Researchers are creating the first comprehensive functional map of genes expressed in human cells

Researchers are creating the first comprehensive functional map of genes expressed in human cells

The Human Genome Project was an ambitious initiative to sequence each piece of human DNA. The project brought together researchers from research institutions around the world, including the Whitehead Institute, and was finally completed in 2003. Now, over two decades later, Whitehead Institute member Jonathan Weissman and colleagues have gone beyond the sequence to present the first comprehensive functional map of expressed genes in human cells. Data from this project, published online June 9 in Cell, binds each gene to its job in the cell, and is the culmination of years of collaboration around the single-cell sequencing method Perturb-seq.

The data is available on the Weissman Labs website for other researchers to use.

It is a great resource in the way that the human genome is a great resource, in that one can go in and do discovery-based research. Instead of defining in advance which biology to look at, you have this map of genotype-phenotype relationships and you can go in and screen the database without having to do any experiments. “

Jonathan Weissman, Professor of Biology, Massachusetts Institute of Technology (MIT) and Researcher, Howard Hughes Medical Institute

The screen made it possible for the researchers to delve into various biological issues. They used it to explore the cellular effects of genes with unknown functions, to study the response of mitochondria to stress, and to screen for genes that cause chromosomes to be lost or gain chromosomes, a phenotype that has proven difficult to study in the past. “I think this amount of data will enable all sorts of analyzes that we have not even devised yet by people coming from other parts of biology and suddenly they only have this available to draw on,” says former Weissman Lab postdoc Tom Norman, a co-senior author of the magazine.

Pioneering Perturb-seq

The project benefits from the Perturb-seq method, which makes it possible to follow the effect of switching on or off genes with an unprecedented depth. This method was first published in 2016 by a group of researchers including Weissman and fellow MIT professor Aviv Regev, but could only be used on small sets of genes and at high cost.

The massive Perturb-seq map was made possible by basic work by Joseph Replogle, a doctoral student at Weissman’s lab and co-author of this article. Replogle, in collaboration with Norman, who now leads a lab at the Memorial Sloan Kettering Cancer Center, Britt Adamson, assistant professor at the Department of Molecular Biology at Princeton University, and a group at 10x Genomics, set out to create a new version of Perturb- seq that could be scaled up. The researchers published a proof of concept paper in Nature’s biotechnology year 2020.

The Perturb-seq method uses CRISPR / Cas9 genome editing to introduce genetic changes into cells and then uses single-cell RNA sequencing to capture information about the RNAs expressed as a result of a given genetic change. Because RNA controls all aspects of how cells behave, this method can help decode the many cellular effects of genetic changes.

Since their first proof of concept paper, Weissman, Regev and others have used this sequencing method on a smaller scale. For example, researchers used Perturb-seq 2021 to explore how human and viral genes interact during the course of an infection with HCMV, a common herpes virus.

In the new study, Replogle and colleagues, including Reuben Saunders, a doctoral student in Weissman’s lab and co-author of the thesis, scaled the method to the entire genome. Using human blood cancer cell lines as well as non-cancerous cells derived from the retina, he performed Perturb-seq over more than 2.5 million cells and used data to build a comprehensive map linking genotypes to phenotypes.

To delve into the data

After completing the screen, the researchers decided to use their new data set and investigate some biological issues. “The advantage of Perturb-seq is that it allows you to get a large amount of data in an impartial way,” said Tom Norman. “No one really knows what the limits are for what you can get out of that type of dataset. Now the question is, what are you really doing with it?”

The first, most obvious application was to examine genes with unknown functions. Because the screen also read out phenotypes of many known genes, the researchers were able to use data to compare unknown genes with known ones and look for similar transcriptional results, which could indicate that the gene products worked together as part of a larger complex.

The mutation of a gene is called C7orf26 especially stood out. Researchers noticed that genes whose removal led to a similar phenotype were part of a protein complex called the Integrator that played a role in creating small nuclear RNAs. The integrator complex consists of many smaller subunits – previous studies have suggested 14 individual proteins -; and the researchers were able to confirm that C7orf26 consists of a fifteenth component of the complex.

They also discovered that the 15 subunits worked together in smaller modules to perform specific functions within the Integrator complex. “From this thousand-foot-high picture of the situation, it was not so clear that these different modules were so functionally distinct,” Saunders said.

Another advantage of Perturb-seq is that because the analysis focuses on single cells, the researchers can use the data to look at more complex phenotypes that become cloudy when studied together with data from other cells. “We often take all the cells where ‘gene X’ is knocked down and cut them together to see how they changed,” Weissman said. “But sometimes when you knock down a gene, different cells that lose the same gene behave differently, and that behavior can be missed by the average.”

The researchers found that a subset of genes whose removal led to different cell-to-cell results were responsible for chromosome segregation. Their removal caused cells to lose a chromosome or pick up an extra, a condition called aneuploidy. “You could not predict what the transcriptional response to losing this gene was because it was due to the secondary effect of which chromosome you got or lost,” Weissman said. “We realized that we could then reverse this and create this composite phenotype and look for signatures of chromosomes that are obtained and lost. In this way, we have made the first comprehensive screening for factors required for proper DNA segregation.”

I think the aneuploidy study is the most interesting application of this data to date. It captures a phenotype that you can only get with a single cell reading. You can not go after it in any other way. “

Tom Norman, co-senior author of the magazine

The researchers also used their data set to study how mitochondria responded to stress. Mitochondria, developed from free-living bacteria, carry 13 genes in their genomes. Within nuclear DNA, about 1000 genes are in some way related to mitochondrial function. “People have long been interested in how nuclear and mitochondrial DNA is coordinated and regulated under different cellular conditions, especially when a cell is stressed,” Replogle said.

The researchers found that when they disrupted different mitochondrial-related genes, the nuclear genome responded similarly to many different genetic changes. However, the mitochondrial responses were much more variable.

“There’s still an open question as to why mitochondria still have their own DNA,” Replogle said. “A big picture of our work is that an advantage of having a separate mitochondrial genome can be to have localized or very specific genetic regulation in response to different stress factors.

“If you have one mitochondria that is broken, and another that is broken in a different way, those mitochondria may react differently,” Weissman said.

In the future, the researchers hope to be able to use Perturb-seq on different types of cells in addition to the cancer cell line they started in. They also hope to continue exploring their map of gene functions and hope that others will do the same. “This is truly the culmination of many years of work by the authors and other contributors, and I’m really pleased to see it continue to succeed and expand,” Norman said.

Source:

Journal reference:

Replogle, JM, et al. (2022) Mapping of information-rich genotype-phenotype landscapes with scaled-down Perturb-seq. Cell. doi.org/10.1016/j.cell.2022.05.013.

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