Biography
Dr Cheetham has a long-term interest with understanding the functions of the noncoding genome. He conducted his Honours project with Professor John Mattick at the Institute for Molecular Bioscience, UQ. His research focussed on differentiating human protein-coding and noncoding RNAs. For his PhD, he joined the group of Professor Andrea Brand at the University of Cambridge, on a Herchel Smith Studentship. There he studied the functions of long noncoding RNAs (lncRNAs) in neural development and the mechanisms through which they act. Dr Cheetham expanded the application of DamID including developing a novel method to map lncRNA-chromatin associations in vivo and developing Targeted DamID for the mammalian cells.
Following his PhD Dr Cheetham has joined Professor Geoff Faulkner’s group to continue to investigate the roles of “junk” DNA in human biology. In 2019 Dr Cheetham was awarded an NHMRC Peter Doherty Early Career Fellowship commencing in 2019.
Research Links:
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Publications:
2020 | |
Ewing, Adam D; Smits, Nathan; Sanchez-Luque, Francisco J; Faivre, Jamila; Brennan, Paul M; Richardson, Sandra R; Cheetham, Seth W; Faulkner, Geoffrey J Nanopore Sequencing Enables Comprehensive Transposable Element Epigenomic Profiling (Journal Article) Molecular Cell, 2020, ISSN: 1097-2765. (Abstract | Links | BibTeX | Altmetric) @article{ewing_nanopore_2020, title = {Nanopore Sequencing Enables Comprehensive Transposable Element Epigenomic Profiling}, author = {Adam D Ewing and Nathan Smits and Francisco J Sanchez-Luque and Jamila Faivre and Paul M Brennan and Sandra R Richardson and Seth W Cheetham and Geoffrey J Faulkner}, url = {http://www.sciencedirect.com/science/article/pii/S1097276520307310}, doi = {10.1016/j.molcel.2020.10.024}, issn = {1097-2765}, year = {2020}, date = {2020-01-01}, urldate = {2020-11-30}, journal = {Molecular Cell}, abstract = {Transposable elements (TEs) drive genome evolution and are a notable source of pathogenesis, including cancer. While CpG methylation regulates TE activity, the locus-specific methylation landscape of mobile human TEs has to date proven largely inaccessible. Here, we apply new computational tools and long-read nanopore sequencing to directly infer CpG methylation of novel and extant TE insertions in hippocampus, heart, and liver, as well as paired tumor and non-tumor liver. As opposed to an indiscriminate stochastic process, we find pronounced demethylation of young long interspersed element 1 (LINE-1) retrotransposons in cancer, often distinct to the adjacent genome and other TEs. SINE-VNTR-Alu (SVA) retrotransposons, including their internal tandem repeat-associated CpG island, are near-universally methylated. We encounter allele-specific TE methylation and demethylation of aberrantly expressed young LINE-1s in normal tissues. Finally, we recover the complete sequences of tumor-specific LINE-1 insertions and their retrotransposition hallmarks, demonstrating how long-read sequencing can simultaneously survey the epigenome and detect somatic TE mobilization.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Transposable elements (TEs) drive genome evolution and are a notable source of pathogenesis, including cancer. While CpG methylation regulates TE activity, the locus-specific methylation landscape of mobile human TEs has to date proven largely inaccessible. Here, we apply new computational tools and long-read nanopore sequencing to directly infer CpG methylation of novel and extant TE insertions in hippocampus, heart, and liver, as well as paired tumor and non-tumor liver. As opposed to an indiscriminate stochastic process, we find pronounced demethylation of young long interspersed element 1 (LINE-1) retrotransposons in cancer, often distinct to the adjacent genome and other TEs. SINE-VNTR-Alu (SVA) retrotransposons, including their internal tandem repeat-associated CpG island, are near-universally methylated. We encounter allele-specific TE methylation and demethylation of aberrantly expressed young LINE-1s in normal tissues. Finally, we recover the complete sequences of tumor-specific LINE-1 insertions and their retrotransposition hallmarks, demonstrating how long-read sequencing can simultaneously survey the epigenome and detect somatic TE mobilization. | |
Cheetham, Seth W; Faulkner, Geoffrey J; Dinger, Marcel E Overcoming challenges and dogmas to understand the functions of pseudogenes (Journal Article) Nature Reviews Genetics, 21 (3), pp. 191–201, 2020, ISSN: 1471-0064, (Number: 3 Publisher: Nature Publishing Group). (Abstract | Links | BibTeX | Altmetric) @article{cheetham_overcoming_2020, title = {Overcoming challenges and dogmas to understand the functions of pseudogenes}, author = {Seth W Cheetham and Geoffrey J Faulkner and Marcel E Dinger}, url = {https://www.nature.com/articles/s41576-019-0196-1}, doi = {10.1038/s41576-019-0196-1}, issn = {1471-0064}, year = {2020}, date = {2020-01-01}, urldate = {2020-11-30}, journal = {Nature Reviews Genetics}, volume = {21}, number = {3}, pages = {191--201}, abstract = {Pseudogenes are defined as regions of the genome that contain defective copies of genes. They exist across almost all forms of life, and in mammalian genomes are annotated in similar numbers to recognized protein-coding genes. Although often presumed to lack function, growing numbers of pseudogenes are being found to play important biological roles. In consideration of their evolutionary origins and inherent limitations in genome annotation practices, we posit that pseudogenes have been classified on a scientifically unsubstantiated basis. We reflect that a broad misunderstanding of pseudogenes, perpetuated in part by the pejorative inference of the ‘pseudogene’ label, has led to their frequent dismissal from functional assessment and exclusion from genomic analyses. With the advent of technologies that simplify the study of pseudogenes, we propose that an objective reassessment of these genomic elements will reveal valuable insights into genome function and evolution.