Scientific discoveries are often obliged by the persuasive need to explore. Particularly, innovation of new tools and techniques in science are all consequences of a major expedition in the search for answers to some fundamental questions. The invention of Split-DamID (SpDamID) is one such story of ‘necessity driven invention’. It all started towards the end of 2010 when Raphael (Rafi) Kopan (Washington University, St. Louis) and Steve Blacklow (Harvard Medical School, Boston) discovered that dimerization of Notch receptor on the cis-regulatory elements of target genes is required for activation1-2. Back in late 90’s Rafi’s lab in series of landmark papers, inaugurated the importance of Notch intracellular localization in gene expression and cell fate decisions3-5.
Matthew Hass, a postdoctoral fellow in Kopan laboratory, decided to tease apart the role of monomeric and dimeric Notch binding to promoters and its influence on cell fate. For most part of 2010-2012 Matt had tried almost all DNA-protein binding assay but couldn’t come up with a strategy to solve this puzzle. Most of DNA-protein interaction tools including ChiP (chromatin immunoprecipitation) failed to distinguish between the binding of one or two (Notch) molecules at same cis-element.
In scientific research, the quest to explore a hypothesis often opens other doors with bigger opportunity. When Matt was juggling with this paradox one day Rafi forwarded him a 2001 paper6 form van Steensel and Steve Henikoff (inventors of DamID). In this seminal paper Van Steensel demonstrated the implication of bacterial protein DNA adenine methyltransferase (Dam) in chromatin profiling. They demonstrated that Dam protein could methylate eukaryotic adenine in GAmTC sequence. This methylation is largely absent in eukaryotic phyla hence this Dam system can be used to profile chromatin in mammalian cells. Rafi suggested to try traditional DamID but that too didn’t work. However, Matt argued that if he can ‘Split’ the ‘Dam’ protein in two halves and molecularly fuse Notch molecules to each half; in that case dimerization of Notch molecule will allow the functional reunion of Dam protein. In coming year Matt made numerous SplitDamID pairs for Notch and associated partners (RBPj, P300, MAML) as well as for other signaling pathways like Wnt/TGF-beta etc7.
Matt perceives it as a fortune that his ChIP experiments did not work, for he couldn’t have developed Split-DamID otherwise. This invention holds an important lesson for fellow scientists- in science set-backs are temporary but the passion is permanent. I was fortunate enough to witness and contribute to the development of SpDamID and I am confident about the applicability of this technique to understand the role of transcription factor (TF-TF) pairs in gene regulation and diseases.
Science is not just about generating knowledge, but also the process of generating knowledge, and very few things delight as much as the creative ways to do so. To me, best ideas are the ones originating from the most basic concepts, which challenge your ability to simplify and put together. Rafi introduced me to such competencies and kept reminding me that the key to do good science is to always wear your most creative hat!
Link to original paper: http://www.cell.com/molecular-cell/fulltext/S1097-2765(15)00539-0
About the author: Ankur Sharma is a post-doctoral fellow at Genome Institute of Singapore. He is employing single cell approaches to dissect the individuality of cancer cells. Twitter: @asharmaiisc https://twitter.com/asharmaiisc Blog: RANDOM MUSINGS FROM A CANCER RESEARCHER. https://anksharma28.wordpress.com
Photo source: Ankur Sharma
Further Reading:
1) Structural and mechanistic insights into cooperative assembly of dimeric Notch transcription complexes. Nat. Struct. Mol. Biol. 2010; 17: 1312–1317
2) Notch dimerization is required for leukemogenesis and T-cell development. Genes Dev. 2010; 2395–2407
3) Signalling downstream of activated mammalian Notch. Nature 1995; 355-358
4) Notch-1 signalling requires ligand-induced proteolytic release of intracellular domain. Nature 1998; 382-386
5) A presenilin-1-dependent γ-secretase-like protease mediates release of Notch intracellular domain. Nature 1999; 518-522
6) Chromatin profiling using targeted DNA adenine methyltransferase. Nature genetics 2001; 304-308
7) SpDamID: Marking DNA Bound by Protein Complexes Identifies Notch-Dimer Responsive Enhancers. Molecular cell 2015; 685-697
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