137 results on '"Bayne, Elizabeth"'
Search Results
2. GRK5 Controls SAP97-Dependent Cardiotoxic β1 Adrenergic Receptor-CaMKII Signaling in Heart Failure
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Xu, Bing, Li, Minghui, Wang, Ying, Zhao, Meimi, Morotti, Stefano, Shi, Qian, Wang, Qingtong, Barbagallo, Federica, Teoh, Jian-Peng, Reddy, Gopireddy R, Bayne, Elizabeth F, Liu, Yongming, Shen, Ao, Puglisi, Jose L, Ge, Ying, Li, Ji, Grandi, Eleonora, Nieves-Cintron, Madeline, and Xiang, Yang K
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Medical Physiology ,Biomedical and Clinical Sciences ,Cardiovascular ,Heart Disease ,Aetiology ,2.1 Biological and endogenous factors ,Animals ,Apoptosis ,Calcium-Calmodulin-Dependent Protein Kinase Type 2 ,Cells ,Cultured ,Cyclic AMP-Dependent Protein Kinases ,Discs Large Homolog 1 Protein ,Disease Models ,Animal ,Excitation Contraction Coupling ,G-Protein-Coupled Receptor Kinase 5 ,Guanine Nucleotide Exchange Factors ,Heart Failure ,Humans ,Male ,Mice ,Inbred C57BL ,Mice ,Knockout ,Myocardial Contraction ,Myocytes ,Cardiac ,Receptors ,Adrenergic ,beta-1 ,beta-Arrestin 1 ,calmodulin ,heart failure ,myocyte ,cardiac ,myocardium ,signaling pathways ,myocyte ,cardiac ,Cardiorespiratory Medicine and Haematology ,Clinical Sciences ,Cardiovascular System & Hematology ,Cardiovascular medicine and haematology ,Clinical sciences - Abstract
RationaleCardiotoxic β1 adrenergic receptor (β1AR)-CaMKII (calmodulin-dependent kinase II) signaling is a major and critical feature associated with development of heart failure. SAP97 (synapse-associated protein 97) is a multifunctional scaffold protein that binds directly to the C-terminus of β1AR and organizes a receptor signalosome.ObjectiveWe aim to elucidate the dynamics of β1AR-SAP97 signalosome and its potential role in chronic cardiotoxic β1AR-CaMKII signaling that contributes to development of heart failure.Methods and resultsThe integrity of cardiac β1AR-SAP97 complex was examined in heart failure. Cardiac-specific deletion of SAP97 was developed to examine β1AR signaling in aging mice, after chronic adrenergic stimulation, and in pressure overload hypertrophic heart failure. We show that the β1AR-SAP97 signaling complex is reduced in heart failure. Cardiac-specific deletion of SAP97 yields an aging-dependent cardiomyopathy and exacerbates cardiac dysfunction induced by chronic adrenergic stimulation and pressure overload, which are associated with elevated CaMKII activity. Loss of SAP97 promotes PKA (protein kinase A)-dependent association of β1AR with arrestin2 and CaMKII and turns on an Epac (exchange protein directly activated by cAMP)-dependent activation of CaMKII, which drives detrimental functional and structural remodeling in myocardium. Moreover, we have identified that GRK5 (G-protein receptor kinase-5) is necessary to promote agonist-induced dissociation of SAP97 from β1AR. Cardiac deletion of GRK5 prevents adrenergic-induced dissociation of β1AR-SAP97 complex and increases in CaMKII activity in hearts.ConclusionsThese data reveal a critical role of SAP97 in maintaining the integrity of cardiac β1AR signaling and a detrimental cardiac GRK5-CaMKII axis that can be potentially targeted in heart failure therapy. Graphical Abstract: A graphical abstract is available for this article.
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- 2020
3. High-Throughput Extracellular Matrix Proteomics of Human Lungs Enabled by Photocleavable Surfactant and diaPASEF.
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Bayne, Elizabeth F., Buck, Kevin M., Towler, Anna G., Yanlong Zhu, Pergande, Melissa R., Tianhua Zhou, Price, Scott, Rossler, Kalina J., Morales-Tirado, Vanessa, Lloyd, Sarah, Fei Wang, Yupeng He, Yu Tian, and Ying Ge
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- 2024
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4. Distinct hypertrophic cardiomyopathy genotypes result in convergent sarcomeric proteoform profiles revealed by top-down proteomics
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Tucholski, Trisha, Cai, Wenxuan, Gregorich, Zachery R., Bayne, Elizabeth F., Mitchell, Stanford D., McIlwain, Sean J., de Lange, Willem J., Wrobbel, Max, Karp, Hannah, Hite, Zachary, Vikhorev, Petr G., Marston, Steven B., Lal, Sean, Li, Amy, dos Remedios, Cristobal, Kohmoto, Takushi, Hermsen, Joshua, Ralphe, J. Carter, Kamp, Timothy J., Moss, Richard L., and Ge, Ying
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- 2020
5. SUMOylation regulates Lem2 function in centromere clustering and silencing
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Strachan, Joanna, primary, Leidecker, Orsolya, additional, Spanos, Christos, additional, Le Coz, Clementine, additional, Chapman, Elliott, additional, Arsenijevic, Ana, additional, Zhang, Haidao, additional, Zhao, Ning, additional, Spoel, Steven H., additional, and Bayne, Elizabeth H., additional
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- 2023
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6. Lactate and Immunomagnetic-purified hiPSC-derived cardiomyocytes generate comparable engineered cardiac tissue constructs
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Rossler, Kalina J., primary, De Lange, Willem J., additional, Mann, Morgan W., additional, Aballo, Timothy J., additional, Melby, Jake A., additional, Zhang, Jianhua, additional, Kim, Gina, additional, Bayne, Elizabeth F., additional, Zhu, Yanlong, additional, Farrell, Emily T., additional, Kamp, Timothy J., additional, Ralphe, J. Carter, additional, and Ge, Ying, additional
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- 2023
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7. High-throughput Extracellular Matrix Proteomics of Human Lungs Enabled by Photocleavable Surfactant and diaPASEF
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Bayne, Elizabeth F, primary, Buck, Kevin M, additional, Towler, Anna G, additional, Zhu, Yanlong, additional, Pergande, Melissa R, additional, Zhou, Tianhua, additional, Price, Scott J, additional, Rossler, Kalina J, additional, Morales-Tirado, Vanessa, additional, Lloyd, Sarah, additional, Wang, Fei, additional, He, Yupeng, additional, Tian, Yu, additional, and Ge, Ying, additional
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- 2023
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8. Lactate and Immunomagnetic-purified iPSC-derived Cardiomyocytes Generate Comparable Engineered Cardiac Tissue Constructs
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Rossler, Kalina J, primary, de Lange, Willem, additional, Mann, Morgan W, additional, Aballo, Timothy J, additional, Melby, Jake A., additional, Zhang, Jianhua, additional, Kim, Gina, additional, Bayne, Elizabeth F, additional, Zhu, Yanlong, additional, Farrell, Emily T., additional, Kamp, Timothy J, additional, Ralphe, John Carter, additional, and Ge, Ying, additional
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- 2023
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9. Molecular characterisation of a viral suppressor of RNA silencing
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Bayne, Elizabeth H.
