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Francis Stewart - Epigenetic regulation and genomic engineering

  • 1986: PhD University of N.S.W., Australia
  • Postdoctoral work at the Deutsches Krebsforschungszentrum, Heidelberg
  • 1991-2001: Group leader at EMBL, Heidelberg
  • since 2001: Professor of Genomics, TU Dresden

 

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Previous and current research

Our work focuses on two complementary aspects of genomics,
(i) mechanisms of epigenetic regulation in eukaryotic chromatin and
(ii) technologies of genetic engineering.

EPIGENETIC REGULATION IN CHROMATIN.

Although the complete DNA sequence of an organism encodes the primary information, additional information is added by epigenetic regulation. In eukaryotic chromatin, epigenetic regulation is conveyed by covalent modifications of DNA (DNA methylation) and histone tails (acetylation, phosphorylation, methylation, ubiquitinylation). Much attention worldwide is now focused on the histone tails and the proposition that patterns of covalent modifications serve as an epigenetic code. Our approach to unravelling epigenetic mechanisms and hierarchies is based on complementary uses of the yeast, S. cerevisiae and the mouse as experimental systems. We apply advanced reverse genetic strategies, some of which were developed by us, to analyze select classes of epigenetic regulators in both organisms. In yeast, we are using protein-tagging and mass spectrometry to characterize complexes containing epigenetic regulators. Amongst other complexes that we have identified in the proteomic environment of chromatin, we have recently identified a new histone methyltransferase activity for lysine 4 of histone 3.

In mice, we are studying two candidate histone methyltransferases by knock-out and conditional strategies using Cre/lox, as well applying proteomic approaches to characterize the complexes. A future aspect of our mouse work is directed towards use of ES cell differentiation in culture as a model for epigenetic decisions and stem cell manipulations.

GENOMIC ENGINEERING

We have developed several aspects of genetic engineering technology using site specific and homologous recombination. We aim at more fluent manipulation of mammalian cells, particularly ES cells and in mice. Most recent work involves exploration and implementation of a novel homologous recombination system that we discovered in E.coli phages. This permits fluent engineering of BACs in E.coli, and may offer new routes for directly engineering eukaryotic cells.

Future prospects and goals

Further work on epigenetic regulators in eukaryotes will be accompanied by advanced engineering strategies to examine roles of epigenetic regulation in mammalian development, stem cells, ageing and disease.

Selected Publications

You can find the complete list of publications (with web links) here.

2012 - 2014

will be posted soon

2011

Skarnes, W. C., Rosen, B., West, A. P., Koutsourakis, M., Bushell, W., Iyer, V., Cox, R., Jackson, D., Severin, J., Biggs, P., Thomas, M., Mujica, A., Harrow, J., Fu, J., Nefedov, M., de Jong, P., Stewart, A. F. and Bradley, A. (2011). "A conditional knockout resource for genome-wide analysis of mouse gene function." Nature  474: 62-263.

Hofemeister, H., Ciotta, G., Fu, J., Seibert, P. M., Schulz, A., Maresca, M., Sarov, M., Anastassiadis, K. and Stewart, A. F. (2011). "Recombineering, transfection, Western, IP and ChIP methods for protein tagging via gene targeting or BAC transgenesis." Methods.

2010

Ciotta, G., Hofemeister, H., Maresca, M., Fu, J., Sarov, M., Anastassiadis, K. and Stewart, A. F. (2010). "Recombineering BAC transgenes for protein tagging." Methods.

Fu, J., Teucher, M., Anastassiadis, K., Skarnes, W. and Stewart, A. F. (2010). "A Recombineering Pipeline to Make Conditional Targeting Constructs." Methods Enzymol 477C: 125-144.

Anastassiadis, K., Glaser, S., Kranz, A., Bernhardt, K. and Stewart, A. F. (2010). "A Practical Summary of Site-Specific Recombination, Conditional Mutagenesis, and Tamoxifen Induction of CreERT2." Methods Enzymol 477C: 109-123.

Kranz, A., Fu, J., Duerschke, K., Weidlich, S., Naumann, R., Stewart, A. F. and Anastassiadis, K. (2010). "An improved Flp deleter mouse in C57Bl/6 based on Flpo recombinase." Genesis.

