Next Steps for Functional Genomics Proceedings of a Workshop (2020) / Chapter Skim
Currently Skimming:
3 Case Studies on Building Functional Genomics Tools in Diverse Systems
3 Case Studies on Building Functional Genomics Tools in Diverse Systems
Pages 17-38
The Chapter Skim interface presents what we've algorithmically identified as the most significant single chunk of text within every page in the chapter.
Select key terms on the right to highlight them within pages of the chapter.
From page 17... ...
The session's speakers were Andrea Sweigart from the University of Georgia, who spoke about her work with monkeyflowers; Rachel Dutton of the University of California, San Diego, who described her work with microbial communities living on cheese rinds and the tools she has developed to study those communities; Zoe Donaldson of the University of Colorado Boulder, whose presentation dealt with the neurogenetics of sociality in voles; Dominique Bergmann of Stanford University who explained how she has moved among diverse species of plants, using what is learned with one to work on another and piecing together a broader picture than would be possible using just one species; and Steven Henikoff of the Fred Hutchinson 17
|
From page 18... ...
Much of the phenotypic diversity in the group is driven by divergence in pollinator attraction and mating systems. One highly studied group of monkeyflowers is the species complex Mimulus guttatus, which Sweigart illustrated with a slide showing the flowers from half a dozen members of this group (see Figure 3-1)
|
From page 19... ...
It is found on high alpine rocky outcrops, on dunes along the Pacific, in serpentine soils, in abandoned copper mines, and even in 60°C thermal soils in Yellowstone National Park. These varied habitats require significant adaptation, Sweigart said.
|
From page 20... ...
"So we've realized as a community," she said, "that we're only going to get so far with this kind of classic approach of brute-force positional cloning." Fortunately, she said, her team has been awarded a grant through the National Science Foundation's Enabling Discovery through GEnomic Tools (EDGE) program to develop robust, repeatable transgenic techniques for use in many genotypes and species and mutant libraries to be of use in multiple species.
|
From page 21... ...
MICROBIAL COMMUNITIES AND THEIR INTERACTIONS ON CHEESE RINDS Rachel Dutton began studying microbial communities living on cheese rinds, she said, because of a conviction that a vast amount of biology was being missed by studying individual organisms in isolation. "My background is in E
|
From page 22... ...
. "You're making large random mutant libraries," she explained, "but each of the transposons in the library has a random barcode, so you can associate each insertion in the genome with a barcode sequence and follow the population changes in the library just by sequencing barcode abundances." The library they used had a pool of about 150,000 mutants, which represented about 15 different insertions and every non-essential gene in the genome, she said.
|
From page 23... ...
They calculated the gene fitness data based on the barcode abundance of the inoculums versus growth on their in vitro medium, which is a cheese curd agar. What they found was a large number of genes that were required in the grow-alone condition and a somewhat smaller set of genes that were
|
From page 24... ...
coli is growing by itself, it will die." However, if it is growing in a community, the data imply that some other members of the community are providing it with the required amino acids. When they looked at their data in more detail and examined individual pair-wise contributions to the fitness effects, they found that it was only the fungal species that were producing this cross-feeding effect.
|
From page 25... ...
"Females will become behaviorally receptive to males, will mate sometimes with multiple males, and then go off and raise the offspring by themselves," Donaldson said. The question Donaldson asked was what genetic differences underlie this behavioral difference and makes the brains of the prairie voles, but not the meadow voles, capable of forming long-term pair bonds.
|
From page 26... ...
"We fished out the gene from a phage library and found that the coding region of this gene was nearly identical between these species," she said. In contrast, there is a length of repetitive DNA upstream of the gene that is nearly absent in the promiscuous species but more than 600 base pairs long in the monogamous species.
|
From page 27... ...
"This wound up being a silver lining," she said, "because now instead of having to look at the entire brain and the complexity of gene expression within multiple brain regions, I was able to focus on what was going on within these three separate brain regions." Also, she added, the similarity in gene expression patterns in other regions of the brain implied that there were other regulatory elements outside of that 3.5-kb region that were driving the gene expression in those other parts of the brain. When Donaldson examined how patterns of gene expression differed among the three types of transgenic mice in those three relevant brain regions, she found that the patterns differed in the same direction as the patterns in the three voles -- that is, the promiscuous species and the two versions of the monogamous species with the shorter and longer repeats.
|
From page 28... ...
"The question then," Donaldson said, "is how we begin to parse out the pleiotropic effects of these genes, and that is where my lab is currently making a lot of effort to develop ways to go in and selectively manipulate gene expression in adult animals in specific brain regions." She described one success that her lab has had in this effort, which involved injecting short-hairpin RNAs (shRNAs) in particular areas of the brain to decrease the expression of vasopressin receptors.
|
From page 29... ...
To deal with that complexity, she said, researchers typically work with models, both model organisms and "models that extract features that are common to many developmental decisions, but do it in a very simple way." Her lab works with the model organism Arabidopsis, and focuses on a specialized cell type, the stomatal guard cell, a pair of which form a valve, which allows carbon dioxide into the plant, and water vapor and oxygen
|
From page 30... ...
The two extra cells line up on either side of the guard cells and are called "subsidiary cells." The grasses are more resilient during drought, and one reason appears to be the performance of their stomata. And this is something that studies in Arabidopsis can say nothing about, she commented.
|
From page 31... ...
Bergmann's group was surprised to find that the transcription factor was similar to one used in Arabidopsis to make a precursor to the stomatal guard cells. However, in Brachypodium it appears to switch its function to make the subsidiary cells.
|
From page 32... ...
LOW-COST, HIGH-RESOLUTION CHROMATIN PROFILING Unlike the first four speakers in the session who had focused on one or a few particular organisms, the final speaker, Steven Henikoff of the Fred Hutchinson Cancer Research Center, described some epigenomics tools his lab has developed that can be applied to a wide variety of organisms. In particular, he spoke about methods to perform low-cost, high-resolution chromatin profiling.
|
From page 33... ...
In particular, Henikoff's lab modified a chromatin immunoclevage method to create what they call "CUT&RUN" (cleavage under targets and release using nuclease) (Skene and Henikoff, 2017)
|
From page 34... ...
The reason for that, he explained, is that unlike ChIP, where one solubilizes the entire cellular contents and then grabs the antibody like pulling a needle out of a haystack, "with CUT&RUN we leave the haystack behind." The lower background means that fewer reads are needed to get a clear signal. To illustrate, he showed ChIP-seq data from ENCODE that was generated with 56 million reads and comparable data from CUT&RUN with only 7.5 million reads.
|
From page 35... ...
Donaldson agreed that bespoke methods are typically required to make germline transgenesis work in diverse species. Thus, she said, her preference is often just to use a workaround.
|
From page 36... ...
She proposed a funding model where a researcher could study these genes of unknown function, assuming they had a plan outlining a research plan. She hopes this would prevent researchers from immediately getting dismissed when trying to study a challenging but important area of research, such as gene hits with unknown function.
|
From page 37... ...
" It will be difficult to discover these new ways without allowing researchers to, in a way, go back 50 years to when none of the current tools were available and "everyone had to do these fairly slow, painstaking ways of interrogating gene function." Bergmann reiterated that researchers will need time and funding to follow such a path. Another audience member offered a different perspective on the unknown, saying, "even in model organisms, there are tens of thousands of transcripts that are expressed at high levels that also make proteins, and they're considered either lcnRNAs (long non-coding RNAs)
|
Key Terms
This material may be derived from roughly machine-read images, and so is provided only to facilitate research.
More
information on Chapter Skim is available.