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572.88 - Published
- 2004
10. Integrated proteomics reveals alterations in sarcomere composition and developmental processes during postnatal swine heart development
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Aballo, Timothy J., primary, Roberts, David S., additional, Bayne, Elizabeth F., additional, Zhu, Wuqiang, additional, Walcott, Gregory, additional, Mahmoud, Ahmed I., additional, Zhang, Jianyi, additional, and Ge, Ying, additional
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- 2023
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11. Top-down Proteomics of Myosin Light Chain Isoforms Define Chamber-Specific Expression in the Human Heart
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Bayne, Elizabeth F., primary, Rossler, Kalina J., additional, Gregorich, Zachery R., additional, Aballo, Timothy J., additional, Roberts, David S., additional, Chapman, Emily A., additional, Guo, Wei, additional, Ralphe, J. Carter, additional, Kamp, Timothy J., additional, and Ge, Ying, additional
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- 2023
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12. Top-down proteomics of myosin light chain isoforms define chamber-specific expression in the human heart.
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Bayne, Elizabeth F., Rossler, Kalina J., Gregorich, Zachery R., Aballo, Timothy J., Roberts, David S., Chapman, Emily A., Guo, Wei, Palecek, Sean P., Ralphe, J. Carter, Kamp, Timothy J., and Ge, Ying
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HEART , *MYOSIN , *MYOCARDIUM , *MOLECULAR motor proteins , *CARDIAC contraction , *AMINO acid sequence , *PROTEOMICS - Abstract
Myosin functions as the "molecular motor" of the sarcomere and generates the contractile force necessary for cardiac muscle contraction. Myosin light chains 1 and 2 (MLC-1 and -2) play important functional roles in regulating the structure of the hexameric myosin molecule. Each of these light chains has an 'atrial' and 'ventricular' isoform, so called because they are believed to exhibit chamber-restricted expression in the heart. However, recently the chamber-specific expression of MLC isoforms in the human heart has been questioned. Herein, we analyzed the expression of MLC-1 and -2 atrial and ventricular isoforms in each of the four cardiac chambers in adult non-failing donor hearts using top-down mass spectrometry (MS)-based proteomics. Strikingly, we detected an isoform thought to be ventricular, MLC-2v (gene: MYL2), in the atria and confirmed the protein sequence using tandem MS (MS/MS). For the first time, a putative deamidation post-translation modification (PTM) located on MLC-2v in atrial tissue was localized to amino acid N13. MLC-1v (MYL3) and MLC-2a (MYL7) were the only MLC isoforms exhibiting chamber-restricted expression patterns across all donor hearts. Importantly, our results unambiguously show that MLC-1v, not MLC-2v, is ventricle-specific in adult human hearts. Moreover, we found elevated MLC-2 phosphorylation in male hearts compared to female hearts across each cardiac chamber. Overall, top-down proteomics allowed an unbiased analysis of MLC isoform expression throughout the human heart, uncovering previously unexpected isoform expression patterns and PTMs. [Display omitted] • Top-down proteomics comprehensively characterized cardiac myosin light chains (MLC). • Strikingly, an MLC ventricular isoform, MLC-2v, was detected in the atrial tissues. • Deamidated MLC-2v was detected in atrial tissue and localized to residue Asn13. • Both MLC-1v and MLC-2a exhibit chamber-specific expression. • Sex-differences detected for phosphorylation of MLC-2v and MLC-2a. [ABSTRACT FROM AUTHOR]
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- 2023
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13. SUMOylation regulates Lem2 function in centromere clustering and silencing
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Strachan, Joanna, primary, Leidecker, Orsolya, additional, Spanos, Christos, additional, Coz, Clementine Le, additional, Chapman, Elliott, additional, Arsenijevic, Ana, additional, Zhang, Haidao, additional, Zhao, Ning, additional, and Bayne, Elizabeth H., additional
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- 2022
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14. Structural analysis of Stc1 provides insights into the coupling of RNAi and chromatin modification
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He, Chao, Pillai, Sreerekha S., Taglini, Francesca, Li, Fudong, Ruan, Ke, Zhang, Jiahai, Wu, Jihui, Shi, Yunyu, and Bayne, Elizabeth H.
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- 2013
15. Separable roles for RNAi in regulation of transposable elements and viability in the fission yeast Schizosaccharomyces japonicus
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Chapman, Elliott, primary, Taglini, Francesca, additional, and Bayne, Elizabeth H., additional
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- 2022
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16. Comparative Functional Genomics of the Fission Yeasts
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Rhind, Nicholas, Chen, Zehua, Yassour, Moran, Thompson, Dawn A., Haas, Brian J., Habib, Naomi, Wapinski, Ilan, Roy, Sushmita, Lin, Michael F., Heiman, David I., Young, Sarah K., Furuya, Kanji, Guo, Yabin, Pidoux, Alison, Chen, Huei Mei, Robbertse, Barbara, Goldberg, Jonathan M., Aoki, Keita, Bayne, Elizabeth H., Berlin, Aaron M., Desjardins, Christopher A., Dobbs, Edward, Dukaj, Livio, Fan, Lin, FitzGerald, Michael G., French, Courtney, Gujja, Sharvari, Hansen, Klavs, Keifenheim, Dan, Levin, Joshua Z., Mosher, Rebecca A., Müller, Carolin A., Pfiffner, Jenna, Priest, Margaret, Russ, Carsten, Smialowska, Agata, Swoboda, Peter, Sykes, Sean M., Vaughn, Matthew, Vengrova, Sonya, Yoder, Ryan, Zeng, Qiandong, Allshire, Robin, Baulcombe, David, Birren, Bruce W., Brown, William, Ekwall, Karl, Kellis, Manolis, Leatherwood, Janet, Levin, Henry, Margalit, Hanah, Martienssen, Rob, Nieduszynski, Conrad A., Spatafora, Joseph W., Friedman, Nir, Dalgaard, Jacob Z., Baumann, Peter, Niki, Hironori, Regev, Aviv, and Nusbaum, Chad
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- 2011
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17. Splicing Factors Facilitate RNAi-Directed Silencing in Fission Yeast
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Bayne, Elizabeth H., Portoso, Manuela, Kagansky, Alexander, Kos-Braun, Isabelle C., Urano, Takeshi, Ekwall, Karl, Alves, Flavia, Rappsilber, Juri, and Allshire, Robin C.