Maresca, M., Erler, A., Fu, J., Friedrich, A., Zhang, Y. and Stewart, A. F. (2010). "Single-stranded heteroduplex intermediates in lambda Red homologous recombination." BMC Mol Biol 11: 54.

Anastassiadis, K., Rostovskaya, M., Lubitz, S., Weidlich, S. and Stewart, A. F. (2010). "Precise conditional immortalization of mouse cells using tetracycline-regulated SV40 large T-antigen." Genesis.

2009

Buchanan, L., Durand-Dubief, B., Roguev, A., Sakalar, C., Wilhelm, B., Stralfors, A., Shevchenko, A., Aasland, R., Shevchenko, A., Ekwall, K. and Stewart, A. F. (2009). "The S.pombe JmjC-protein, Msc1, prevents H2A.Z localization in centromeric and subtelomeric chromatin domains." PLoS Genetics 5: e1000726.

Anastassiadis, K., J. Fu, C. Patsch, S. Hu, S. Weidlich, K. Duerschke, F. Buchholz, F. Edenhofer and A. F. Stewart (2009). "Dre recombinase, like Cre, is a highly efficient site-specific recombinase in E. coli, mammalian cells and mice." Dis Model Mech 2: 508-15.

Cambridge, S. B., D. Geissler, F. Calegari, K. Anastassiadis, M. T. Hasan, A. F. Stewart, W. B. Huttner, V. Hagen and T. Bonhoeffer (2009). "Doxycycline-dependent photoactivated gene expression in eukaryotic systems." Nat Methods 6: 527-31.

Ding, L., M. Paszkowski-Rogacz, A. Nitzsche, M. M. Slabicki, A. K. Heninger, I. de Vries, R. Kittler, M. Junqueira, A. Shevchenko, H. Schulz, N. Hubner, M. X. Doss, A. Sachinidis, J. Hescheler, R. Iacone, K. Anastassiadis, A. F. Stewart, M. T. Pisabarro, A. Caldarelli, I. Poser, M. Theis and F. Buchholz (2009). "A genome-scale RNAi screen for Oct4 modulators defines a role of the Paf1 complex for embryonic stem cell identity." Cell Stem Cell 4: 403-15.

Erler, A., S. Wegmann, C. Elie-Caille, C. R. Bradshaw, M. Maresca, R. Seidel, B. Habermann, D. J. Muller and A. F. Stewart (2009). "Conformational adaptability of Redbeta during DNA annealing and implications for its structural relationship with Rad52." J Mol Biol 391: 586-98.

Glaser, S., S. Lubitz, K. L. Loveland, K. Ohbo, L. Robb, F. Schwenk, J. Seibler, D. Roellig, A. Kranz, K. Anastassiadis and A. F. Stewart (2009). "The histone 3 lysine 4 methyltransferase, Mll2, is only required briefly in development and spermatogenesis." Epigenetics Chromatin 2: 5.

2008

Fu, J., S. C. Wenzel, O. Perlova, J. Wang, F. Gross, Z. Tang, Y. Yin, A. F. Stewart, R. Muller and Y. Zhang (2008). "Efficient transfer of two large secondary metabolite pathway gene clusters into heterologous hosts by transposition." Nucleic Acids Res 36: e113.

Poser, I., M. Sarov, J. R. Hutchins, J. K. Heriche, Y. Toyoda, A. Pozniakovsky, D. Weigl, A. Nitzsche, B. Hegemann, A. W. Bird, L. Pelletier, R. Kittler, S. Hua, R. Naumann, M. Augsburg, M. M. Sykora, H. Hofemeister, Y. Zhang, K. Nasmyth, K. P. White, S. Dietzel, K. Mechtler, R. Durbin, A. F. Stewart, J. M. Peters, F. Buchholz and A. A. Hyman (2008). "BAC TransgeneOmics: a high-throughput method for exploration of protein function in mammals." Nat Methods 5: 409-15.