}, note = {Number: 3 Publisher: Nature Publishing Group}, keywords = {}, pubstate = {published}, tppubtype = {article} } Pseudogenes are defined as regions of the genome that contain defective copies of genes. They exist across almost all forms of life, and in mammalian genomes are annotated in similar numbers to recognized protein-coding genes. Although often presumed to lack function, growing numbers of pseudogenes are being found to play important biological roles. In consideration of their evolutionary origins and inherent limitations in genome annotation practices, we posit that pseudogenes have been classified on a scientifically unsubstantiated basis. We reflect that a broad misunderstanding of pseudogenes, perpetuated in part by the pejorative inference of the ‘pseudogene’ label, has led to their frequent dismissal from functional assessment and exclusion from genomic analyses. With the advent of technologies that simplify the study of pseudogenes, we propose that an objective reassessment of these genomic elements will reveal valuable insights into genome function and evolution. | |
2019 | |
Sanchez-Luque, Francisco J; Kempen, Marie-Jeanne H C; Gerdes, Patricia; Vargas-Landin, Dulce B; Richardson, Sandra R; Troskie, Robin-Lee; Jesuadian, Samuel J; Cheetham, Seth W; Carreira, Patricia E; Salvador-Palomeque, Carmen; García-Cañadas, Marta; Muñoz-Lopez, Martin; Sanchez, Laura; Lundberg, Mischa; Macia, Angela; Heras, Sara R; Brennan, Paul M; Lister, Ryan; Garcia-Perez, Jose L; Ewing, Adam D; Faulkner, Geoffrey J LINE-1 Evasion of Epigenetic Repression in Humans (Journal Article) Molecular Cell, 0 (0), 2019, ISSN: 1097-2765. (Abstract | Links | BibTeX | Altmetric) @article{sanchez-luque_line-1_2019, title = {LINE-1 Evasion of Epigenetic Repression in Humans}, author = {Francisco J Sanchez-Luque and Marie-Jeanne H C Kempen and Patricia Gerdes and Dulce B Vargas-Landin and Sandra R Richardson and Robin-Lee Troskie and Samuel J Jesuadian and Seth W Cheetham and Patricia E Carreira and Carmen Salvador-Palomeque and Marta Garc\'{i}a-Ca\~{n}adas and Martin Mu\~{n}oz-Lopez and Laura Sanchez and Mischa Lundberg and Angela Macia and Sara R Heras and Paul M Brennan and Ryan Lister and Jose L Garcia-Perez and Adam D Ewing and Geoffrey J Faulkner}, url = {https://www.cell.com/molecular-cell/abstract/S1097-2765(19)30396-X}, doi = {10.1016/j.molcel.2019.05.024}, issn = {1097-2765}, year = {2019}, date = {2019-01-01}, urldate = {2019-06-24}, journal = {Molecular Cell}, volume = {0}, number = {0}, abstract = {textlessh2textgreaterSummarytextless/h2textgreatertextlessptextgreaterEpigenetic silencing defends against LINE-1 (L1) retrotransposition in mammalian cells. However, the mechanisms that repress young L1 families and how L1 escapes to cause somatic genome mosaicism in the brain remain unclear. Here we report that a conserved Yin Yang 1 (YY1) transcription factor binding site mediates L1 promoter DNA methylation in pluripotent and differentiated cells. By analyzing 24 hippocampal neurons with three distinct single-cell genomic approaches, we characterized and validated a somatic L1 insertion bearing a 3ʹ transduction. The source (donor) L1 for this insertion was slightly 5ʹ truncated, lacked the YY1 binding site, and was highly mobile when tested textitin vitro. Locus-specific bisulfite sequencing revealed that the donor L1 and other young L1s with mutated YY1 binding sites were hypomethylated in embryonic stem cells, during neurodifferentiation, and in liver and brain tissue. These results explain how L1 can evade repression and retrotranspose in the human body.textless/ptextgreater}, keywords = {}, pubstate = {published}, tppubtype = {article} } textlessh2textgreaterSummarytextless/h2textgreatertextlessptextgreaterEpigenetic silencing defends against LINE-1 (L1) retrotransposition in mammalian cells. However, the mechanisms that repress young L1 families and how L1 escapes to cause somatic genome mosaicism in the brain remain unclear. Here we report that a conserved Yin Yang 1 (YY1) transcription factor binding site mediates L1 promoter DNA methylation in pluripotent and differentiated cells. By analyzing 24 hippocampal neurons with three distinct single-cell genomic approaches, we characterized and validated a somatic L1 insertion bearing a 3ʹ transduction. The source (donor) L1 for this insertion was slightly 5ʹ truncated, lacked the YY1 binding site, and was highly mobile when tested textitin vitro. Locus-specific bisulfite sequencing revealed that the donor L1 and other young L1s with mutated YY1 binding sites were hypomethylated in embryonic stem cells, during neurodifferentiation, and in liver and brain tissue. These results explain how L1 can evade repression and retrotranspose in the human body.textless/ptextgreater | |