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- 2008
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18. Multiomics Method Enabled by Sequential Metabolomics and Proteomics for Human Pluripotent Stem-Cell-Derived Cardiomyocytes
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Bayne, Elizabeth F., primary, Simmons, Aaron D., additional, Roberts, David S., additional, Zhu, Yanlong, additional, Aballo, Timothy J., additional, Wancewicz, Benjamin, additional, Palecek, Sean P., additional, and Ge, Ying, additional
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- 2021
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19. A Multi-omics Method Enabled by Sequential Metabolomics and Proteomics for Human Pluripotent Stem Cell-derived Cardiomyocytes
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Bayne, Elizabeth F., primary, Simmons, Aaron D., additional, Roberts, David S., additional, Zhu, Yanlong, additional, Aballo, Timothy J., additional, Wancewicz, Benjamin, additional, Palecek, Sean P., additional, and Ge, Ying, additional
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- 2021
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20. Novel Strategies to Address the Challenges in Top-Down Proteomics
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Melby, Jake A., primary, Roberts, David S., additional, Larson, Eli J., additional, Brown, Kyle A., additional, Bayne, Elizabeth F., additional, Jin, Song, additional, and Ge, Ying, additional
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- 2021
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21. GRK5 Controls SAP97-Dependent Cardiotoxic β 1 Adrenergic Receptor-CaMKII Signaling in Heart Failure
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Xu, Bing, primary, Li, Minghui, additional, Wang, Ying, additional, Zhao, Meimi, additional, Morotti, Stefano, additional, Shi, Qian, additional, Wang, Qingtong, additional, Barbagallo, Federica, additional, Teoh, Jian-Peng, additional, Reddy, Gopireddy R., additional, Bayne, Elizabeth F., additional, Liu, Yongming, additional, Shen, Ao, additional, Puglisi, Jose L., additional, Ge, Ying, additional, Li, Ji, additional, Grandi, Eleonora, additional, Nieves-Cintron, Madeline, additional, and Xiang, Yang K., additional
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- 2020
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22. MASH Explorer: A Universal Software Environment for Top-Down Proteomics
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Wu, Zhijie, primary, Roberts, David S., additional, Melby, Jake A., additional, Wenger, Kent, additional, Wetzel, Molly, additional, Gu, Yiwen, additional, Ramanathan, Sudharshanan Govindaraj, additional, Bayne, Elizabeth F., additional, Liu, Xiaowen, additional, Sun, Ruixiang, additional, Ong, Irene M., additional, McIlwain, Sean J., additional, and Ge, Ying, additional
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- 2020
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23. Mkt1 is required for RNAi-mediated silencing and establishment of heterochromatin in fission yeast
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Taglini, Francesca, primary, Chapman, Elliott, primary, van Nues, Rob, primary, Theron, Emmanuelle, primary, and Bayne, Elizabeth H, primary
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- 2019
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24. Cell-to-cell movement of Potato Potexvirus X is dependent on suppression of RNA silencing
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Bayne, Elizabeth H., Rakitina, Daria V., Morozov, Sergey Y., and Baulcombe, David C.
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- 2005
25. Starting out
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Bayne, Elizabeth
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- 2002
26. DegrAAAded into Silence
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Bayne, Elizabeth H., White, Sharon A., and Allshire, Robin C.
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Biological sciences - Abstract
To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2007.05.004 Byline: Elizabeth H. Bayne (1), Sharon A. White (1), Robin C. Allshire (1) Abstract: In fission yeast, RNA interference (RNAi)-dependent heterochromatin formation silences transgenes inserted at centromeres. In this issue, demonstrate that the RNAi machinery directly targets transgene transcripts. Furthermore, they link transgene silencing to a protein complex resembling the TRAMP complex of budding yeast, which promotes transcript degradation via the exosome. Thus, RNAi-independent transcript degradation may also contribute to heterochromatin gene silencing. Author Affiliation: (1) Wellcome Trust Centre for Cell Biology, University of Edinburgh, Kings Buildings, Michael Swann Building, Mayfield Road, Edinburgh, EH9 3JR, UK
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- 2007
27. Mkt1 is required for RNAi-mediated silencing and establishment of heterochromatin in fission yeast.
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Taglini, Francesca, Chapman, Elliott, van Nues, Rob, Theron, Emmanuelle, and Bayne, Elizabeth H
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- 2020
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28. Global regulation of heterochromatin spreading by Leo1
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Verrier Laure, Taglini Francesca, Barrales Ramon R., Webb Shaun, Urano, Takeshi, Braun Sigurd, and Bayne Elizabeth H.
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Chromatin Immunoprecipitation ,Histones ,genome regulation ,Gene Expression Regulation, Fungal ,Gene Order ,Schizosaccharomyces ,lcsh:QH301-705.5 ,Binding Sites ,epigenetics ,Research ,fungi ,Inverted Repeat Sequences ,heterochromatin ,High-Throughput Nucleotide Sequencing ,RNA-Binding Proteins ,Acetylation ,fission yeast ,Leo1 ,lcsh:Biology (General) ,Genome, Fungal ,Research Article ,Protein Binding - Abstract
Heterochromatin plays important roles in eukaryotic genome regulation. However, the repressive nature of heterochromatin combined with its propensity to self-propagate necessitates robust mechanisms to contain heterochromatin within defined boundaries and thus prevent silencing of expressed genes. Here we show that loss of the PAF complex (PAFc) component Leo1 compromises chromatin boundaries, resulting in invasion of heterochromatin into flanking euchromatin domains. Similar effects are seen upon deletion of other PAFc components, but not other factors with related functions in transcription-associated chromatin modification, indicating a specific role for PAFc in heterochromatin regulation. Loss of Leo1 results in reduced levels of H4K16 acetylation at boundary regions, while tethering of the H4K16 acetyltransferase Mst1 to boundary chromatin suppresses heterochromatin spreading in leo1Δ cells, suggesting that Leo1 antagonises heterochromatin spreading by promoting H4K16 acetylation. Our findings reveal a previously undescribed role for PAFc in regulating global heterochromatin distribution.
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- 2015
29. A systematic genetic screen identifies new factors influencing centromeric heterochromatin integrity in fission yeast
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Bayne, Elizabeth H, Bijos, Dominika A, White, Sharon A, de Lima Alves, Flavia, Rappsilber, Juri, and Allshire, Robin C
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Research ,Gene Expression Regulation, Fungal ,Heterochromatin ,Centromere ,Schizosaccharomyces ,fungi ,RNA Interference ,Schizosaccharomyces pombe Proteins - Abstract
Background Heterochromatin plays important roles in the regulation and stability of eukaryotic genomes. Both heterochromatin components and pathways that promote heterochromatin assembly, including RNA interference, RNAi, are broadly conserved between the fission yeast Schizosaccharomyces pombe and humans. As a result, fission yeast has emerged as an important model system for dissecting mechanisms governing heterochromatin integrity. Thus far, over 50 proteins have been found to contribute to heterochromatin assembly at fission yeast centromeres. However, previous studies have not been exhaustive, and it is therefore likely that further factors remain to be identified. Results To gain a more complete understanding of heterochromatin assembly pathways, we have performed a systematic genetic screen for factors required for centromeric heterochromatin integrity. In addition to known RNAi and chromatin modification components, we identified several proteins with previously undescribed roles in heterochromatin regulation. These included both known and newly characterised splicing-associated proteins, which are required for proper processing of centromeric transcripts by the RNAi pathway, and COP9 signalosome components Csn1 and Csn2, whose role in heterochromatin assembly can be explained at least in part by a role in the Ddb1-dependent degradation of the heterochromatin regulator Epe1. Conclusions This work has revealed new factors involved in RNAi-directed heterochromatin assembly in fission yeast. Our findings support and extend previous observations that implicate components of the splicing machinery as a platform for RNAi, and demonstrate a novel role for the COP9 signalosome in heterochromatin regulation. Electronic supplementary material The online version of this article (doi:10.1186/s13059-014-0481-4) contains supplementary material, which is available to authorized users.
