A. Francis Stewart

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A. Francis Stewart
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BornOctober 1956 (age 67)
NationalityAustralian
EducationUniversity of New South Wales (BSc Hons 1, PhD)
OrganizationGerman Cancer Research Center (DKFZ); European Molecular Biology Laboratories (EMBL), Technische Universität Dresden, Max Planck Institute of Molecular Cell Biology and Genetics, Shandong University, St Johns College, University of New South Wales
Known forGenetic engineering (conditional mutagenesis, recombineering); Epigenetics (histone 3 lysine 4 methylation)
Spouse(s)Michelle Meredyth-Stewart (MBBS, Dr. med.)
ChildrenAlex, Nick, Ian, Vivian

A. Francis Stewart (born 13 October 1956)[citation needed] is an Australian professor of biochemistry, genetics and mammalian developmental biology who spent most of his working life in Germany. He made contributions to Genetics by developing conditional mutagenesis (conditional gene knockout) and recombineering technologies. He made contributions to chromatin and epigenetics by discovering components of the histone 3 lysine 4 methyltransferase system. He was a group leader at EMBL (European Molecular Biology Laboratories), Heidelberg, 1991-2001 and a professor at TU Dresden University since 2001. He founded Gene Bridges GmbH in 2000 and GenArc Directions GmbH in 2019.

Biography and research

Francis Stewart is the son of Donald Stewart, who was Australia’s first qualified aeronautical engineer (University of Queensland, 1942) and became managing director of Hamersley Iron (Pilbara Iron) and Peko-Wallsend.

Francis Stewart was educated at The King’s School Parramatta and University of New South Wales, receiving a PhD in biochemistry in 1986 for cloning and sequencing the bovine milk protein genes in the lab of Prof. Anthony Mackinlay. On an Alexander-von-Humboldt Stipendium (Alexander-von-Humboldt Foundation), he pursued post-doctoral work in the lab of Prof. Günther Schütz at the German Cancer Research Center, Heidelberg, where he made two notable contributions to chromatin research; (I) proof that Topoisomerase 1 facilitates transcriptional elongation by relaxing positive supercoils ahead of the transcribing polymerase.[1]; and (II) the first description of rapid, replication-independent, nucleosome disassembly caused by transcription factor binding, and rapid reassembly upon unbinding [2]. Based on these accomplishments, Stewart began his independent career in 1991 as a group leader at EMBL to explore the idea that eukaryotic transcription is not only regulated by transcription factors but also by epigenetic mechanisms based on chromatin. To pursue this challenge in mammalian development, he realised that new methodologies were required. The Stewart lab developed genetic engineering technologies that have not only facilitated his, and other, epigenetic investigations but also contributed to biomedical research well beyond the original intentions.

Classical genetics is restricted to revealing the first developmental phenotype of a mutation. Conditional mutagenesis utilizes site specific recombinases (SSRs) to bypass this limitation. The Stewart lab enabled conditional mutagenesis in several ways. With his first PhD student, Colin Logie, they pioneered (and patented) the induction of an intended genetic change in a living cell by delivery of a ligand [3]. Using SSR-steroid ligand binding domain fusion proteins (SSR-LBDs), genetic enquiry can be directed to investigate function at any time. Together with their use as robust gene switches for inducible transgene expression, SSR-LBDs have enhanced functional enquiry in many eukaryotic systems including mice, fish, gene therapy, stem and cancer cells in culture, organoids, plants and yeast. In most mammalian applications, tamoxifen is the small molecule and the SSR-LBD is Cre fused to a mutated estrogen LBD [4]. By linking a genetic change to the time when a small molecule is administered, SSR-LBDs are particularly useful for explorations of adult diseases, including cancer and auto-immunity, as well as post-developmental issues such as homeostasis and aging.

The early years of conditional mutagenesis in the mouse were based on the remarkable properties of Cre recombinase. To expand the technology, a second SSR was required. Stewart was the first to realise that the other available SSR, FLP recombinase, was inadequate because this yeast enzyme is thermolabile. After his efforts to identify other SSRs encountered the same limitation, Frank Buchholz and Stewart used molecular evolution to establish a thermostable FLP [5], which is now in widespread use. With two SSRs, Stewart initiated a new class of allele design that was switchable (from null to conditional). This design was adopted by the International Knockout Mouse Consortium for systematic mutagenesis of the mouse genome [6] Amongst several advantages, the switchable allele design pioneered a new way to conclude that an allele is a null (that is, a complete loss of function), which is a critical genetic conclusion.