Aughey, Gabriel N; Cheetham, Seth W; Southall, Tony D DamID as a versatile tool for understanding gene regulation (Journal Article) Development, 146 (6), 2019, ISSN: 1477-9129. (Abstract | Links | BibTeX | Altmetric) @article{aughey_damid_2019, title = {DamID as a versatile tool for understanding gene regulation}, author = {Gabriel N Aughey and Seth W Cheetham and Tony D Southall}, doi = {10.1242/dev.173666}, issn = {1477-9129}, year = {2019}, date = {2019-01-01}, journal = {Development}, volume = {146}, number = {6}, abstract = {The interaction of proteins and RNA with chromatin underlies the regulation of gene expression. The ability to profile easily these interactions is fundamental for understanding chromatin biology in vivo DNA adenine methyltransferase identification (DamID) profiles genome-wide protein-DNA interactions without antibodies, fixation or protein pull-downs. Recently, DamID has been adapted for applications beyond simple assaying of protein-DNA interactions, such as for studying RNA-chromatin interactions, chromatin accessibility and long-range chromosome interactions. Here, we provide an overview of DamID and introduce improvements to the technology, discuss their applications and compare alternative methodologies.}, keywords = {}, pubstate = {published}, tppubtype = {article} } The interaction of proteins and RNA with chromatin underlies the regulation of gene expression. The ability to profile easily these interactions is fundamental for understanding chromatin biology in vivo DNA adenine methyltransferase identification (DamID) profiles genome-wide protein-DNA interactions without antibodies, fixation or protein pull-downs. Recently, DamID has been adapted for applications beyond simple assaying of protein-DNA interactions, such as for studying RNA-chromatin interactions, chromatin accessibility and long-range chromosome interactions. Here, we provide an overview of DamID and introduce improvements to the technology, discuss their applications and compare alternative methodologies. | |
2018 | |
Cheetham, Seth W; Brand, Andrea H RNA-DamID reveals cell-type-specific binding of roX RNAs at chromatin-entry sites (Journal Article) Nature Structural & Molecular Biology, 25 (1), pp. 109–114, 2018, ISSN: 1545-9985. (Abstract | Links | BibTeX | Altmetric) @article{cheetham_rna-damid_2018, title = {RNA-DamID reveals cell-type-specific binding of roX RNAs at chromatin-entry sites}, author = {Seth W Cheetham and Andrea H Brand}, doi = {10.1038/s41594-017-0006-4}, issn = {1545-9985}, year = {2018}, date = {2018-01-01}, journal = {Nature Structural & Molecular Biology}, volume = {25}, number = {1}, pages = {109--114}, abstract = {Thousands of long noncoding RNAs (lncRNAs) have been identified in eukaryotic genomes, many of which are expressed in spatially and temporally restricted patterns. Nonetheless, the roles of the majority of these transcripts are still unknown. One of the mechanisms by which lncRNAs function is through the modulation of chromatin states. To assess the functions of lncRNAs, we developed RNA-DamID, a novel approach that detects lncRNA-genome interactions in a cell-type-specific manner in vivo with high sensitivity and accuracy. Identifying the cell-type-specific genome occupancy of lncRNAs is vital to understanding their mechanisms of action in development and disease. We used RNA-DamID to investigate targeting of the lncRNAs in the Drosophila dosage-compensation complex (DCC) and show that initial targeting is cell-type specific.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Thousands of long noncoding RNAs (lncRNAs) have been identified in eukaryotic genomes, many of which are expressed in spatially and temporally restricted patterns. Nonetheless, the roles of the majority of these transcripts are still unknown. One of the mechanisms by which lncRNAs function is through the modulation of chromatin states. To assess the functions of lncRNAs, we developed RNA-DamID, a novel approach that detects lncRNA-genome interactions in a cell-type-specific manner in vivo with high sensitivity and accuracy. Identifying the cell-type-specific genome occupancy of lncRNAs is vital to understanding their mechanisms of action in development and disease. We used RNA-DamID to investigate targeting of the lncRNAs in the Drosophila dosage-compensation complex (DCC) and show that initial targeting is cell-type specific. | |
Cheetham, Seth W; Gruhn, Wolfram H; van den Ameele, Jelle; Krautz, Robert; Southall, Tony D; Kobayashi, Toshihiro; Surani, Azim M; Brand, Andrea H Targeted DamID reveals differential binding of mammalian pluripotency factors (Journal Article) Development, 145 (20), 2018, ISSN: 1477-9129. (Abstract | Links | BibTeX | Altmetric) @article{cheetham_targeted_2018, title = {Targeted DamID reveals differential binding of mammalian pluripotency factors}, author = {Seth W Cheetham and Wolfram H Gruhn and Jelle van den Ameele and Robert Krautz and Tony D Southall and Toshihiro Kobayashi and Azim M Surani and Andrea H Brand}, doi = {10.1242/dev.170209}, issn = {1477-9129}, year = {2018}, date = {2018-01-01}, journal = {Development}, volume = {145}, number = {20}, abstract = {The precise control of gene expression by transcription factor networks is crucial to organismal development. The predominant approach for mapping transcription factor-chromatin interactions has been chromatin immunoprecipitation (ChIP). However, ChIP requires a large number of homogeneous cells and antisera with high specificity. A second approach, DamID, has the drawback that high levels of Dam methylase are toxic. Here, we modify our targeted DamID approach (TaDa) to enable cell type-specific expression in mammalian systems, generating an inducible system (mammalian TaDa or MaTaDa) to identify genome-wide protein/DNA interactions in 100 to 1000 times fewer cells than ChIP-based approaches. We mapped the binding sites of two key pluripotency factors, OCT4 and PRDM14, in mouse embryonic stem cells, epiblast-like cells and primordial germ cell-like cells (PGCLCs). PGCLCs are an important system for elucidating primordial germ cell development in mice. We monitored PRDM14 binding during the specification of PGCLCs, identifying direct targets of PRDM14 that are key to understanding its crucial role in PGCLC development. We show that MaTaDa is a sensitive and accurate method for assessing cell type-specific transcription factor binding in limited numbers of cells.