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- 2014
30. The Discovery, Distribution, and Evolution of Viruses Associated with Drosophila melanogaster
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Webster, Claire L., primary, Waldron, Fergal M., additional, Robertson, Shaun, additional, Crowson, Daisy, additional, Ferrari, Giada, additional, Quintana, Juan F., additional, Brouqui, Jean-Michel, additional, Bayne, Elizabeth H., additional, Longdon, Ben, additional, Buck, Amy H., additional, Lazzaro, Brian P., additional, Akorli, Jewelna, additional, Haddrill, Penelope R., additional, and Obbard, Darren J., additional
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- 2015
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31. The discovery, distribution and evolution of viruses associated with Drosophila melanogaster
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Webster, Claire L, primary, Waldron, Fergal M, additional, Robertson, Shaun, additional, Crowson, Daisy, additional, Ferrari, Giada, additional, Quintana, Juan F, additional, Brouqui, Jean-Michel, additional, Bayne, Elizabeth H, additional, Longdon, Ben, additional, Buck, Amy H, additional, Lazzaro, Brian P, additional, Akorli, Jewelna, additional, Haddrill, Penelope R, additional, and Obbard, Darren J, additional
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- 2015
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32. A systematic genetic screen identifies new factors influencing centromeric heterochromatin integrity in fission yeast
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Bayne, Elizabeth H, primary, Bijos, Dominika A, additional, White, Sharon A, additional, Alves, Flavia de Lima, additional, Rappsilber, Juri, additional, and Allshire, Robin C, additional
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- 2014
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33. Raf1 Is a DCAF for the Rik1 DDB1-Like Protein and Has Separable Roles in siRNA Generation and Chromatin Modification
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Buscaino, Alessia, primary, White, Sharon A., additional, Houston, Douglas R., additional, Lejeune, Erwan, additional, Simmer, Femke, additional, de Lima Alves, Flavia, additional, Diyora, Piyush T., additional, Urano, Takeshi, additional, Bayne, Elizabeth H., additional, Rappsilber, Juri, additional, and Allshire, Robin C., additional
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- 2012
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34. Stc1: A Critical Link between RNAi and Chromatin Modification Required for Heterochromatin Integrity
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Bayne, Elizabeth H., primary, White, Sharon A., additional, Kagansky, Alexander, additional, Bijos, Dominika A., additional, Sanchez-Pulido, Luis, additional, Hoe, Kwang-Lae, additional, Kim, Dong-Uk, additional, Park, Han-Oh, additional, Ponting, Chris P., additional, Rappsilber, Juri, additional, and Allshire, Robin C., additional
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- 2010
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35. Analysis of small RNA in fission yeast; centromeric siRNAs are potentially generated through a structured RNA
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Djupedal, Ingela, primary, Kos-Braun, Isabelle C, additional, Mosher, Rebecca A, additional, Söderholm, Niklas, additional, Simmer, Femke, additional, Hardcastle, Thomas J, additional, Fender, Aurélie, additional, Heidrich, Nadja, additional, Kagansky, Alexander, additional, Bayne, Elizabeth, additional, Wagner, E Gerhart H, additional, Baulcombe, David C, additional, Allshire, Robin C, additional, and Ekwall, Karl, additional
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- 2009
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36. Reducing Health Disparities in Childhood Asthma Through a Sustained Long-Term Social Marketing Campaign
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Bayne, Elizabeth, primary, Miller, Kristy, additional, and Goldman, Patricia, additional
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- 2009
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37. RNA-directed transcriptional gene silencing in mammals
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Bayne, Elizabeth H., primary and Allshire, Robin C., additional
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- 2005
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38. Starting out: Elizabeth Bayne finds her outlook changed by working on an elderly care ward. (perspectives)
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Bayne, Elizabeth
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Geriatric nursing -- Personal narratives ,Patients -- Care and treatment ,Aged patients -- Care and treatment ,Nursing students -- Personal narratives ,Health ,Health care industry ,Care and treatment ,Personal narratives - Abstract
I was in the first semester of my second year and about to have my first adult ward experience--on an elderly ward. I was panic stricken and not very excited. [...]
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- 2002
39. Investigating the role of Raf2 in heterochromatin formation in fission yeast
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Arsenijevic, Ana, Allshire, Robin, and Bayne, Elizabeth
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Raf2 ,heterochromatin formation ,fission yeast ,RNAi ,CLRC ,DNMT1 ,heterochromatin islands - Abstract
In fission yeast, heterochromatin formation requires RNAi and the histone H3K9 methyltransferase complex CLRC which contains Raf2, a protein with unknown function. Raf2 has an RFTS domain which is also found in DNMT1 in higher eukaryotes, and by this domain DNMT1 binds to ubiquitinated and methylated histone H3. Interestingly, the same pattern of H3 ubiquitination is conserved from fission yeast to mammals, along with the well-known H3K9 methylation, so we hypothesised that Raf2 might interact with H3 via the RFTS domain to tether CLRC to chromatin. Our in vitro experiments provided evidence of Raf2 interacting with unmodified H3, supporting this hypothesis. Using protein modelling of Raf2 to the published DNMT1 structures, we identified residues of Raf2 RFTS domain that could be involved in H3 binding. My analyses showed that mutating some of these residues in Raf2 leads to defects in heterochromatin formation. While one of the residues is still a good candidate for mediating the possible Raf2-H3 interaction, mutating others seemed to have phenotypes that were different than expected, and after performing corrected protein modelling, I found that they were probably not mediating H3 interaction. Additionally, ubiquitination of H3K14 has recently been implicated as a prerequirement for H3K9 methylation at heterochromatin. Based on a genetic screen, we identified Ubp14 as a putative deubiquitinating enzyme of H3K14, and by using genetic approaches and protein analyses we provide evidence that this might be the enzyme responsible for the removal of this mark. One of the Raf2 mutations (raf2W72A) that impaired heterochromatin assembly probably independently of H3 interaction, showed different effects at different heterochromatic loci, similar to what was previously reported for mutants of the FACT complex. Mass spectrometry data showed that wild-type Raf2, but not the mutant, potentially interacts with both FACT components Spt16 and Pob3, suggesting that the Raf2 RFTS domain may mediate interaction with FACT and possibly help recruit it to heterochromatin. I also found decreased Spt16 binding to one heterochromatic locus in the strain bearing this Raf2 mutation, confirming that the role of Raf2 could involve recruiting FACT to chromatin. Additionally, this mutant showed a strong defect in silencing of all types of heterochromatin islands. Interestingly, mass spectrometry data suggested a potential interaction between Raf2 and Ccr4-Not complex component Caf1 which is required for proper heterochromatin formation at the island regions. A different Raf2 mutation (raf2Q171Y) was found to impair the transition of di- to trimethylated H3 at the centromere, which is a phenotype resembling other mutants such as the deletion of the ATPase Abo1, which I also identified as a potential interactor by mass spectrometry. Both aforementioned Raf2 mutants have severe silencing defects at subtelomeric heterochromatin, and Raf2 has been implicated in mediating recruitment of CLRC to telomeres by interacting with Shelterin component Ccq1. I showed that this interaction is perturbed in both of these mutants, which provides evidence that RFTS domain mediates this interaction and the establishment of subtelomeric heterochromatin domains. In this work, I suggest several potential functions of Raf2 that have not previously been described, including interaction with H3 and/or Abo1 to tether CLRC to chromatin, association with the FACT complex to recruit it to heterochromatic loci and the interaction with the Ccr4-Not complex to regulate heterochromatin islands. Future work will aim to describe these functions in more detail, but there is evidence that Raf2 is involved in different interactions and that it might serve as a "hub" to regulate heterochromatin assembly through association with different factors.