Designer nucleases, primarily CRISPR/Cas9, have revolutionized genome engineering. They are phenomenally efficient at site directed mutagenesis but suffer from the same limitation as classical genetics; that is, only the first mutational impact can be observed. Conditional mutagenesis is still required to bypass this limitation.

In the 1990’s, building recombinant DNA constructs for gene targeting was challenging and constructs including loxP sites (and later also FRT sites) for conditional mutagenesis even more so. To facilitate the building of complex constructs, Youming Zhang and Stewart pioneered a new recombinant DNA engineering method based on homologous recombination (HR) in E.coli mediated by phage proteins [7]. This technology, termed ‘recombineering’, not only facilitated the construction of complex alleles for conditional mutagenesis but also broke through the restrictive size limitations inherent in classical recombinant and PCR methods. Thereby fluent engineering of large plasmids (>15kb) like BACs (bacterial artificial chromosomes) became routine, which opened access to the comprehensive BAC resources created by the genome sequencing projects. Recombineered BACs not only provide more reliable transgenesis than small transgenes but also facilitated further sophistications of allele design and complex genome engineering exercises such as humanizations.[8][9]. In particular, converting BACs into transposons overturned the widely held dogma that transposons are severely limited to small cargos [10]. BAC transposons are now a premier vector for transgenesis.

While exploring recombineering methodology, Stewart realised that a simple protocol adjustment (using a nonreplicating R6K plasmid as PCR template) completely eliminated unwanted background so only the intended HR product survived, which eliminated the need for step-by-step, clone-by-clone, quality checking. This paved the way to high throughput DNA engineering for genome-scale projects, including high throughput protein tagging[11], the mouse genome conditional knock-out project [6] and gRNA expression libraries for Cas9 applications [12].

Recombineering is now both a cornerstone recombinant DNA methodology and the premier method for E.coli genome engineering including genome minimization, the complete E.coli knock-out library, tagging for systematic proteomics, and codon reassignment (by others) with further applications in synthetic biology and bioprospecting [13]. Recombineering continues to dominate bacterial genome engineering because most bacteria have no non-homologous end joining (NHEJ) machinery and Cas9 applications are lethal.

Genetic innovations in the Stewart lab have been facilitated by accompanying research on fundamental mechanisms including (i) quantitative biochemistry for mathematical modelling of SSR action [14] (ii) identification of a new paradigm for HR operating directly at the replication fork [15], (iii) investigations of single strand annealing proteins (SSAPs) and their role in the initiation of HR by DNA clamping [16], including the proposition that all SSAPs are ancestrally related thereby comprising a new protein superfamily - the RAD52 superfamily [17]. Evidence securing this proposition is recently published [18]

Stewart’s innovations are now integrated into molecular biology and biomedical research. However his motivations arose from the complex challenges posed by mammalian developmental epigenetics. Stewart chose a focus on Trithorax-Group (trxG) and Polycomb-group proteins|Polycomb-Group (PcG) action. These groups were genetically defined in Drosophila to oppose each other in the regulation of homeotic gene expression during fly development and were likely sources of the chromatin-based transcriptional regulatory mechanisms that he was seeking. While identifying mammalian trxG/PcG homologues, Rein Aasland and Stewart discovered several chromatin domains including the PHD finger [19], which is now known to bind methylated histone 3 lysine 4 (H3K4) epitopes. A yeast homologue for the trxG protein, Ash2, was also identified. Tagging and purifying the protein complex associated with Ash2 retrieved Set1, which includes the same SET domain as fly Trithorax. The purified yeast Set1/Ash2 complex was the first histone 3 lysine 4 methyltransferase (H3K4 MT) complex and the first connection between trxG action and H3K4 methylation [20]. The following year several papers linked PcG action with H3K27 methylation. Thereby the genetically defined opposition between trxG and PcG action found a biochemical basis in the mutual exclusion of methylation at H3K4 or H3K27, which sharply tuned the focus of epigenetic, and more recently cancer, research onto the lysine methylation status of the H3 tail (on key nucleosomes).

In addition to H3K4 methylation studies in yeast [21], Stewart has been systematically evaluating the six mammalian H3K4 MTs in the mouse using the tools developed by his lab [22]. With their publication of the Mll3 and Mll4 developmental nulls [23], the full set of six is completed. MLL3 and MLL4 are the largest known nuclear proteins (~5,000 amino acids) whose genes, as revealed by the international cancer genome sequencing projects, are mutated in almost all cancers. An explanation for this extraordinary mutagenic frequency is lacking.