}, keywords = {}, pubstate = {published}, tppubtype = {article} } The precise control of gene expression by transcription factor networks is crucial to organismal development. The predominant approach for mapping transcription factor-chromatin interactions has been chromatin immunoprecipitation (ChIP). However, ChIP requires a large number of homogeneous cells and antisera with high specificity. A second approach, DamID, has the drawback that high levels of Dam methylase are toxic. Here, we modify our targeted DamID approach (TaDa) to enable cell type-specific expression in mammalian systems, generating an inducible system (mammalian TaDa or MaTaDa) to identify genome-wide protein/DNA interactions in 100 to 1000 times fewer cells than ChIP-based approaches. We mapped the binding sites of two key pluripotency factors, OCT4 and PRDM14, in mouse embryonic stem cells, epiblast-like cells and primordial germ cell-like cells (PGCLCs). PGCLCs are an important system for elucidating primordial germ cell development in mice. We monitored PRDM14 binding during the specification of PGCLCs, identifying direct targets of PRDM14 that are key to understanding its crucial role in PGCLC development. We show that MaTaDa is a sensitive and accurate method for assessing cell type-specific transcription factor binding in limited numbers of cells. | |
2017 | |
Gloss, Brian S; Signal, Bethany; Cheetham, Seth W; Gruhl, Franziska; Kaczorowski, Dominik C; Perkins, Andrew C; Dinger, Marcel E High resolution temporal transcriptomics of mouse embryoid body development reveals complex expression dynamics of coding and noncoding loci (Journal Article) Scientific Reports, 7 (1), pp. 6731, 2017, ISSN: 2045-2322. (Abstract | Links | BibTeX | Altmetric) @article{gloss_high_2017, title = {High resolution temporal transcriptomics of mouse embryoid body development reveals complex expression dynamics of coding and noncoding loci}, author = {Brian S Gloss and Bethany Signal and Seth W Cheetham and Franziska Gruhl and Dominik C Kaczorowski and Andrew C Perkins and Marcel E Dinger}, url = {https://www.nature.com/articles/s41598-017-06110-5}, doi = {10.1038/s41598-017-06110-5}, issn = {2045-2322}, year = {2017}, date = {2017-01-01}, urldate = {2017-12-11}, journal = {Scientific Reports}, volume = {7}, number = {1}, pages = {6731}, abstract = {Cellular responses to stimuli are rapid and continuous and yet the vast majority of investigations of transcriptional responses during developmental transitions typically use long interval time courses; limiting the available interpretive power. Moreover, such experiments typically focus on protein-coding transcripts, ignoring the important impact of long noncoding RNAs. We therefore evaluated coding and noncoding expression dynamics at unprecedented temporal resolution (6-hourly) in differentiating mouse embryonic stem cells and report new insight into molecular processes and genome organization. We present a highly resolved differentiation cascade that exhibits coding and noncoding transcriptional alterations, transcription factor network interactions and alternative splicing events, little of which can be resolved by long-interval developmental time-courses. We describe novel short lived and cycling patterns of gene expression and dissect temporally ordered gene expression changes in response to transcription factors. We elucidate patterns in gene co-expression across the genome, describe asynchronous transcription at bidirectional promoters and functionally annotate known and novel regulatory lncRNAs. These findings highlight the complex and dynamic molecular events underlying mammalian differentiation that can only be observed though a temporally resolved time course.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Cellular responses to stimuli are rapid and continuous and yet the vast majority of investigations of transcriptional responses during developmental transitions typically use long interval time courses; limiting the available interpretive power. Moreover, such experiments typically focus on protein-coding transcripts, ignoring the important impact of long noncoding RNAs. We therefore evaluated coding and noncoding expression dynamics at unprecedented temporal resolution (6-hourly) in differentiating mouse embryonic stem cells and report new insight into molecular processes and genome organization. We present a highly resolved differentiation cascade that exhibits coding and noncoding transcriptional alterations, transcription factor network interactions and alternative splicing events, little of which can be resolved by long-interval developmental time-courses. We describe novel short lived and cycling patterns of gene expression and dissect temporally ordered gene expression changes in response to transcription factors. We elucidate patterns in gene co-expression across the genome, describe asynchronous transcription at bidirectional promoters and functionally annotate known and novel regulatory lncRNAs. These findings highlight the complex and dynamic molecular events underlying mammalian differentiation that can only be observed though a temporally resolved time course. | |
