News item

A “tour de force” to systematically characterize developmental enhancers in Drosophila: cataloguing 7,705 enhancer candidates

Evgeny Kvon and his colleagues in the Stark Lab at IMP have developed a high-throughput pipeline to assess enhancer activities in fly embryos by "in situ" hybridization.  They analyzed nearly 8000 candidates providing a genome-wide snapshot of the organization of the DNA regulatory regions. Using this pipeline they found that the fly genome is packed with developmental enhancers, which are highly dynamic and mostly organized locally. The paper was published online in Nature this week: Genome-scale functional characterization of Drosophila developmental enhancers "in vivo".

Drosophila embryos

Development of multicellular organism is a fascinating process: a single cell gives rise to multiple different cell types, tissues and ultimately an organism. This process is tightly controlled and relies on the spatial and temporal regulation of genes, giving rise to unique expression patterns. DNA regulatory elements, such as enhancers, are fundamental to the establishment of these expression patterns.  It is known that enhancers are DNA sequences that encode all the information to transcribe a gene in a specific place at a specific time. However, despite this defined sequence-to-function relationship, it is still not clear how this information is encoded in the DNA and deciphered by the cell, i.e. the regulatory code remains unknown, the Stark Lab at the IMP is focused on decrypting it.

Evgeny Kvon studied genetics at Novosibirsk State University in Russia and after finishing his degree in 2008 he was extremely interested in gene regulation by small RNAs, through a friend he learned about the RNA Community at the Vienna Biocenter and decided to apply for a PhD here. Alex Stark had joined the campus around that time as a Group Leader and Evgeny was attracted by Alex’s excitement for regulatory genomics and decided to join his lab (he was the first PhD Student in the Stark lab!).  Evgeny´s first project was to try to analyze cell type specific gene expression, but towards the end of his first year nothing seemed to work. At that time he had his first thesis committee meeting - which was instrumental – and by discussing his ideas and aims it was decided to switch projects. Evgeny was curious about the fact that most of the unique DNA in different animal species lies within non-coding regions, and therefore set as a goal to characterize the regulatory landscape of the fly genome.

The Dickson lab (formerly IMP, now at the HHMI Janelia Farm Research Campus) was working on a genome-wide library of transgenic Drosophila lines containing short fragments of genomic DNA controlling GAL4 expression (Opens external link in new windowVT - Vienna Tiles). The library comprises around 8000 overlapping 2Kb non-coding DNA fragments. This was an ideal collection to try to map and study enhancer activity, but it was also a huge challenge - imagine walking into a library and having to read 8000 random books and try to catalogue them. Evgeny started to test each line individually, hoping to characterize 1000, but quickly realized that at that speed he would never finish.  He and Alex Stark had many discussions on how to optimize the protocols to be able to scale it up.  The first step was to collect and fixate Drosophila embryos, in the traditional (very laborious) protocol each line is processed individually in a specific container (basket with a mesh).  Alex and Evgeny approached the in-house workshop, which was then able to build a contraption that allowed them to process multiple lines at once. The next step was to scale-up the staining protocol (even if you are a pipette master there is a limit to what you can do singlehandedly); Evgeny and Alex talked to the genomics facility and the facility acquired its first robot, which allowed for the automation of most of the liquid handling steps (this was so useful that the same facility now has 4 robots, which most groups use).  The last automation step was to scale-up the image acquisition. The Bio-optics team helped them pick and optimize a slide scanner system that was able to work 24/7, making it possible for them to acquire images from 100 to 200 lines per week. 

After a long troubleshooting and optimization phase (which took almost two years!), Evgeny managed to - within 6 months - screen the library of approximately 8000 transgenic lines for enhancer activity. Each slide image comprised 400 embryos of a given line (enhancer candidate), depicting all stages of embryogenesis. Evgeny manually annotated the enhancer activity patterns for each of the slides!

Their systematic analysis provides a global understanding of the organization of an animal regulatory genome, namely it suggests that during development a protein-coding gene is regulated, on average, by four enhancers, meaning that there could be 50,000 to 100,000 developmental enhancers in Drosophila and the human genome may contain several million enhancers. They also found that most enhancers act on neighboring genes and that their expression activity is very dynamic during development.

In this story I really admire Evgeny’s perseverance and hard work, the whole project was conceived by Evgeny and Alex Stark, but it was a huge collaborative effort (which is very nice to see!). Technicians in the lab and other students helped with the screen and made major contributions: Gerald Stampfel and Tomas Kazmar helped with the image analysis and Tomas developed enhancers.starklab.org; and J. Omar Yáñez-Cuna analyzed the sequences of the enhancers identified in the screen. Besides the Stark lab, it is also an example of how the Vienna Biocenter works, if you have a great idea you knock on a couple of doors and everyone will try to help you make it work.

It is worth mention that in-between Evgeny worked on other parallel projects that were also published: he is co-first author in a paper in Genes and Development and co-author in three other papers. Evgeny graduated and he is now doing a post-doc in the Genomics Division research group run by Eddy Rubin, Len Pennacchio, and Axel Visel at the Lawrence Berkeley National Laboratory.  As the coordinator of the PhD Programme it is extremely fulfilling to witness Evgeny’s progress, which I will continue to follow with interest – well done!

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Kvon EZ, Kazmar T, Stampfel G, Yáñez-Cuna JO, Pagani M , Schernhuber K, Dickson BJ, Stark A (2014) Genome-scale functional characterization of Drosophila developmental enhancers in vivoNature, Available online 1 June 2014, 10.1038/nature13395