Although the H3K4 system is a ubiquitous feature of transcription and central to epigenetic regulation, a unifying understanding remains to be found. The recent finding that MLL1 is required for intestinal stem cell maintenance [24] is revealing. MLL1 also plays this role in two other classic stem cells; hematopoietic stem cells and muscle satellite cells. Current work is focused on the proposition that the MLLs present an overlapping system that secures the gene expression status of stem cells and then subsequent decisions during lineage commitment, potentially by clustering and tethering.

Stewart was elected an EMBO Fellow in 2007. In 2010 he was awarded the annual International Society for Transgenic Technology Prize. He was a Beaufort Visiting Scholar at St John’s College, Cambridge (2019-2020).

TU Dresden

In 1999, Stewart was invited by Kai Simons to join his move from EMBL to Dresden. Simons was assembling a new Max Planck Institute (of Molecular Cell Biology and Genetics; MPI-CBG) in Dresden with co-directors Wieland Huttner, Tony Hyman and Marino Zerial. Simons invited Stewart to start an interdisciplinary research initiative at Technische Universität Dresden (TUD) utilizing Stewart’s molecular bioengineering concept. TUD’s first extramural research center, Biotechnology Center (Biotec) was established with Federal funding and Stewart the first appointment. With contributions from Stewart, Biotec has won major grants to foster three new research institutes; Center for Regenerative Therapies Dresden (CRTD; 2005); B Cube - Center for Molecular Bioengineering (bioprospecting; 2008) and Physics of Life (PoL; 2019). Together the four institutes secured more than €300 million of research funding to TUD and now comprise 57 research groups in a new campus close to the MPI-CBG. Concomitantly, TUD was promoted to be one of the eleven German Universities of Excellence in 2012, renewed in 2019. In 2002, Stewart co-established the first international Masters course at TUD ‘Molecular Bioengineering’ and initiated the Dresden Genome Center, which is now one of the four designated national NGS DNA sequencing facilities. He is chair of the advisory board. Stewart established the Mass Spectrometry Facility, which like the Genome Center is one of the Technology Platforms in the Dresden Concept. The Dresden Concept is serving as a new model in Germany for city-based sharing of advanced expertise and equipment, which is a substantial departure from former German academic practice.

Cricket

The German Cricket Federation (Deutscher Cricket Bund, DCB) was founded in 1988 and in 1989 Stewart was selected in the first German XI, to play v Denmark. In 1992 he became the second captain (and main selector) and led Germany to victory in the 2nd European Cricketer Cup in Worksop, Nottinghamshire, defeating Italy, Belgium, Switzerland, Austria and then France in the final. As victors, Germany played the MCC at Lord's|Lord's Cricket Ground the next day (July 17, 1992) and the German flag was raised over the visitors end of the members pavillion. In 2007, Stewart founded the Dresden Cricket Club, joining the rugby club to form Rugby Cricket Dresden (RCD) in 2008.