2016 | |
Marshall, Owen J; Southall, Tony D; Cheetham, Seth W; Brand, Andrea H Cell-type-specific profiling of protein-DNA interactions without cell isolation using targeted DamID with next-generation sequencing (Journal Article) Nature Protocols, 11 (9), pp. 1586–1598, 2016, ISSN: 1750-2799. (Abstract | Links | BibTeX | Altmetric) @article{marshall_cell-type-specific_2016, title = {Cell-type-specific profiling of protein-DNA interactions without cell isolation using targeted DamID with next-generation sequencing}, author = {Owen J Marshall and Tony D Southall and Seth W Cheetham and Andrea H Brand}, doi = {10.1038/nprot.2016.084}, issn = {1750-2799}, year = {2016}, date = {2016-01-01}, journal = {Nature Protocols}, volume = {11}, number = {9}, pages = {1586--1598}, abstract = {This protocol is an extension to: Nat. Protoc. 2, 1467-1478 (2007); doi:10.1038/nprot.2007.148; published online 7 June 2007The ability to profile transcription and chromatin binding in a cell-type-specific manner is a powerful aid to understanding cell-fate specification and cellular function in multicellular organisms. We recently developed targeted DamID (TaDa) to enable genome-wide, cell-type-specific profiling of DNA- and chromatin-binding proteins in vivo without cell isolation. As a protocol extension, this article describes substantial modifications to an existing protocol, and it offers additional applications. TaDa builds upon DamID, a technique for detecting genome-wide DNA-binding profiles of proteins, by coupling it with the GAL4 system in Drosophila to enable both temporal and spatial resolution. TaDa ensures that Dam-fusion proteins are expressed at very low levels, thus avoiding toxicity and potential artifacts from overexpression. The modifications to the core DamID technique presented here also increase the speed of sample processing and throughput, and adapt the method to next-generation sequencing technology. TaDa is robust, reproducible and highly sensitive. Compared with other methods for cell-type-specific profiling, the technique requires no cell-sorting, cross-linking or antisera, and binding profiles can be generated from as few as 10,000 total induced cells. By profiling the genome-wide binding of RNA polymerase II (Pol II), TaDa can also identify transcribed genes in a cell-type-specific manner. Here we describe a detailed protocol for carrying out TaDa experiments and preparing the material for next-generation sequencing. Although we developed TaDa in Drosophila, it should be easily adapted to other organisms with an inducible expression system. Once transgenic animals are obtained, the entire experimental procedure-from collecting tissue samples to generating sequencing libraries-can be accomplished within 5 d.}, keywords = {}, pubstate = {published}, tppubtype = {article} } This protocol is an extension to: Nat. Protoc. 2, 1467-1478 (2007); doi:10.1038/nprot.2007.148; published online 7 June 2007The ability to profile transcription and chromatin binding in a cell-type-specific manner is a powerful aid to understanding cell-fate specification and cellular function in multicellular organisms. We recently developed targeted DamID (TaDa) to enable genome-wide, cell-type-specific profiling of DNA- and chromatin-binding proteins in vivo without cell isolation. As a protocol extension, this article describes substantial modifications to an existing protocol, and it offers additional applications. TaDa builds upon DamID, a technique for detecting genome-wide DNA-binding profiles of proteins, by coupling it with the GAL4 system in Drosophila to enable both temporal and spatial resolution. TaDa ensures that Dam-fusion proteins are expressed at very low levels, thus avoiding toxicity and potential artifacts from overexpression. The modifications to the core DamID technique presented here also increase the speed of sample processing and throughput, and adapt the method to next-generation sequencing technology. TaDa is robust, reproducible and highly sensitive. Compared with other methods for cell-type-specific profiling, the technique requires no cell-sorting, cross-linking or antisera, and binding profiles can be generated from as few as 10,000 total induced cells. By profiling the genome-wide binding of RNA polymerase II (Pol II), TaDa can also identify transcribed genes in a cell-type-specific manner. Here we describe a detailed protocol for carrying out TaDa experiments and preparing the material for next-generation sequencing. Although we developed TaDa in Drosophila, it should be easily adapted to other organisms with an inducible expression system. Once transgenic animals are obtained, the entire experimental procedure-from collecting tissue samples to generating sequencing libraries-can be accomplished within 5 d. | |