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- 2023
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40. Investigating the role SUMO plays in heterochromatin formation in the fission yeast Schizosaccharomyces pombe
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Zhao, Ning, Bayne, Elizabeth, and Marston, Adele
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SUMO ,heterochromatin formation ,Schizosaccharomyces pombe ,H3K9 methylation ,RNA interference ,heterochromatin silencing - Abstract
In eukaryotic cells, DNA is organized by histones and associated proteins into a complex that is called chromatin. The fundamental subunit of chromatin is the nucleosome that is composed of eight histone proteins and a segment of DNA. The chromatin is divided into euchromatin and heterochromatin based on the appearance of the chromosomes. Euchromatin is an open configuration reflecting regions that are allowed to be replicated and transcribed. In contrast, heterochromatin corresponds to genetically silent chromosome regions, which have an altered chromatin structure and decreased recombination frequencies. Heterochromatin domains are normally found close to the nuclear periphery, and contain transposable elements and repetitive sequences. It has also shown that heterochromatin is essential to chromosome structures such as centromeres and telomeres, associated with some chromatin proteins and different states of histone modifications. Moreover, heterochromatin regulates gene expression via epigenetic silencing mechanisms during the process of cell development and differentiation. Thus, it is crucial to study heterochromatin as it is important to maintain genome stability. My PhD project is to investigate the role SUMO plays in heterochromatin formation in fission yeast. SUMO is a small protein modifier that is crucial for numerous cellular processes in eukaryotic cells. SUMO is attached to the substrates via the action of three enzymes, including SUMO E1 activating enzyme, E2 conjugating enzyme, and E3 ligase enzyme, that are analogous to ubiquitin pathway. SUMO has also been defined as a key mediator of genome stability, cell proliferation etc. Once SUMO targets the substrates, it can exert effects by altering the interaction with partner proteins and DNA, changing cellular localization, or disturbing protein stability by competing with ubiquitination. In S. pombe, the pmt3+ gene that encodes SUMO causes defects in heterochromatin silencing at the endogenous loci, however, the mechanism of the molecular action remains to be determined. In this study I have first confirmed the previously published silencing defect associated with the deletion of SUMO in fission yeast cells. Moreover, I found a specific defect in in tri-methylated H3K9. Further exploration indicates that SUMO deletion is affecting heterochromatin silencing through Clr4 chromodomain (CD), probably due to Clr4 binding too tightly to the chromatin. This is the first time it has been found that SUMO is affecting the H3K9 methyltransferase Clr4 binding to the chromatin, I hope this will advance other studies in dissecting the role of SUMO in regulation of heterochromatin formation.
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- 2023
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41. A molecular dissection of the spindle assembly checkpoint signalling in Cryptococcus neoformans
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Aktar, Koly, Hardwick, Kevin, and Bayne, Elizabeth
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Cryptococcus neoformans ,aneuploidy ,cell division control ,cell cycle checkpoints ,Mad1 ,Mad2 ,Titan cell viability - Abstract
Cryptococcosis is a severe fungal infection caused by an opportunistic fungal pathogen, Cryptococcus neoformans which has been medically significant for more than half of the last century. This yeast displays noticeable ploidy shifts during in vivo pulmonary infection. These polyploid cells often generate aneuploid progeny which has been repeatedly reported as one of the main virulence factors for disease progression. Given this capability of escaping equal chromosome segregation during mitosis, they are possibly escaping several cell cycle controls including the spindle assembly checkpoint. The spindle assembly checkpoint is undescribed in this fungal pathogen. Therefore, I aimed to understand how this checkpoint signalling contribute to cell division in C. neoformans. My current aim is to study one of the critical spindle assembly checkpoint proteins, Mad1, which remains undescribed in this fungal pathogen. Deletion of mad1 and mad2 in Cryptococcus showed sensitivity to anti-microtubules drugs. Microscopy and microfluidics data revealed that the mad1 and mad2 mutants were unable to maintain mitotic arrest in response to such drugs. Both proteins were also found to be important for Titan cell viability. Mad1 showed localisation to unattached kinetochores of arrested cells. Purified Mad1 complexes showed interactions with other checkpoint proteins Bub1, Mad2, Cdc20 and Mps1, by co-immunoprecipitation and mass spectrometry. I believe that several of these interactions are driven by phosphorylation. I found Mad1 to be phosphorylated by recombinant Mps1 kinase. I have generated several Mad1 phospho-mutants and some show defects in checkpoint signalling. Thus, Mad1 protein-protein interactions could be regulated by kinases such as Mps1, Cdk, Plk1 or Bub1 kinase and this may affect Mad1 interaction with Cdc20 (the APC/C co-activator). This study leads to a plausible molecular explanation of Mad1 contribution in MCC assembly (Mitotic checkpoint complex). The precise in vivo functions of Mad1 and more details of the underlying molecular mechanisms of spindle assembly checkpoint signalling in this understudied pathogenic fungus will be discussed.
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- 2023
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42. Investigating the role of RNAi and epigenetic modifications in the basidiomycetes yeast Cryptococcus deneoformans
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Scoynes, Charlotte, Bayne, Elizabeth, and Buck, Amy
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Cryptococcosis ,centromeres ,epigenetic silencing pathway ,heterochromatin ,RNA interference ,RNAi ,Cryptococcus deneoformans - Abstract
The basidiomycete yeasts from the Cryptococcus neoformans/gattii species complex are major fungal pathogens and are particularly prevalent in the developing world. The species within the complex are rapidly evolving, with several species loosing genes encoding proteins required for silencing by the RNA interference (RNAi) pathway which correlate with an increase virulence. The species C. deneoformans, however, has retained all five of the core RNAi components (Rdp1, Ago1, Ago2, Dcr1, Dcr2), and has been shown to have a functional RNAi pathway involved in the silencing of transposable elements (TEs). Centromeric TEs have also been shown to coincide with DNA methylation in C. deneoformans, and also with H3K9 methylation in neighbouring species C. neoformans. Here I look at the relationship between these three potential mechanisms of silencing in C. deneoformans, RNAi, DNA methylation and H3K9 methylation, focussing on how RNAi interacts with both methylation marks in TE regulation. Identification of the H3K9 methyltransferase Clr4 and H3K9me2-ChIP confirmed the presence of H3K9 methylation at the centromeres in C. deneoformans. Analysis of strains with deletions of core RNAi components revealed wild-type levels of centromeric H3K9 methylation, confirming that RNAi is not required for maintenance of this heterochromatin mark. Analysis of transcript levels at RNAi target sites showed no difference between wild-type and RNAi deficient strains. This suggests that RNAi silences targets through a post-transcriptional gene silencing (PTGS) method out with RNA degradation. To investigate the role of RNAi in suppressing transposon activity, mutation rate assays were carried out by screening for spontaneous 5-FOA resistance that results from disruption of the URA3 or URA5 genes. Strains lacking both H3K9 methylation and DNA methylation (clr4Δdnmt5Δ) had the highest drug resistance rates. PCR screening determined if 5-FOA resistance was due to transposon insertion into URA3 or URA5, and both T1 and T2 DNA transposon insertions were identified. The rate of inserts identified within rdp1Δ and clr4Δdnmt5Δ strains was significantly higher than in WT, showing increased transposon mobility in both strains. Analysis of DNA transposable element expression showed large variance between replicate cultures but suggested that T3 may be regulated by an RNAi-independent mechanism, unlike T1 and T2 where suppression appears dependent on Rdp1. Analysis of retrotransposon copy numbers showed no significant increase in any strains tested when compared to WT. Overall this shows a potential role for H3K9 and/or DNA methylation in controlling transposon mobility alongside RNAi. Finally, analysis was carried out into the roles of both Argonaute proteins within C. deneoformans, as Ago2 is frequently lost within the species complex, and is not present within neighbouring species C. neoformans. Mass spectrometry of tagged proteins showed that each Ago binds to a different subset of proteins, suggesting a different role for each protein within the RNAi pathway. Deletion of Gwo1, the main Ago1 interactor, increases the interaction of Ago1 with Ago2. The work undertaken here contributes to the further understanding of the interaction between RNAi and the DNA and H3K9 methylation silencing pathway in C. deneoformans and shed lights on the different roles of the two Argonaute proteins in this species.