Webpage

References

  1. ↑ Stewart AF, Herrera RE, Nordheim A. Rapid induction of c-fos transcription reveals quantitative linkage of RNA polymerase II and DNA topoisomerase I enzyme activities. Cell. 1990;60(1):141-9. Epub 1990/01/12. doi: 10.1016/0092-8674(90)90724-s. PubMed PMID: 2153054. 167 citations.
  2. ↑ Reik A, Schütz G, Stewart AF. Glucocorticoids are required for establishment and maintenance of an alteration in chromatin structure: induction leads to a reversible disruption of nucleosomes over an enhancer. The EMBO Journal. 1991;10(9):2569-76. Epub 1991/09/01. doi: 10.1002/j.1460-2075.1991.tb07797.x. PubMed PMID: 1678348; PubMed Central PMCID: PMCPMC452954. 212 citations.
  3. ↑ Logie C, Stewart AF. Ligand-regulated site-specific recombination. Proceedings of the National Academy of Sciences of the United States of America. 1995;92(13):5940-4. Epub 1995/06/20. PubMed PMID: 7597057; PubMed Central PMCID: PMC41617. 224 citations.
  4. ↑ Schwenk F, Kuhn R, Angrand PO, Rajewsky K, Stewart AF. Temporally and spatially regulated somatic mutagenesis in mice. Nucleic Acids Research. 1998;26(6):1427-32. Epub 1998/04/29. PubMed PMID: 9490788; PubMed Central PMCID: PMC147429. 234 citations.
  5. ↑ Buchholz F, Angrand PO, Stewart AF. Improved properties of FLP recombinase evolved by cycling mutagenesis. Nature Biotechnology. 1998;16(7):657-62. Epub 1998/07/14. doi: 10.1038/nbt0798-657. PubMed PMID: 9661200. 536 citations.
  6. ↑ 6.0 6.1 Skarnes WC, Rosen B, West AP, Koutsourakis M, Bushell W, Iyer V, Cox T, Jackson D, Severin J, Biggs P, Thomas M, Mujica A, Harrow J, Fu J, Nefedov M, de Jong P, Stewart AF, Bradley A. A conditional knockout resource for the genome-wide study of mouse gene function. Nature. 2011;474(7351):337-42. Doi: 10.1038/nature10163|doi: 10.1038/nature10163. PubMed PMID: 21677750; PubMed Central PMCID: PMC3572410. 1704 citations. Two major programs, the EU funded EUCOMM (European Conditional Mouse Mutagenesis) and the NIH funded KOMP (Knock-out Mouse Project) co-operated to generate targeted multi-purpose conditional alleles in most (eventually 16,000) protein coding genes in mouse embryonic stem cells. The majority of the work was performed at WTSI headed by Bill Skarnes and Allan Bradley. Stewart was an author and partner of the EUCOMM proposal. His pioneering work on recombineering and its’ application in high throughput was crucial to the application of gene targeting at genome scale. The project also employed his switchable allele design employing site specific recombinases with a gene trap expression reporter.
  7. ↑ Zhang Y, Buchholz F, Muyrers JP, Stewart AF. A new logic for DNA engineering using recombination in Escherichia coli. Nature Genetics. 1998;20(2):123-8. Epub 1998/10/15. doi: 10.1038/2417. PubMed PMID: 9771703. 1565 citations.
  8. ↑ Testa G, Zhang Y, Vintersten K, Benes V, Pijnappel WW, Chambers I, Smith AJH, Smith AA, Stewart AF. Engineering the mouse genome with bacterial artificial chromosomes to create multipurpose alleles. Nature Biotechnology. 2003;21(4):443-7. doi: 10.1038/nbt804. PubMed PMID: 12627172. 149 citations.
  9. ↑ Baker O, Tsurkan S, Fu J, Klink B, Rump A, Obst M, Kranz A, Schröck E, Anastassiadis K, Stewart AF. The contribution of homology arms to nuclease-assisted genome engineering. Nucleic Acids Research. 2017;45(13):8105-15. Epub 2017/06/06. doi: 10.1093/nar/gkx497. PubMed PMID: 28582546; PubMed Central PMCID: PMC5570031. 32 citations.
  10. ↑ Rostovskaya M, Fu J, Obst M, Baer I, Weidlich S, Wang H, Smith AJH, Anastassiadis K, Stewart AF. Transposon-mediated BAC transgenesis in human ES cells. Nucleic Acids Research. 2012;40(19):e150. doi: 10.1093/nar/gks643. PubMed PMID: 22753106; PubMed Central PMCID: PMC3479164. 138 citations.
  11. ↑ Sarov M, Schneider S, Pozniakovski A, Roguev A, Ernst S, Zhang Y, Hyman AA, Stewart AF. A recombineering pipeline for functional genomics applied to Caenorhabditis elegans. Nature Methods. 2006;3(10):839-44. doi: 10.1038/nmeth933. PubMed PMID: 16990816. 219 citations.
