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Proteomics, Macromolecular Complexes, Transcriptional Regulatory Complexes: The Ranish Group
Many processes in cells involve large molecular complexes consisting of multiple proteins, nucleic acids, and small molecules. Unraveling the composition, structure, and function of these macromolecular machines is especially well suited to the comprehensive and high-throughput methods of systems biology.
Jeff Ranish's group at ISB uses this approach to study the dynamic composition and architecture of large molecular complexes involved in gene transcription. They first devise biochemical strategies to isolate the complexes, and they then employ mass spectrometers to identify and quantify the proteins that make up the complexes. They then track changes in the complexes during such processes as cell growth, division, or differentiation. Recently, he and the members of his group also have been using mass spectrometry to map the architecture of macromolecular complexes. They developed bi-functional cross-linking reagents to covalently connect parts of the complex that are in close proximity. They then identify each of the cross-linked proteins and their point of connection to determine which parts of the protein complex are near each other. Using powerful new software developed for this research, they piece together a model of the complex's overall architecture. One goal of this research is to model the structure of large transcriptional regulatory complexes such as co-activator and chromatin modifying complexes by integrating crosslinking data with information from structural studies, protein-interaction measurements, and other investigations. Understanding the processes that turn genes on and off will be essential in determining how healthy cells function and in preventing and treating disease.
A particular focus of their work has been the gene regulatory complexes which assemble at DNA sequences close to the coding region of a gene and transcribe the gene sequences into messenger RNA which is then used to make proteins. The group has employed quantitative proteomic approaches to the study composition of the complexes and to determine how their composition changes in response to environmental and cellular events. This research has uncovered previously unrecognized components of the gene regulatory complexes and changes in composition that occur, for example, during specific biological processes, such as the differentiation of hematopoietic stem cells into mature red blood cells.
This basic biological research can have immediate applications. For example, the group recently discovered a component of a transcription complex that is also involved in DNA repair processes. When the molecule is mutated, it can cause a rare genetic disease known as tricothiodystrophy disorder (TTD), which is characterized by brittle hair and nails, scaly skin, photosensitivity, developmental defects, and other symptoms. The result suggests that patients with mutations in the molecule have defects in the formation of transcription complexes, which in turn leads to altered patterns of gene expression.
Ranish, who studied transcription complexes at the University of Washington and the Fred Hutchinson Cancer Research Center in Seattle before coming to ISB, learned mass spectrometry specifically to investigate the composition of macromolecular complexes. Recently, he and his colleagues have developed two approaches to systematically study the ensemble of proteins associated with gene regulatory complexes. The first approach is based on selected reaction monitoring (SRM) mass spectrometry in which triple quadrupole mass spectrometers are used to specifically and selectively measure the proteins of interest. The approach provides the exquisite sensitivity that is needed to study these complexes. The second approach, called iMSTIQ, involves the use of synthetic peptides to trigger the acquisition of quantitative data on the peptides of interest in complex samples using a high mass accuracy LTQ-Orbitrap mass spectrometer. Like SRM, iMSTIQ also provides highly sensitive and reproducible quantitative measurements of targeted proteins. The Ranish group is now employing these assays to probe the composition of gene regulatory complexes in chromatin. It is expected that these studies will provide an in depth understanding of how genes are turned on and off, and this information will be essential in determining how cells function. Further, the information will guide efforts to reprogram cell behavior when dysregulation of gene expression results in disease.
Selected Recent Publications:
Akiyoshi B, Nelson CR, Ranish JA, Biggins S. (2009) Quantitative proteomic analysis of purified yeast kinetochores identifies a PP1 regulatory subunit. Genes & Development 23:2887-99.
Ho L, Ronan JL, Wu J, Staahl BT, Chen L, Kuo A, Lessard J, Nesvizhskii AI, Ranish J, Crabtree GR. (2009) An embryonic stem cell chromatin remodeling complex, esBAF, is essential for embryonic stem cell self-renewal and pluripotency. Proceedings of the National Academy of Sciences 106:5181-6.
Picotti P, Lam H, Campbell D, Deutsch EW, Mirzaei H, Ranish J, Domon B, Aebersold R.
(2008) A database of mass spectrometric assays for the yeast proteome. Nature Methods 5:913-4.
Ranish JA, Brand M, Aebersold R. (2007) Using stable isotope tagging and mass spectrometry to characterize protein complexes and to detect changes in their composition. Methods in Molecular Biology 359:17-35.
Kim B, Nesvizhskii AI, Rani PG, Hahn S, Aebersold R, Ranish JA. (2007) The transcription elongation factor TFIIS is a component of RNA polymerase II preinitiation complexes. Proceedings of the National Academy of Sciences 104:16068-73.
Ranish JA, Hahn S, Lu Y, Yi EC, Li XJ, Eng J, Aebersold R. (2004) Identification of TFB5, a new component of general transcription and DNA repair factor IIH. Nature Genetics 36:707-13.
Giglia-Mari G, Coin F, Ranish JA, Hoogstraten D, Theil A, Wijgers N, Jaspers NG, Raams A, Argentini M, van der Spek PJ, Botta E, Stefanini M, Egly JM, Aebersold R, Hoeijmakers JH, Vermeulen W. (2004) A new, tenth subunit of TFIIH is responsible for the DNA repair syndrome trichothiodystrophy group A. Nature Genetics 36:714-9.
Ranish JA, Yi EC, Leslie DM, Purvine SO, Goodlett DR, Eng J, Aebersold R. (2003) The study of macromolecular complexes by quantitative proteomics. Nature Genetics 33:349-55.
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Senior Research Scientist
Wei Yan
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Research Scientist
Jie Luo
Max Robinson
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Postdoctoral Fellow
Mark Gillespie
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Research Associate 3
Bong Kim
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Research Associate 2
Toby Ligon
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Project Coordinator
Darya Kappe
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Visiting Grad Student
Martin Frejno
Nitobe London
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