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- 2022
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43. Molecular and functional characterisation of an extracellular Argonaute protein secreted by a gastrointestinal nematode
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Neophytou, Kyriaki, Buck, Amy, and Bayne, Elizabeth
- Abstract
Parasites manipulate their hosts to promote infection by secreting bioactive molecules including proteins, lipids and RNAs. Helminths (parasitic worms) secrete a plethora of such molecules possessing immunogenic and immunomodulatory properties and some of these are co-packaged within extracellular vesicles (EVs) and can be directly transferred to host cells. We have found that the mouse-infective gastrointestinal nematode Heligmosomoides bakeri, a close relative of the human hookworm Necator americanus and the animal-infective nematodes Teladorsagia circumcincta and Haemonchus contortus, secretes an RNA-binding protein which belongs to the family of Argonaute (AGO) proteins. AGO proteins are at the heart of RNA interference, a mechanism involved in gene regulation. By associating with small RNA guides, AGO proteins are directed to messenger RNA targets. This interaction usually leads to gene silencing. The AGO protein secreted by H. bakeri, termed exWAGO, is found in two forms: a vesicular form (detected inside EVs) and a non-vesicular form that does not co-purify with EVs. One goal in this thesis is to understand whether and how these two forms of exWAGO are different in terms of their sRNA guides and potential targets. It is known that H. bakeri EVs are internalised by mouse host cells, such that the parasite-derived cargo could directly interact with and interfere with host gene expression. The EVs predominantly contain 5'PPP secondary short interfering RNAs (siRNAs) and EV uptake by mouse cells was shown to result in suppression of host genes involved in immunity and inflammation. As AGO proteins coordinate gene silencing mechanisms, we hypothesise that exWAGO is directly involved in mediating changes in host gene targets. The goal in this thesis is to build understanding on the putative role of exWAGO in mediating cross-species gene silencing by identifying the RNA and protein interaction partners of exWAGO. A further goal is to determine the importance of exWAGO in parasite survival by blocking it through vaccination and testing the consequence to subsequent infections. To identify the small RNAs (sRNAs) that associate with exWAGO we immunopurified exWAGO and generated sRNA libraries from three different sample types: adult worms (to detect the intra-parasite exWAGO), exWAGO from the excretory-secretory products that is found in EVs, and exWAGO from the excretory-secretory products that does not co-purify with EVs. sRNA sequencing analyses show that the intra-parasite exWAGO and the two extracellular forms of exWAGO bind 22-23G 5'PPP secondary siRNAs originating from transposable elements and novel repeats, but are depleted from microRNAs (miRNAs), Y-RNAs, transfer RNAs and ribosomal RNAs. This suggests that exWAGO is bound specifically to secondary siRNAs, and we hypothesise that exWAGO is required for the export of these sequences from the parasite. To study the binding selectivity of exWAGO, we used gel shift assays and found that exWAGO binds 5'PPP guide RNAs with high affinity in contrast to the mammalian mouse AGO2 (mAGO2). Direct comparison of the secondary siRNA sequences bound by the vesicular and non-vesicular exWAGO indicates that the two exWAGO forms have some overlap in which secondary siRNAs they bind, however there are differences in the relative abundance of different siRNAs and there are some siRNAs only found in one exWAGO form. This suggests that the vesicular and non-vesicular forms of exWAGO might target different genes and have different functional properties. Identification of the host transcripts targeted by exWAGO is crucial in understanding the role that exWAGO might play in cross-species gene silencing. To examine this, we developed a method to immunopurify the exWAGO protein in vivo from the gut of H. bakeri-infected mice and sequenced the RNAs with which it associates. Our initial dataset using the mAGO2 as a positive control suggests that we can successfully detect gene targets with this method. The preliminary results indicate that RNAs associated with exWAGO map to intronic regions in the mouse transcriptome, in contrast to mAGO2 where reads map to 3' untranslated regions and coding regions (consistent with location of mAGO2 target sites). Further experiments are required to test if the putative targets of exWAGO identified are true targets. Development of a bioinformatics pipeline to examine this complex dataset would also allow us to test if the method enabled formation of guide-target chimeric reads, hence permitting direct identification of guide sRNA-host target interactions. To understand the mechanism by which exWAGO might mediate gene silencing in host cells, we tested whether exWAGO possesses the ability to cleave "slice" targets in vitro. Our results show that exWAGO does not possess slicer activity, consistent with its lack of a catalytic motif required by other AGOs for slicer activity. To explore what other gene regulation mechanism(s) might be employed by exWAGO inside host cells we set out to identify the protein interactors of exWAGO in vitro and in vivo using liquid chromatography-tandem mass spectrometry. The proteomic analysis identified some putative interactors that have been reported to interact with other known mammalian AGO proteins as well as some proteins with no previous literature linking these to gene silencing. From these data we can now formulate hypotheses that exWAGO might be localised to specific compartments in host cells to perform its function. We also discovered a putative candidate protein that might be involved in the internalisation of exWAGO inside host cells and in host AGO trafficking. Further experiments are required to validate the protein interactions identified. Finally, to understand the importance of exWAGO in infection and test if exWAGO can serve as a vaccine candidate, we immunised mice with recombinant exWAGO protein. Vaccination with recombinant exWAGO protein led to a strong induction of IgG1 antibodies against the protein and resulted in decreased egg and worm burdens (59.0% and 66.7% respectively, calculated as average across three experiments). These data suggest that blocking exWAGO in vivo reduces the ability of the parasite to survive. As exWAGO is highly conserved amongst Clade V gastrointestinal worms, including the human hookworm and sheep parasites, we propose that exWAGO could be a vaccine candidate for these nematodes. In summary, this thesis presents a first insight into the mechanistic basis of exWAGO in terms of what sRNA guides it binds, what its putative target host genes are, and what proteins it interacts with. We also explore the properties and characteristics of the two extracellular forms of exWAGO for the first time. By understanding if and how exWAGO may facilitate communication between parasite sRNAs and host targets, we expand our understanding of how helminths manipulate their hosts. This thesis offers a putative intervention for gastrointestinal worm infections via exWAGO vaccination, but further studies could also lead to development of other therapeutic strategies or molecular tools based on exWAGO.
