from its replicationdependent counterparts. With the aim of determining H3.3 function, novel knockin/ conditional knock-out mouse models were established and characterized. In these models, one of the two genes coding for H3.3, H3f3a or H3f3b has been modified to code for an N-terminal FLAG-FLAG-HA tagged H3.3A or H3.3B which can be depleted upon Cre expression. Nucleosome resolution genome-wide mapping FH-H3.3A and FH-H3.3B determined that H3.3A and H3.3B were similarly enriched at promoter regions and their enrichment levels positively correlated with high expression and gene body enrichment. They were also found enriched in telomeres and some repetitive DNA sequences. In a subset of these repetitive regions H3.3A and H3.3B showed differential enrichment properties. As double H3.3-KO mouse generation resulted lethal, mouse embryonic fibroblasts (MEFs) were isolated from FH-H3.3 mice and transformed. Using a combination of Cre recombinase mediated knock-out and RNA interference technology, a new cellular model was established where H3.3 expression was essentially depleted. Although H3.3 enrichment profiles were indicative of a role in active transcription, whole transcriptome analysis upon single H3.3 depletion in livers and an almost complete H3.3 depletion in MEFs yielded very few differentially regulated genes. Interestingly, H3.3 depleted MEFs showed a high increase in mitotic defects and abnormal nuclear structures. Thus, an important yet often understudied role for H3.3 in genomic maintenance during mitotic progression was highlighted.