1,721,038 research outputs found
Ccdc62/Erap75扮演輔助因子角色可增强雌激素受體媒介之前列腺細胞目標基因之表達及作用
No full textHuman prostate cancer (PCa) and prostate epithelial cells predominantly express estrogen receptor (ER ), but not estrogen receptor (ER ). ER might utilize various ER coregulators to mediate the E2 signaling pathway in PCa. Here we identified CCDC62/ERAP75 as a novel ER coactivator . CCDC62/ERAP75 is widely expressed in PCa cell lines and has low expression in MCF7 cells. Both in vitro and in vivo interaction assays using mammalian two-hybrid, GST pull-down , and co-immunoprecipitation methods proved that ER can interact with the C terminus of CCDC62/ERAP75 via the LBD. The first LXXLL motif within CCDC62/ERAP75 is required for the interaction between ER and CCDC62/ERAP75. Electrophoretic mobility shift assay showed that CCDC62/ERAP 75 can be recruited by the estrogen response element (ERE)- ER complex in the presence of ligand. Furthermore, a chromatin immunoprecipitation assay demonstrated the hormone -dependent recruitment of CCDC62/ ERAP75 within the promoter of the estrogen- responsive gene cyclin D1. In addition, using siRNA against response element (ERE)-ER complex in the presence of ligand. Furthermore, a chromatin immunoprecipitation assay demonstrated the hormone-dependent recruitment of CCDC62/ ERAP75 within the promoter of the estrogen- responsive gene cyclin D1. In addition, using siRNA against and receptor function
Potensiyel olarak DNA’ya bağlanan CXXC5 proteininin ön karakterizasyonu.
No full text17β-estradiol (E2), the main circulating estrogen hormone, is involved in the physiological and pathophysiological regulation of various tissue notably mammary tissue functions. E2 is responsible for the cellular proliferation, differentiation and/or death in target tissue. The E2 effect is mediated by the nuclear receptors, estrogen receptor α and β, as ligand-dependent transcription factors. Upon binding of E2, ER is converted to an active form and regulates the expression of target genes primarily through genomic signaling pathways leading to cellular responses. The binding of E2-ER to specific DNA sequences estrogen response elements (EREs) initiates the transcription through the ERE-dependent signaling pathway. The interaction of the E2-ER complex with the other transcription factors that are already bound to their cognate response elements mediates the transcriptional events via the ERE-independent signaling pathway. Previous genome-wide analysis studies of our laboratory suggested that the CXXC5 gene expression is regulated by E2-ERα through the ERE-dependent signaling pathway. CXXC5 is a member of the ZF-CXXC domain protein family that contains a highly conserved CXXC domain and a nuclear localization signal. The ZF-CXXC family proteins, through their CXXC domain, bind to non-methylated CpG dinucleotides in CpG islands of transcriptionally active DNA regions. This binding prevents cytosine methylation and leads to the formation of a nucleation site for the direct or indirect recruitment of histone modifying proteins to DNA for transcription regulation. Although studies on functional features of CXXC5 are scarce, the protein appears to participate as a transcription factor, an epigenetic regulator and/or a co-modulator in the transcriptional regulation of cellular events in response to various signaling pathways. We therefore predict that CXXC5, as a non-methylated CpG dinucleotide binding protein, also plays a fundamental role in E2-mediated cellular events. CXXC5 is located on 5q31.2, oriented on the forward strand and encompasses 35- kb DNA. There are three exons and the parts of the second and third exons generate a 1447 bp long mRNA with 969 bp long open reading frame that encodes a 322 amino-acid long protein with a molecular mass of approximately 33 kDa. To begin examining the structure-function of CXXC5, we initially verified our genome-wide findings that CXXC5 is an estrogen responsive gene using MCF7 cells derived from a breast adenocarcinoma. We found by RT-qPCR that CXXC5 is indeed an E2- and ER-responsive gene. We subsequently cloned the ORF of the CXXC5 transcript into a mammalian expression vector. We examined the synthesis and intracellular location of endogenous and exogenously introduced CXXC5 using western blot, immunocytochemistry and short interfering RNA approaches in cell models. We observed that CXXC5 is synthesized at varying amounts in cell lines of breast carcinomas. We also found that CXXC5 localized in the nucleus and shows a diffuse distribution in interphase appearing to overlap with DNA. However during cell division, CXXC5 displayed a distinct nuclear staining that does not overlap with DNA. This suggests that the intra-nuclear distribution of CXXC5 is cell-cycle dependent. Based on our homology modeling and the presence of CXXC domain we predicted that CXXC5 as other protein members of the ZF-CXXC family is a non-methylated CpG dinucleotide binding protein. To verify this prediction, we analyzed the interactions of CXXC5 and CpG dinucleotide containing DNA fragments by electrophoretic mobility shift assay using whole cell extracts or recombinant protein obtained from a bacterial protein expression system. However, we did not observe any binding of CXXC5 to DNA. These findings suggest either that CXXC5, in contrast to our prediction, is not a DNA binding protein; or that bacterial expression is not an appropriate system for CXXC5 to obtain a functional protein. We are currently addressing these issues using various eukaryotic expression systems. Inclusion of studies on the identification of protein interaction partners of CXXC5 will be an important aspect of future studies aimed at the dissection of structural and functional features of the protein.M.S. - Master of Scienc
Östrojen yanıt geni ypel2’ nin klonlanması ve proteinin ilkin karakterizasyonu .