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- 2022
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44. Functional analysis of proteasome-associated ubiquitin ligases in plants
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Wang, Zhishuo, Spoel, Steven, and Bayne, Elizabeth
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ubiquitin-proteasome system ,UPS ,cell signalling regulation ,proteasome-associated ubiquitin ligases ,substrate degradation - Abstract
Degradation of intracellular proteins by the ubiquitin-proteasome system (UPS) is a sophisticated mechanism that begins with anchoring ubiquitin molecules to a substrate and ends with proteasome-dependent proteolysis. Initiation of ubiquitination by E3 ligases is a key step in this pathway that selectively labels unstable or damaged proteins. The ubiquitinated substrate is then recognised by proteasome-associated ubiquitin receptors and subsequently degraded by the proteasome. Recent studies have identified several E3 ligases that surprisingly associate with the proteasome as accessory proteins. As substrates are already modified by ubiquitin when they arrive at the proteasome, it is unclear what the role of these proteasome-associated ligases are. In this study, the role of proteasome-associated ubiquitin ligases in proteasomal substrate degradation was characterised and the functional significance of ubiquitin chain remodelling at the proteasome were explored in planta. In Arabidopsis thaliana, HECT-type Ubiquitin Protein Ligases (UPLs) have been identified as proteasome-associated ubiquitin ligases that are required for salicylic acid (SA)-induced plant immunity. Accordingly, the mechanism behind regulation of plant immune response by UPLs is further studied in Chapter 3. Here, it is shown that UPLs control SA-dependent transcriptional reprogramming via regulating homeostasis of the SA-responsive coactivator NPR1. SA-induced accumulation of NPR1 was impaired in upl mutants, which resulted in diminished expression of immune genes. Additionally, proteasome-associated UPLs facilitated polyubiquitination of NPR1, and thereby promoted its proteasomal turnover. This process was indispensable for clearing inactive NPR1 from chromatin. Thus, UPL-mediated remodelling of NPR1-attached ubiquitin chains at the proteasome is required for maximum transcriptional activity of NPR1. In Chapter 4 I show that proteasome-associated UPLs also target other transcription activators, including the developmental and ethylene-responsive EIN3 activator. I demonstrate that by physically interacting with UPL3, the SCFEBF2 ubiquitin ligase complex directly escorted EIN3 to the proteasome. Subsequent 'eleventh-hour' ubiquitin chain remodelling by proteasome-associated UPL3/4 was required for processive degradation of EIN3 by the proteasome and was critical for removal of EIN3 from its target gene promoters. Besides targeting substrates destined for the proteasome, I show in Chapter 5 that UPL3 and UPL4 are also involved in polyubiquitination of other E3 ligases. UPL3/4 catalysed ubiquitination of the immune-responsive U-box E3 ligase, PUB22, and controlled its proteasomal turnover. Mutation of PUB22 and its homologues, PUB23 and PUB24, supressed the disease susceptibility phenotype of the upl3 upl4 mutant, indicating that UPL3/4 also regulate immunity via modulating homeostasis of PUB ligases. Overall, my findings indicate that unstable hormone-responsive transcriptional activators are sequentially polyubiquitinated by relays of ubiquitin ligases in which HECT-type ligases prevent the stalling of proteasome-bound substrates. On the other hand, HECT-type ligases also target other E3 ligases for degradation, thereby indirectly influencing substrate levels of these E3 ligases. Thus, my findings demonstrate that proteasomes unexpectedly influence the ubiquitination and stability of both E3 ligases and their substrates to regulate transcriptional programmes in plants.
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- 2022
- Full Text
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45. Structural analysis of Stc1 provides insights into the coupling of RNAi and chromatin modification.
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Chao He, Pillai, Sreerekha S., Taglini, Francesca, Fudong Li, Ke Ruan, Jiahai Zhang, Jihui Wu, Yunyu Shi, and Bayne, Elizabeth H.
- Subjects
YEAST ,PROTEIN structure ,ARGONAUTE proteins ,CHROMATIN ,ZINC-finger proteins - Abstract
The article presents a study which investigates the function and structure of Stc1, a protein believed to mediate in fission yeast. The study shows that one end of the Stc1 protein has an unusual form of protein structure type called as a tandem zinc finger domain. It reveals that the domain ties Argonaute 1, while the other end of Stc1 associates with the machinery of chromatin modification.
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- 2013
- Full Text
- View/download PDF
46. Investigating the role of S-nitrosylation in plant antiviral defense
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Monteiro, Muriel, Molnar, Attila, and Bayne, Elizabeth
- Subjects
571.2 ,S-nitrosylation ,antiviral response ,TuMV ,gsnor1-3 - Abstract
S-Nitrosylation, a post-translational modification, involves specific reversible incorporation of nitric oxide (NO) moiety to protein cysteine thiol to form an S-nitrosothiol (SNO). The cellular level of S-nitrosylation is maintained by S-nitroso glutathione reductase (GSNOR), an enzyme involved in the turnover of NO reservoir, S-nitroso glutathione (GSNO). Loss of function of AtGSNOR1 leads to increased SNO level and compromised immune response against bacterial and fungal pathogens. Similar consequences are observed in plants with excessive NO production. However, not much is known about the role of S-nitrosylation in antiviral response in plants which we aim to tackle in this study. Arabidopsis mutants with varying level of nitric oxide (NO) and SNO: gsnor1-3, gsnor1-3R, nox1, TRXh5 (nox1) were selected. These mutants along with controls (Col-0, eIF(iso)4E‐1) were inoculated with a Green Fluorescent Protein (GFP)-tagged potyvirus, Turnip mosaic virus (TuMV-GFP) to study susceptibility to viral infection. The susceptibility assay demonstrated higher viral resistance in plants with increased SNO levels. This observation contrasts with the observation made with bacterial and fungal pathogens. Multiple facets associated with both host and pathogen were explored, to understand the underlying mechanism behind TuMV resistance in plants with higher SNO level. To achieve this, we focused on gsnor1-3 mutants due to clearly displayed delayed viral infection, infection progression pattern and ease of viral inoculation. This SNO mutant displayed slower onset of viral infection in rub-inoculated leaves, however viral replication rate and cell to cell movement was not hampered. We found that the systemic movement of TuMV was slower and delayed in gsnor1-3 plants. This was confirmed by evaluation of gsnor1-3 morphology that showed variation in plasmodesmata number, vascular pattern, and phloem transport rate, suggesting cumulative effect of host factors on delayed viral movement.
- Published
- 2021
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47. Proteolysis-dependent regulation of telomerase
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Degtev, Dmitrii, Makovets, Svetlana, and Bayne, Elizabeth
- Subjects
572.8 ,telomerase regulation ,Saccharomyces cerevisiae ,telomerase degradation ,oncogenesis regulation ,TOM1 ,Est2 - Abstract
Eukaryotes maintain their genomes in the form of linear chromosomes. Because of the inability of the replication machinery to copy linear DNA molecules to the very end, the chromosomes were predicted to become shorter with every round of replication. This phenomenon was called "the end replication problem" by Watson and Olovnikov. Later, it has been discovered that eukaryotes have evolved telomeres, non-coding DNA repeats at the chromosomal ends, and telomerase, a ribonucleoprotein that extends the telomeres to overcome "the end replication problem". Telomerase is known to be downregulated in humans through the development resulting in progressive telomere shortening and replicative senescence with age. This is believed to be one of the major tumour suppressor mechanisms. However, most cancer cells reactivate telomerase to become immortal. Thus, understating telomerase regulation is one of the central questions in the field of cancer biology. Telomerase complex formation involves several component maturation and assembly steps. In Saccharomyces cerevisiae, the steady-state levels of the catalytic subunit Est2 are significantly reduced when its interaction with the telomerase RNA component TLC1 is impaired. I have found that Est2 not bound to TLC1 undergoes degradation in a proteasome-dependent manner. Loss of Tom1, an E3-ubiquitin ligase, leads to an increase in the Est2 levels accompanied by a decrease in the Est2 degradation rate. Consistent with these findings, tom1 mutants have longer telomeres. Furthermore, Tom1 physically interacts with Est2, specifically through recognizing its RNA binding domain. Disruption of TOM1 does inhibit proteolysis of Est2 but does not stop it completely, suggesting the existence of an additional, Tom1-independent degradation pathway of Est2. Interestingly, the Est2 levels are reduced in cells grown at elevated temperatures. This decrease is caused by the temperature-triggered degradation of Est2. This degradation resembles proteolysis through the protein quality control system, however, the exact regulator of this mechanism is yet unknown. I propose that regulation of the telomerase through the proteolysis contributes to telomere length homeostasis indirectly through controlling the levels of Est2. The human Est2 homologue hTERT is also degraded in a proteasome-dependent manner. Thus, the proteolysis-dependent regulation of the telomerase might be evolutionarily conserved. RNA-free hTERT is believed to have extra-telomeric roles and this regulation might balance its telomeric and extra-telomeric functions.