Bell, Charles C; Amaral, Paulo P; Kalsbeek, Anton; Magor, Graham W; Gillinder, Kevin R; Tangermann, Pierre; di Lisio, Lorena; Cheetham, Seth W; Gruhl, Franziska; Frith, Jessica; Tallack, Michael R; Ru, Ke-Lin; Crawford, Joanna; Mattick, John S; Dinger, Marcel E; Perkins, Andrew C The Evx1/Evx1as gene locus regulates anterior-posterior patterning during gastrulation (Journal Article) Scientific Reports, 6 , pp. 26657, 2016, ISSN: 2045-2322. (Abstract | Links | BibTeX | Altmetric) @article{bell_evx1/evx1as_2016, title = {The Evx1/Evx1as gene locus regulates anterior-posterior patterning during gastrulation}, author = {Charles C Bell and Paulo P Amaral and Anton Kalsbeek and Graham W Magor and Kevin R Gillinder and Pierre Tangermann and Lorena di Lisio and Seth W Cheetham and Franziska Gruhl and Jessica Frith and Michael R Tallack and Ke-Lin Ru and Joanna Crawford and John S Mattick and Marcel E Dinger and Andrew C Perkins}, doi = {10.1038/srep26657}, issn = {2045-2322}, year = {2016}, date = {2016-01-01}, journal = {Scientific Reports}, volume = {6}, pages = {26657}, abstract = {Thousands of sense-antisense mRNA-lncRNA gene pairs occur in the mammalian genome. While there is usually little doubt about the function of the coding transcript, the function of the lncRNA partner is mostly untested. Here we examine the function of the homeotic Evx1-Evx1as gene locus. Expression is tightly co-regulated in posterior mesoderm of mouse embryos and in embryoid bodies. Expression of both genes is enhanced by BMP4 and WNT3A, and reduced by Activin. We generated a suite of deletions in the locus by CRISPR-Cas9 editing. We show EVX1 is a critical downstream effector of BMP4 and WNT3A with respect to patterning of posterior mesoderm. The lncRNA, Evx1as arises from alternative promoters and is difficult to fully abrogate by gene editing or siRNA approaches. Nevertheless, we were able to generate a large 2.6 kb deletion encompassing the shared promoter with Evx1 and multiple additional exons of Evx1as. This led to an identical dorsal-ventral patterning defect to that generated by micro-deletion in the DNA-binding domain of EVX1. Thus, Evx1as has no function independent of EVX1, and is therefore unlikely to act in trans. We predict many antisense lncRNAs have no specific trans function, possibly only regulating the linked coding genes in cis.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Thousands of sense-antisense mRNA-lncRNA gene pairs occur in the mammalian genome. While there is usually little doubt about the function of the coding transcript, the function of the lncRNA partner is mostly untested. Here we examine the function of the homeotic Evx1-Evx1as gene locus. Expression is tightly co-regulated in posterior mesoderm of mouse embryos and in embryoid bodies. Expression of both genes is enhanced by BMP4 and WNT3A, and reduced by Activin. We generated a suite of deletions in the locus by CRISPR-Cas9 editing. We show EVX1 is a critical downstream effector of BMP4 and WNT3A with respect to patterning of posterior mesoderm. The lncRNA, Evx1as arises from alternative promoters and is difficult to fully abrogate by gene editing or siRNA approaches. Nevertheless, we were able to generate a large 2.6 kb deletion encompassing the shared promoter with Evx1 and multiple additional exons of Evx1as. This led to an identical dorsal-ventral patterning defect to that generated by micro-deletion in the DNA-binding domain of EVX1. Thus, Evx1as has no function independent of EVX1, and is therefore unlikely to act in trans. We predict many antisense lncRNAs have no specific trans function, possibly only regulating the linked coding genes in cis. | |
2014 | |
Otsuki, Leo; Cheetham, Seth W; Brand, Andrea H Freedom of expression: cell-type-specific gene profiling (Journal Article) Wiley Interdisciplinary Reviews. Developmental Biology, 3 (6), pp. 429–443, 2014, ISSN: 1759-7692. (Abstract | Links | BibTeX | Altmetric) @article{otsuki_freedom_2014, title = {Freedom of expression: cell-type-specific gene profiling}, author = {Leo Otsuki and Seth W Cheetham and Andrea H Brand}, doi = {10.1002/wdev.149}, issn = {1759-7692}, year = {2014}, date = {2014-01-01}, journal = {Wiley Interdisciplinary Reviews. Developmental Biology}, volume = {3}, number = {6}, pages = {429--443}, abstract = {Cell fate and behavior are results of differential gene regulation, making techniques to profile gene expression in specific cell types highly desirable. Many methods now enable investigation at the DNA, RNA and protein level. This review introduces the most recent and popular techniques, and discusses key issues influencing the choice between these such as ease, cost and applicability of information gained. Interdisciplinary collaborations will no doubt contribute further advances, including not just in single cell type but single-cell expression profiling.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Cell fate and behavior are results of differential gene regulation, making techniques to profile gene expression in specific cell types highly desirable. Many methods now enable investigation at the DNA, RNA and protein level. This review introduces the most recent and popular techniques, and discusses key issues influencing the choice between these such as ease, cost and applicability of information gained. Interdisciplinary collaborations will no doubt contribute further advances, including not just in single cell type but single-cell expression profiling. | |
2013 | |
Cheetham, Seth W; Brand, Andrea H Insulin Finds Its Niche (Journal Article) Science, 340 (6134), pp. 817–818, 2013, ISSN: 0036-8075, 1095-9203. (Abstract | Links | BibTeX | Altmetric) @article{cheetham_insulin_2013, title = {Insulin Finds Its Niche}, author = {Seth W Cheetham and Andrea H Brand}, url = {http://science.sciencemag.org/content/340/6134/817}, doi = {10.1126/science.1238525}, issn = {0036-8075, 1095-9203}, year = {2013}, date = {2013-05-01}, urldate = {2017-12-11}, journal = {Science}, volume = {340}, number = {6134}, pages = {817--818}, abstract = {Coordination of organ growth and metabolism in response to changing environmental conditions is essential for physiological homeostasis. A central metabolic control mechanism in multicellular organisms is insulin signaling. Under conditions of elevated blood sugar, insulin promotes the