  12. ↑ Lackner A, Sehlke R, Garmhausen M, Giuseppe Stirparo G, Huth M, Titz-Teixeira F, van der Lelij P, Ramesmayer J, Thomas HF, Ralser M, Santini L, Galimberti E, Sarov M, Stewart AF, Smith A, Beyer A, Leeb M. Cooperative genetic networks drive embryonic stem cell transition from naïve to formative pluripotency. The EMBO Journal. 2021;40(8):e105776. Epub 2021/03/10. doi: 10.15252/embj.2020105776. PubMed PMID: 33687089; PubMed Central PMCID: PMC8047444. 19 citations. This is a major paper from the SYBOSS (System Biology of Stem Cells) consortium (https://cordis.europa.eu/project/id/242129), which was an €11 million EU Integrated Project with 11 European partners. Stewart was the SYBOSS co-ordinator and wrote the paper with Smith, Beyer and Leeb.
  13. ↑ Fu J, Bian X, Hu S, Wang H, Huang F, Seibert PM, Plaza A, Xia L, Müller R, Stewart AF, Zhang Y. Full-length RecE enhances linear-linear homologous recombination and facilitates direct cloning for bioprospecting. Nature Biotechnology. 2012;30(5):440-6. doi: 10.1038/nbt.2183. PubMed PMID: 22544021. 443 citations.
  14. ↑ Ringrose L, Lounnas V, Ehrlich L, Buchholz F, Wade R, Stewart AF. Comparative kinetic analysis of FLP and cre recombinases: mathematical models for DNA binding and recombination. Journal of Molecular Biology. 1998;284(2):363-84. Epub 1998/11/14. doi: 10.1006/jmbi.1998.2149. PubMed PMID: 9813124. 157 citations.
  15. ↑ Maresca M, Erler A, Fu J, Friedrich A, Zhang Y, Stewart AF. Single-stranded heteroduplex intermediates in lambda Red homologous recombination. BMC Molecular Biology. 2010;11:54. Doi: 10.1186/1471-2199-11-54|doi: 10.1186/1471-2199-11-54. PubMed PMID: 20670401; PubMed Central PMCID: PMC2918612. 142 citations.
  16. ↑ Ander M, Subramaniam S, Fahmy K, Stewart AF, Schäffer E. A Single-Strand Annealing Protein Clamps DNA to Detect and Secure Homology. PLoS Biology. 2015;13(8):e1002213. doi: 10.1371/journal.pbio.1002213. PubMed PMID: 26271032; PubMed Central PMCID: PMC4535883. 23 citations.
  17. ↑ Erler A, Wegmann S, Elie-Caille C, Bradshaw CR, Maresca M, Seidel R, Habermann B, Muller D, Stewart AF. Conformational adaptability of Redbeta during DNA annealing and implications for its structural relationship with Rad52. Journal of Molecular Biology. 2009;391(3):586-98. doi: 10.1016/j.jmb.2009.06.030. PubMed PMID: 19527729. 77 citations.
  18. ↑ Al-Fatlawi A, Schroeder M, Stewart AF. The Rad52 SSAP superfamily and new insight into homologous recombination. Commun Biol. 2023;6(1):87. Epub 2023/01/24. doi: 10.1038/s42003-023-04476-z. PubMed PMID: 36690694; PubMed Central PMCID: PMCPMC9870868. 1 citation.
  19. ↑ Aasland R, Gibson TJ, Stewart AF. The PHD finger: implications for chromatin-mediated transcriptional regulation. Trends in Biochemical Sciences. 1995;20(2):56-9. Epub 1995/02/01. PubMed PMID: 7701562. 1091 citations.
  20. ↑ Roguev A, Schaft D, Shevchenko A, Pijnappel WW, Wilm M, Aasland R, Stewart AF. The Saccharomyces cerevisiae Set1 complex includes an Ash2 homologue and methylates histone 3 lysine 4. The EMBO Journal. 2001;20(24):7137-48. Epub 2001/12/18. doi: 10.1093/emboj/20.24.7137. PubMed PMID: 11742990; PubMed Central PMCID: PMC125774. 694 citations.
  21. ↑ Choudhury R, Singh S, Arumugam S, Roguev A, Stewart AF. The Set1 complex is dimeric and acts with Jhd2 demethylation to convey symmetrical H3K4 trimethylation. Genes & Development. 2019;33(9-10):550-64. Epub 2019/03/08. doi: 10.1101/gad.322222.118. PubMed PMID: 30842216; PubMed Central PMCID: PMC6499330. 24 citations.
  22. ↑ Denissov S, Hofemeister H, Marks H, Kranz A, Ciotta G, Singh S, Anastassiadis K, Stunnenberg HG, Stewart AF. Mll2 is required for H3K4 trimethylation on bivalent promoters in embryonic stem cells, whereas Mll1 is redundant. Development. 2014;141(3):526-37. doi: 10.1242/dev.102681. PubMed PMID: 24423662. 259 citations.
  23. ↑ Ashokkumar D, Zhang Q, Much C, Bledau AS, Naumann R, Alexopoulou D, Dahl A, Goveas N, Fu J, Anastassiadis K, Stewart AF, Kranz A. MLL4 is required after implantation, whereas MLL3 becomes essential during late gestation. Development. 2020;147(12). Epub 2020/05/23. doi: 10.1242/dev.186999. PubMed PMID: 32439762. 16 citations.
  24. ↑ Goveas N, Waskow C, Arndt K, Heuberger J, Zhang Q, Alexopoulou D, Dahl A, Birchmeier W, Anastassiadis K, Stewart AF, Kranz A. MLL1 is required for maintenance of intestinal stem cells. PLoS Genetics. 2021;17(12):e1009250. Epub 2021/12/04. doi: 10.1371/journal.pgen.1009250. PubMed PMID: 34860830; PubMed Central PMCID: PMC8641872. 4 citations.

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