No full text17β-estradiol (E2), the main circulating estrogen in the body, is involved in physiological regulation of many tissue and organ functions, including mammary tissue. E2 is also involved in target tissue malignancies. E2 regulates cellular proliferation, differentiation and death in target tissues. The lasting effects of E2 on cells are mediated by estrogen receptor and β that are the products of distinct genes and act as transcription factors. Upon binding to E2, the activated ER regulates the expression of E2 target genes through ERE (estrogen response element)-dependent and ERE-independent signaling pathways. The ERE-dependent signaling pathway refers to transcription events initiated by the interaction of E2-ER with ERE sequences. The transcription regulation involving the functional interactions of E2-ER with other transcription factors bound to their cognate response elements on DNA is called as the ERE-independent signaling pathway. In a microarray study conducted in our laboratory to identify genes involved in ERE-dependent and ERE-independent signaling pathways, YPEL2, a member of the highly conserved Yippee-like (YPEL) gene family, is suggested to be an E2 responsive gene regulated through the ERE-dependent signaling pathway. The YPEL gene family, named after Drosophila Yippee protein, has 100 members which share an extremely high amino-acid sequence identity in 68 species ranging from yeast, C.elegans, flies, plants to mammals. The members of the human YPEL genes, YPEL1-5, encode putative zinc binding small proteins with molecular vi masses ranging from 13,500 to 17,500 Da. Although structures and functions of Ypel proteins are yet unclear, a limited number of studies suggests the involvement of Ypel proteins in development, cell cycle progression and mitosis, as well as cellular senescence and death. Our analyses using various bioinformatics tools suggest that Ypel proteins share a high degree of structural and functional properties that might be important for basic cellular processes. Our bioinformatics analyses also suggest that each YPEL gene is spatiotemporally regulated by different repertoire of transcription factors which may be activated by distinct signaling pathway in response to different internal and external clues. To analyze the synthesis and intracellular localization of Ypel2, we initially cloned cDNAs of all five members of the human YPEL family, using a cDNA library from ER-positive MCF7 cell line derived from a breast adenocarcinoma, for comparisons. We then showed that the un-liganded ER regulates basal mRNA levels of YPEL2. Moreover, the expression of YPEL2, as well as YPEL3, is repressed by E2. These findings are consistent with our prediction that YPEL2 and YPEL3 are E2 and ER responsive genes. We found that Ypel1, 2 and/or 3 are synthesized in COS7, derived from transformed African green monkey kidney fibroblast-like cells, and localized to a region just outside of the nucleus, however we could not detect any endogenous Ypel protein in MCF7 cells. On the other hand, we observed that over-expressions of YPEL1-5 lead to the leakage of DNA from the nucleus into the cytoplasm in a pattern that overlaps with the localization of each Ypel protein in COS7 and MCF7 cells, in the latter the over-expression of Ypel1-5 is associated with a gross deterioration of the nuclear lamina integrity. Future studies will address the regulation of YPEL2 expression as well as the functions of Ypel2 in cell models.M.S. - Master of Scienc
CXXC5’in E2 tarafından düzenlenen hücre proliferasyonundaki fonksiyonel önemi.