storage of glucose in tissues such as muscle, fat, and liver. Classically, the role of insulin signaling is systemic. In mammals, insulin is produced by pancreatic beta cells and released into the bloodstream in response to increased concentrations of blood glucose, inducing global changes in growth and metabolism. Intriguingly, recent studies have demonstrated that insulin signaling can also occur locally, over a short range. Why have local insulin signaling? Local signals allow organ-specific, rather than organismal responses to changing environmental conditions (see the figure). This allows the modulation of the growth and development of individual tissues to be separately controlled, and raises the question of whether this phenomenon could be exploited for therapeutic strategies. Many of these recent findings have arisen from research in invertebrates; however, there are striking parallels in mammals. Localized insulin signaling allows organ-specific rather than organism-level responses to the environmental conditions. Localized insulin signaling allows organ-specific rather than organism-level responses to the environmental conditions.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Coordination of organ growth and metabolism in response to changing environmental conditions is essential for physiological homeostasis. A central metabolic control mechanism in multicellular organisms is insulin signaling. Under conditions of elevated blood sugar, insulin promotes the storage of glucose in tissues such as muscle, fat, and liver. Classically, the role of insulin signaling is systemic. In mammals, insulin is produced by pancreatic beta cells and released into the bloodstream in response to increased concentrations of blood glucose, inducing global changes in growth and metabolism. Intriguingly, recent studies have demonstrated that insulin signaling can also occur locally, over a short range. Why have local insulin signaling? Local signals allow organ-specific, rather than organismal responses to changing environmental conditions (see the figure). This allows the modulation of the growth and development of individual tissues to be separately controlled, and raises the question of whether this phenomenon could be exploited for therapeutic strategies. Many of these recent findings have arisen from research in invertebrates; however, there are striking parallels in mammals. Localized insulin signaling allows organ-specific rather than organism-level responses to the environmental conditions. Localized insulin signaling allows organ-specific rather than organism-level responses to the environmental conditions. | |
Cheetham, S W; Gruhl, F; Mattick, J S; Dinger, M E Long noncoding RNAs and the genetics of cancer (Journal Article) British Journal of Cancer, 108 (12), pp. 2419–2425, 2013, ISSN: 1532-1827. (Abstract | Links | BibTeX | Altmetric) @article{cheetham_long_2013, title = {Long noncoding RNAs and the genetics of cancer}, author = {S W Cheetham and F Gruhl and J S Mattick and M E Dinger}, doi = {10.1038/bjc.2013.233}, issn = {1532-1827}, year = {2013}, date = {2013-01-01}, journal = {British Journal of Cancer}, volume = {108}, number = {12}, pages = {2419--2425}, abstract = {Cancer is a disease of aberrant gene expression. While the genetic causes of cancer have been intensively studied, it is becoming evident that a large proportion of cancer susceptibility cannot be attributed to variation in protein-coding sequences. This is highlighted by genome-wide association studies in cancer that reveal that more than 80% of cancer-associated SNPs occur in noncoding regions of the genome. In this review, we posit that a significant fraction of the genetic aetiology of cancer is exacted by noncoding regulatory sequences, particularly by long noncoding RNAs (lncRNAs). Recent studies indicate that several cancer risk loci are transcribed into lncRNAs and these transcripts play key roles in tumorigenesis. We discuss the epigenetic and other mechanisms through which lncRNAs function and how they contribute to each stage of cancer progression, understanding of which will be crucial for realising new opportunities in cancer diagnosis and treatment. Long noncoding RNAs play important roles in almost every aspect of cell biology from nuclear organisation and epigenetic regulation to post-transcriptional regulation and splicing, and we link these processes to the hallmarks and genetics of cancer. Finally, we highlight recent progress and future potential in the application of lncRNAs as therapeutic targets and diagnostic markers.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Cancer is a disease of aberrant gene expression. While the genetic causes of cancer have been intensively studied, it is becoming evident that a large proportion of cancer susceptibility cannot be attributed to variation in protein-coding sequences. This is highlighted by genome-wide association studies in cancer that reveal that more than 80% of cancer-associated SNPs occur in noncoding regions of the genome. In this review, we posit that a significant fraction of the genetic aetiology of cancer is exacted by noncoding regulatory sequences, particularly by long noncoding RNAs (lncRNAs). Recent studies indicate that several cancer risk loci are transcribed into lncRNAs and these transcripts play key roles in tumorigenesis. We discuss the epigenetic and other mechanisms through which lncRNAs function and how they contribute to each stage of cancer progression, understanding of which will be crucial for realising new opportunities in cancer diagnosis and treatment. Long noncoding RNAs play important roles in almost every aspect of cell biology from nuclear organisation and epigenetic regulation to post-transcriptional regulation and splicing, and we link these processes to the hallmarks and genetics of cancer. Finally, we highlight recent progress and future potential in the application of lncRNAs as therapeutic targets and diagnostic markers. | |