- Published
- 2021
- Full Text
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48. Investigating mechanisms of RNAi-dependent heterochromatin establishment in Schizosaccharomyces pombe
- Author
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Kapitonova, Ekaterina, Bayne, Elizabeth, and Heun, Patrick
- Subjects
579 ,chromatin ,euchromatin ,Schizosaccharomyces pombe ,S. pombe ,centromeric heterochromatin formation ,heterochromatin establishment - Abstract
Heterochromatin is a condensed conformation of eukaryotic DNA, which plays an essential role in genome homeostasis. In depth research across various species showed that RNA interference (RNAi) is one of the pathways that is important for heterochromatin formation. To date RNAi-driven heterochromatin assembly has been extensively studied in the fission yeast Schizosaccharomyces pombe. At centromeres in S. pombe, RNAi is required for heterochromatin establishment and maintenance. Unlike heterochromatin maintenance, RNAi-driven heterochromatin establishment at the centromeres is not very well understood. The interconnectedness between the RNAi and chromatin modification pathways that are required to establish heterochromatin makes it difficult to elucidate which pathway acts first. Recent studies also showed that heterochromatin establishment requires additional factors dispensable for maintenance. Thus exploring novel factors required for the de novo heterochromatin formation can help us to understand the order and the mechanism of heterochromatin establishment. In this study I developed and tested two assays ⎯ plasmid-based and cross-based assays ⎯ designed to identify novel establishment-specific factors genome-wide. While the plasmid based assay proved unreliable, the cross-based establishment assay was shown to effectively identify establishment-specific factors. It also provided new insights into heterochromatin establishment dynamics.
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- 2020
- Full Text
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49. Human neuronal LUHMES cell line as a model system for studying Rett syndrome
- Author
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Shah, Ruth Rama, Bird, Adrian, and Bayne, Elizabeth
- Subjects
616.85 ,Rett syndrome ,MECP2 ,DNA-binding proteins ,mCpG ,LUHMES cells ,MeCP2 mutant cell lines ,MeCP2 protein - Abstract
Rett syndrome (RTT) is a severe neurological disorder that affects approximately 1:10000 girls. Classical RTT is defined by a developmental regression phase and subsequent stabilisation of diagnostic criteria, which include partial or complete loss of spoken language, dyspraxic gait and stereotypic hand movements such as hand mouthing. RTT is a monogenic disorder, with the majority of cases being due to loss-of-function mutations in MeCP2 (methyl-CpG binding protein 2). Due to this clear genotype-phenotype link multiple RTT mouse models have been used to elucidate the molecular details, and consequent neuropathogenesis, of this complex neurological disease, as well as for the development of potential therapeutics for RTT. However, as the molecular details become clearer, the need for a simpler model system becomes evident. Human induced pluripotent stem cells (hiPSCs) generated from RTT patient fibroblasts are an option; however the handling of these cells is laborious, time-consuming and expensive and they often differentiate into a heterogeneous population of cells. To explore an alternative human model system I have been genetically engineering and experimenting with the human dopaminergic LUHMES cell line. LUHMES cells are an immortalised pre-neuronal cell line derived from an 8-week old, female foetus and can readily be differentiated into a homogeneous population of mature, electrically active neurons in just one week. In this thesis I have assessed the phenotypic properties of the wild-type cell line, demonstrated the ease of genetic manipulation of LUHMES cells by CRISPR/Cas9 approaches, generated seven mutant MECP2 LUHMES cell lines and explored the potential of protein therapy as a therapeutic approach for RTT. The LUHMES cell line proves to be extremely easy to handle and robust and has yielded novel molecular insights into the function of MeCP2 in human neurons. In particular, MeCP2-null cells show a striking relationship between the level of gene body methylation and the extent of transcriptional upregulation when compared to wild-type neurons. In contrast neurons that express a form of MeCP2 that can bind to DNA but cannot recruit a transcriptional corepressor complex (the R306C mutant) do not exhibit substantial gene expression alterations, yet do display a consistent decrease in total RNA amount. This decrease in total RNA is recapitulated in MeCP2-null LUHMES-derived neurons and in brain regions from MeCP2-R306C mice. The requirement for functional DNA binding for normal gene-body methylation dependent gene repression is demonstrated by assessing LUHMES cells that overexpress MeCP2-R111G, a protein that cannot bind to DNA. Furthermore, overexpression of the MeCP2-R306C protein highlights the importance of NCoR binding for normal gene repression, but also demonstrates that MeCP2-R306C protein retains some gene repression activity. Thinking more broadly, this cell line also has applications as a model system for a variety of other neurological disorders; as a simplified model system to elucidate molecular and neurological phenotypes, and as a relevant human system that can be cultured in a high-throughput manner for testing therapeutic strategies.
- Published
- 2018
50. Investigating the role of RNA interference in the fission yeast Schizosaccharomyces japonicus
- Author
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Chapman, Elliott, Bayne, Elizabeth, and Finnegan, David
- Subjects
572.8 ,chromatin ,heterochromatin ,RNA interference ,RNAi ,S. japonicus ,Schizosaccharomyces pombe ,Schizosaccharomyces japonicus ,post-transcription - Abstract
RNA interference (RNAi) is a conserved pathway that plays key roles in heterochromatin formation, gene regulation and genome surveillance across a wide range of eukaryotes. One of the most utilised model organisms for studying the RNAi pathway is the fission yeast Schizosaccharomyces pombe. However, this species is somewhat atypical, in that it has not retained the ancestral role for RNAi in the silencing of mobile genetic elements. In contrast, the related fission yeast S. japonicus has a large and diverse retrotransposon complement that appears to give rise to abundant siRNAs. For this reason, we believe that S. japonicus may be a more suitable model for studying the role of RNAi in silencing mobile genetic elements, a function that is conserved in many higher eukaryotes. Functional analysis of the S. japonicus RNAi pathway proved more challenging than expected, as it was generally not possible to recover strains bearing deletions of core RNAi components (Ago1/Clr4/Rdp1/Arb1/Arb2). This suggests that a functional RNAi pathway may be required for viability in S. japonicus, unlike in S. pombe. However, disruption mutants were isolated for the sole Dicer ribonuclease Dcr1, at very low frequency. Analysis of these mutants revealed that disruption of Dcr1 impaired the generation of retrotransposon derived siRNAs, and caused de-repression of retroelement transcript accumulation and mobilisation in an element dependent manner. Surprisingly however, Dcr1 appeared dispensable for the maintenance of H3K9me2 at transposons, suggesting that, in contrast to S. pombe, silencing may occur principally at the post-transcriptional level. It is also possible that the isolated Dcr1 mutants represent rare survivors that are viable due to the presence of suppressor mutations elsewhere in the genome. I utilised my genome wide RNA sequencing data to help improve the annotation of the S. japonicus genome, with a specific focus on the retrotransposon complement. From this, I identified 12 new families of LTR retrotransposon, which increased the annotated retrotransposon complement by around 40% in S. japonicus. Finally, I characterised the integrative preference of the S. japonicus retrotransposon Tj1, and found that it shares characteristics associated with the S. cerevisiae retrotransposons Ty1 and Ty3, mostly integrating upstream of RNA PolIII transcribed tRNA genes. The findings of this work highlight some potentially key differences in the way the RNAi pathway functions across the fission yeast clade, both in terms of its importance for viability and its mode of action. The work undertaken here also contributes to the establishment of S. japonicus as a model for the study of RNA interference and genome regulation.
- Published
- 2018
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