No full text17β-estradiol (E2) as the main circulating estrogen hormone has an important role in the regulation of various tissues including mammary tissue. E2 effects target tissue functions by binding to the nuclear receptors, ERα and β. ERs regulate the expression of target genes. Previous studies conducted in our laboratory indicate that one of these estrogen responsive genes is CXXC5 which is regulated by ERα. CXXC5 has a highly conserved zinc-finger CXXC domain, which makes it a member of zinc-finger CXXC domain protein family. The family binds to non-methylated CpG dinucleotides, specifically in CpG island promoters and alters gene expressions through their enzymatic activities for DNA methylation or epigenetic modifications. However, structural and functional properties of CXXC5 remains largely unknown. In an attempt to decipher the role of CXXC5 in E2-ERα mediated cellular events, we uncovered that CXXC5 do not have an intrinsic transcription activation or repression function but through binding to CpG dinucleotides regulates gene expressions distinctly and mutually modulated by E2 as well. This results in E2-driven cellular proliferation. We therefore suggest that CXXC5 as a CpG binder involves in the regulation of E2-mediated transcriptional activation or repression of genes culminating in the regulation of cellular proliferation.Thesis (M.S.) -- Graduate School of Natural and Applied Sciences. Biology
CXXC5 PROTEİNİNİN HÜCRE DÖNGÜSÜNE GÖRE DÜZENLENMESİ
No full text17β-estradiol (E2) is the main estrogen in circulation and has many physiological effects on various tissues, including the mammary tissue. CXXC5 is an estrogen-responsive gene product that binds to nonmethylated CpG dinucleotides on DNA. CXXC5 synthesis shows fluctuation in the cell cycle. This led to our prediction that the level of CXXC5 synthesis is regulated through the cell cycle. To test this prediction, I investigated the synthesis of CXXC5 in cell cycle-synchronized cells for every 6h up to 36h. I found that the level of CXXC5 synthesis shows alterations with the cell cycle. To investigate cell cycle-dependent expression and/or synthesis of CXXC5, I in this thesis established experimental conditions and protocols. These findings lay the foundations for future studies that aim to further delve into mechanisms of CXXC5 expression, synthesis, and degradation in a cell cycle-dependent manner.17β-östradiol (E2) dolaşımdaki ana östrojendir ve meme dokusu dahil olmak üzere çeşitli dokular üzerinde birçok fizyolojik etkiye sahiptir. CXXC5, DNA üzerindeki metillenmemiş CpG dinükleotitlerine bağlanan östrojene duyarlı bir gen ürünüdür. CXXC5 sentezi, hücre döngüsünde dalgalanma gösterir. Bu, CXXC5 sentez seviyesinin hücre döngüsü boyunca düzenlendiğine dair tahminimize yol açtı. Bu tahmini test etmek için, her 6 ila 36 saat arasında hücre döngüsü ile senkronize edilmiş hücrelerde CXXC5 sentezini araştırdım. CXXC5 sentezi seviyesinin hücre döngüsü ile değişiklikler gösterdiğini buldum. CXXC5'in hücre döngüsüne bağlı ekspresyonunu ve/veya sentezini araştırmak için bu tezde deneysel koşullar ve protokoller oluşturdum. Bu bulgular, hücre döngüsüne bağlı bir şekilde CXXC5 ekspresyonu, sentezi ve bozulması mekanizmalarını daha fazla araştırmayı amaçlayan gelecekteki çalışmaların temellerini atıyor.M.S. - Master of Scienc
CXXC-tipi çinko parmak 5 proteinin (CXXC5) yapısal ve fonksiyonel karakterizasyonu.
No full textEstrogen hormones, particularly 17β-estradiol (E2), are involved in the regulation of physiological and pathophysiological functions of many organs and tissues including breast tissue. The expression of CXXC type zinc finger protein 5 (CXXC5) gene is regulated by E2 through estrogen receptor α. Due to a highly conserved zinc-finger CXXC domain (ZF-CXXC), CXXC5 is considered to be a member of ZF-CXXC family, which binds to non-methylated CpG dinucleotides of transcriptionally active DNA regions. This binding is thought to play critical roles in epigenetic modulation of transcription through the prevention of cytosine methylation and the recruitment of DNA modifying enzymes. The structure and function of CXXC5 and its role in cellular events are yet unclear. However, accumulating evidence is suggesting that CXXC5 is involved in transcriptions as a transcription factor, co-regulator and/or epigenetic factor. In this PhD thesis, I successfully expressed and purified the full-length CXXC5 protein, with which I showed that CXXC5 is a non-methylated CpG DNA binding protein and that the ZF-CXXC domain is indeed responsible for the ability of the protein to interact with DNA. Since proteins exert their functions in the context of dynamically changing interacting protein network, I envisioned that identification of interacting protein partners of CXXC5 would be a critical step in the elucidation of cellular function of the protein. To address this issue, I performed proximity dependent biotinylation assay (BioID) in a cell line model derived from breast adenocarcinoma. Of the identified proteins by Liquid chromatography-tandem mass spectrometry (LC-MS/MS), I validated that CXXC5 protein interacts with MeCP2 (MethylCpG binding protein 2), MAZ (Myc-associated Zinc Finger Protein) and EMD (Emerin) proteins by co-immunoprecipitation assay. I found that the zinc finger domain of CXXC5 is necessary for protein interaction as well. The findings of this study could provide important insights into the mechanism of CXXC5 actions in E2- mediated cellular events.Ph.D. - Doctoral Progra
17beta-östradiol-östrojen reseptör alfa kompleksi aracılığı ile genom çapında oluşan metilasyon ve gen ifadesi değişiklerinin belirlenmesi.