2012 | |
Gascoigne, Dennis K; Cheetham, Seth W; Cattenoz, Pierre B; Clark, Michael B; Amaral, Paulo P; Taft, Ryan J; Wilhelm, Dagmar; Dinger, Marcel E; Mattick, John S Bioinformatics (Oxford, England), 28 (23), pp. 3042–3050, 2012, ISSN: 1367-4811. (Abstract | Links | BibTeX | Altmetric) @article{gascoigne_pinstripe:_2012, title = {Pinstripe: a suite of programs for integrating transcriptomic and proteomic datasets identifies novel proteins and improves differentiation of protein-coding and non-coding genes}, author = {Dennis K Gascoigne and Seth W Cheetham and Pierre B Cattenoz and Michael B Clark and Paulo P Amaral and Ryan J Taft and Dagmar Wilhelm and Marcel E Dinger and John S Mattick}, doi = {10.1093/bioinformatics/bts582}, issn = {1367-4811}, year = {2012}, date = {2012-12-01}, journal = {Bioinformatics (Oxford, England)}, volume = {28}, number = {23}, pages = {3042--3050}, abstract = {MOTIVATION: Comparing transcriptomic data with proteomic data to identify protein-coding sequences is a long-standing challenge in molecular biology, one that is exacerbated by the increasing size of high-throughput datasets. To address this challenge, and thereby to improve the quality of genome annotation and understanding of genome biology, we have developed an integrated suite of programs, called Pinstripe. We demonstrate its application, utility and discovery power using transcriptomic and proteomic data from publicly available datasets. RESULTS: To demonstrate the efficacy of Pinstripe for large-scale analysis, we applied Pinstripe's reverse peptide mapping pipeline to a transcript library including de novo assembled transcriptomes from the human Illumina Body Atlas (IBA2) and GENCODE v10 gene annotations, and the EBI Proteomics Identifications Database (PRIDE) peptide database. This analysis identified 736 canonical open reading frames (ORFs) supported by three or more PRIDE peptide fragments that are positioned outside any known coding DNA sequence (CDS). Because of the unfiltered nature of the PRIDE database and high probability of false discovery, we further refined this list using independent evidence for translation, including the presence of a Kozak sequence or functional domains, synonymous/non-synonymous substitution ratios and ORF length. Using this integrative approach, we observed evidence of translation from a previously unknown let7e primary transcript, the archetypical lncRNA H19, and a homolog of RD3. Reciprocally, by exclusion of transcripts with mapped peptides or significant ORFs (textgreater80 codon), we identify 32 187 loci with RNAs longer than 2000 nt that are unlikely to encode proteins. AVAILABILITY AND IMPLEMENTATION: Pinstripe (pinstripe.matticklab.com) is freely available as source code or a Mono binary. Pinstripe is written in C# and runs under the Mono framework on Linux or Mac OS X, and both under Mono and .Net under Windows. CONTACT: [email protected] or [email protected] SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.}, keywords = {}, pubstate = {published}, tppubtype = {article} } MOTIVATION: Comparing transcriptomic data with proteomic data to identify protein-coding sequences is a long-standing challenge in molecular biology, one that is exacerbated by the increasing size of high-throughput datasets. To address this challenge, and thereby to improve the quality of genome annotation and understanding of genome biology, we have developed an integrated suite of programs, called Pinstripe. We demonstrate its application, utility and discovery power using transcriptomic and proteomic data from publicly available datasets. RESULTS: To demonstrate the efficacy of Pinstripe for large-scale analysis, we applied Pinstripe's reverse peptide mapping pipeline to a transcript library including de novo assembled transcriptomes from the human Illumina Body Atlas (IBA2) and GENCODE v10 gene annotations, and the EBI Proteomics Identifications Database (PRIDE) peptide database. This analysis identified 736 canonical open reading frames (ORFs) supported by three or more PRIDE peptide fragments that are positioned outside any known coding DNA sequence (CDS). Because of the unfiltered nature of the PRIDE database and high probability of false discovery, we further refined this list using independent evidence for translation, including the presence of a Kozak sequence or functional domains, synonymous/non-synonymous substitution ratios and ORF length. Using this integrative approach, we observed evidence of translation from a previously unknown let7e primary transcript, the archetypical lncRNA H19, and a homolog of RD3. Reciprocally, by exclusion of transcripts with mapped peptides or significant ORFs (textgreater80 codon), we identify 32 187 loci with RNAs longer than 2000 nt that are unlikely to encode proteins. AVAILABILITY AND IMPLEMENTATION: Pinstripe (pinstripe.matticklab.com) is freely available as source code or a Mono binary. Pinstripe is written in C# and runs under the Mono framework on Linux or Mac OS X, and both under Mono and .Net under Windows. CONTACT: [email protected] or [email protected] SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online. | |
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