Next Steps for Functional Genomics Proceedings of a Workshop (2020) / Chapter Skim
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7 Interpreting and Validating Results from High-Throughput Screening Approaches
7 Interpreting and Validating Results from High-Throughput Screening Approaches
Pages 87-104
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From page 87... ...
The presenters were Emma Farley of the University of California, San Diego; Philip Benfey of Duke University; Grace Anderson of Octant; and Gary Churchill of The Jackson Laboratory. A panel discussion period followed the presentations.
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From page 88... ...
It is challenging to target KRAS directly due to the exceptionally high binding affinity KRAS has to its endogenous ligand, GTP. With this in mind, Anderson said, they chose to look into downstream methods of targeting the associated pathway.
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From page 89... ...
In fact, only one hit spanned all of them, which was a known pathway reactivation gene, REF. Thus, Anderson noted, most of the boundaries of sensitizers are at the tissue level, an important finding for clinical trial design because most clinical trials were being defined in terms of genotype.
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From page 90... ...
USING FUNCTIONAL GENOMICS TO UNDERSTAND DEVELOPMENT In the next talk Philip Benfey described applying functional genomics tools to study development in a plant -- in particular, the development of the Arabidopsis root. "When I say development, our primary focus is on how you go from an undifferentiated stem cell to a fully differentiated cell and how that cell can function to make an organ that has some real purpose in the world.
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From page 91... ...
His team decided to approach this by combining some of the published datasets with their own datasets, ultimately using data from 80,000 cells to create a "reference transcriptome" that included information on transcription separated both by cell type and age. Ultimately, the goal with this reference transcriptome would be to use it in a way that is similar to how a reference genome is used.
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From page 92... ...
After cloning the genes responsible for SHR and scarecrow, Benfey's team was surprised to find that SHR was not expressed in either of the two lineages from the asymmetric cell division. The SHR protein, a transcription factor, is made internally and then moves out to the adjacent cell layer FIGURE 7-2 Display of asymmetric cell divisions in the Arabidopsis root, where the mature cell divides along the longitudinal axis into two cells with different fates.
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From page 93... ...
They could perform live imaging to watch the levels of both proteins in the different cells of the growing roots and even see the asymmetric cell divisions. The behavior they observed did not completely match up with the prediction of bi-stability from the mathematical model, Benfey said, but perhaps the more important fact is that they were actually able to test the predictions of their mathematical model by watching the expression levels in real time.
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From page 94... ...
The embryo in the top left shows the signaling of fibroblast growth factor (FGF) , which activates the transcription factor ETS, and the embryo on the top right shows the cells on the visible side of the embryo expressing the transcription factor GATA.
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From page 95... ...
In an ideal world, one would take the sequence and then change various nucleotides and see how that affects gene expression, while keeping constant those nucleotides known to be important in order to make it more likely that the resulting variants are actually functional. However, the complexity of the required analysis increases rapidly with the number of nucleotides, which, Farley said, is one of the reasons she chose to work with this enhancer -- because it is very small.
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From page 96... ...
of the barcode, which can be detected, measured, and associated with the appropriate enhancer sequence. After testing millions of enhancer sequences, Farley identified 20,000 that were active at or above WT levels of Otx gene expression.
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From page 97... ...
She worked with the well-known enhancer ZRS, which drives expression of the sonic hedgehog gene in developing limb buds. As she had seen with the sea squirt, the ETS binding sites on the enhancer for sonic hedgehog were low affinity.
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From page 98... ...
HARNESSING GENETIC DIVERSITY TO UNDERSTAND MAINTENANCE OF PLURIPOTENCY IN EMBRYONIC STEM CELLS The session's final speaker, Gary Churchill of The Jackson Laboratory, began his presentation by announcing that he was going to lead off with his conclusion. Touching on some of the themes from the previous talks, he said that if functional genomics is to make the transition from a correlational approach to a causal approach, genetic variation holds great promise as an experimental perturbation.
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From page 99... ...
The inbreds, or collaborative crosses, have reproducible genomes and a high genetic diversity, but fewer recombinations per line. Next, Churchill introduced expressional quantitative trait locus (eQTL)
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From page 100... ...
When they mapped out their data, they found characteristic patterns where there were strong signals of local regulation with a significant amount of distal variation. And that distal variation sometimes seemed to cluster in bands where a single genetic locus appeared to affect the expression of many genes or a single genetic locus seemed to affect the openness of many ATAC peaks in the genome, corresponding to regions in the genome where the chromatin was accessible (see Figure 7-5)
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From page 101... ...
. SOURCE: Gary Churchill presentation, slide 13.
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From page 102... ...
Looking at the genetic pattern at that locus among the eight founder mouse strains at The Jackson Laboratory, he found that of the downstream target genes, many of which are involved in pluripotency, there was high expression in four of the strains and low expression in the other four. Furthermore, the four strains with low expression were "recalcitrant" strains for which the researchers found it difficult to maintain embryonic stem cell lines.
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From page 103... ...
The difficulty will vary depending on the type of data, she said, but with transcriptional regulation studies, for instance, "you could go about asking those questions if you design your experiments right and you design massively parallel reporter assays in the correct way." Benfey had a different take on the question. Much of what researchers do is find something interesting and then go deep into it, learning everything they want to learn, and that is a valuable approach.
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From page 104... ...
Joanna Kelley of Washington State University, playing devil's advocate, asked why, if validation is so important, shouldn't the field put all its effort into "studying the role of transcription factors, ATAC-seq peaks, all of these things, in one organism, to really understand deeply how all of these different functional genomic things that we're measuring work"? In short, why not focus on one organism in depth rather than researchers each trying to validate in their own systems, which presents so many challenges?
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