No full text17β-estradiol (E2), the most potent estrogen hormone, induces cellular responses primarily through Estrogen Receptor-alpha (ERα), which is a transcription factor. Interfering E2 signaling indicates that E2 is mitogenic for cells, exemplified by MCF7 cells derived from breast adenocarcinoma, synthesizing ERα endogenously. Studies used exogenous expression of ERα in ERα-negative cell lines to examine structural/functional properties of the receptor. What was unexpected from these studies is the observation that E2 treatment represses cellular proliferation. However, mechanism(s) of this paradoxical phenomenon remains unknown. Methylation is an important epigenetic DNA modification. Changes in methylation alter gene expressions critical for cellular proliferation/differentiation, embryonic development, genomic imprinting and cancer. We therefore hypothesize that distinct methylation statuses of responsive genes’ regulatory regions underlie differential gene expressions, and hence, proliferative and anti-proliferative effects of E2 in cell models. To test this prediction, we generated a cell model stably expressing ERα in MDAMB231 breast cancer cell line. Of the monoclones synthesizing ERα, the MDA-ERα5, based on expected ERα functions, was selected as the cell model to comparatively assess the E2 effects on changes in methylome and transcriptome profiles to those observed in MCF7 cells. Our studies suggest that cell models have cell-specific methylation patterns for the same genomic region at which E2 induces distinct alterations and differentially modulates gene expressions. However, due to the existence of variations among experimental replicates, establishing a correlation between the methylation statuses to gene expression profile of cell lines appears to be immature. An increase in sample size could circumvent this issue.M.S. - Master of Scienc
Biyolüminesans rezonans enerji transferi (BRET) analizi için transkripsiyon faktörlerinin DNA temelli bir araya getirilmesi.
No full textBioluminescence Resonance Energy Transfer (BRET) is a promising assay for studying molecular dynamics such as protein-protein interactions especially in situ and in vivo since the system requires precise distance between the molecules. BRET technique has been used for identification of molecular interactions in situ, imaging of deep-tissues in animal models and sensing of organic or inorganic molecules in vitro by combining the luciferase derivative that is an obligatory unit for this assay and any fluorescent molecules, such as inorganic fluorescent molecules and fluorescent proteins. Beside the luciferase derivatives must be used as one part of BRET pairs, the overall system would be optimized to adapt desired properties by changing the substrate of the luciferase and fluorescent molecule with suitable excitation and emission wavelength as well as the platform that locates the BRET pairs particularly for sensing studies via BRET technique. As sharing the perspective of Fluorescence Resonance Energy Transfer (FRET) which was described previously and has been used widely in molecular interactions, BRET systems have been established basically on in situ studies; however, BRET could be used in sensing systems more widely thanks to its advantages over FRET technique. Deoxyribonucleic acid (DNA) has been shown as the source of life; nonetheless, it is a remarkable bio-polymer having stable and predictable structure with cheap production of synthetic variants. Basing on these useful properties of DNA, it has been used popularly to develop scaffold to combine molecules with exact distances for any purposes or proof of concept studies using molecular self-assembly principle. In the present study, we aimed to design a BRET assay in which engineered transcription factors are fused to BRET pairs, Renilla luciferase (RLuc) and fluorescent protein, mCherry, and signaled on DNA scaffold as a proof of concept study. We fused complementary DNA (cDNA) of CDC DNA-binding protein that is the engineered form of human estrogen receptor α (hERα) to cDNA of RLuc and cDNA of yeast protein, Gal4 DNA-binding domain to cDNA of mCherry with the common protein purification tag of 6-Histidine (6xHis) in a bacterial expression vector by cloning studies. Upon construction of the plasmids that code for related fusion proteins, we tried to over-express, isolate and purify the fusion proteins for next steps of the study and the final process was confirmed by Western Blot (WB) analysis. As an ongoing study, we will try to verify the binding of proteins to DNA scaffold by biochemical methods. Next, we will try to obtain BRET signal and optimize the overall system. This platform would be adapted to in situ and regarded to be used to study context-dependent transcription machinery, genome editing and protein binding to DNA.M.S. - Master of Scienc
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