Shevchenko, A., A. Roguev, D. Schaft, L. Buchanan, B. Habermann, C. Sakalar, H. Thomas, N. J. Krogan and A. F. Stewart (2008). "Chromatin Central: towards the comparative proteome by accurate mapping of the yeast proteomic environment." Genome Biol 9: R167.

2007

Augui, S., G. J. Filion, S. Huart, E. Nora, M. Guggiari, M. Maresca, A. F. Stewart and E. Heard (2007). "Sensing X chromosome pairs before X inactivation via a novel X-pairing region of the Xic." Science 318: 1632-6.

Lubitz, S., S. Glaser, J. Schaft, A. F. Stewart and K. Anastassiadis (2007). "Increased apoptosis and skewed differentiation in mouse embryonic stem cells lacking the histone methyltransferase Mll2." Mol Biol Cell 18: 2356-66.

2006

Sarov, M., S. Schneider, A. Pozniakovski, A. Roguev, S. Ernst, Y. Zhang, A. A. Hyman and A. F. Stewart (2006). "A recombineering pipeline for functional genomics applied to Caenorhabditis elegans." Nat Methods 3: 839-44.

Sarov, M., S. Schneider, A. Pozniakovski, A. Roguev, S. Ernst, Y. Zhang, A. A. Hyman and A. F. Stewart (2006). "A recombineering pipeline for functional genomics applied to Caenorhabditis elegans." Nat Methods 3: 839-44.

2005

Glaser, S., K. Anastassiadis and A. F. Stewart (2005). "Current issues in mouse genome engineering." Nat Genet 37: 1187-93.

2004

Roguev, A., A. Shevchenko, D. Schaft, H. Thomas and A. F. Stewart (2004). "A comparative analysis of an orthologous proteomic environment in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe." Mol Cell Proteomics 3: 125-32.

Testa, G., J. Schaft, F. van der Hoeven, S. Glaser, K. Anastassiadis, Y. Zhang, T. Hermann, W. Stremmel and A. F. Stewart (2004). "A reliable lacZ expression reporter cassette for multipurpose, knockout-first alleles." Genesis 38: 151-8.

2003

Testa, G., Y. Zhang, K. Vintersten, V. Benes, W. W. Pijnappel, I. Chambers, A. J. Smith, A. G. Smith and A. F. Stewart (2003). "Engineering the mouse genome with bacterial artificial chromosomes to create multipurpose alleles." Nat Biotechnol 21: 443-7.

2002

Anastassiadis, K., J. Kim, N. Daigle, R. Sprengel, H. R. Scholer and A. F. Stewart (2002). "A predictable ligand regulated expression strategy for stably integrated transgenes in mammalian cells in culture." Gene 298: 159-72.

Casanova, E., S. Fehsenfeld, E. Greiner, A. F. Stewart and G. Schutz (2002). "Conditional mutagenesis of CamKIV." Genesis 32: 161-4.

2001

Muyrers, J. P., Y. Zhang and A. F. Stewart (2001). "Techniques: Recombinogenic engineering--new options for cloning and manipulating DNA." Trends Biochem Sci 26: 325-31.

Pijnappel, W. W., D. Schaft, A. Roguev, A. Shevchenko, H. Tekotte, M. Wilm, G. Rigaut, B. Seraphin, R. Aasland and A. F. Stewart (2001). "The S. cerevisiae SET3 complex includes two histone deacetylases, Hos2 and Hst1, and is a meiotic-specific repressor of the sporulation gene program." Genes Dev 15: 2991-3004.

Roguev, A., D. Schaft, A. Shevchenko, W. W. Pijnappel, M. Wilm, R. Aasland and A. F. Stewart (2001). "The Saccharomyces cerevisiae Set1 complex includes an Ash2 homologue and methylates histone 3 lysine 4." EMBO J 20: 7137-48.

Schaft, J., R. Ashery-Padan, F. van der Hoeven, P. Gruss and A. F. Stewart (2001). "Efficient FLP recombination in mouse ES cells and oocytes." Genesis 31: 6-10.

2000

Muyrers, J. P., Y. Zhang, F. Buchholz and A. F. Stewart (2000). "RecE/RecT and Redalpha/Redbeta initiate double-stranded break repair by specifically interacting with their respective partners." Genes Dev 14: 1971-82.

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