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The whole genome is our playground in this.German version/Deutsche Version/span>
From the Lahn to the Charles River
Working at the interface of biology and medicine was almost predetermined for Lucas Jae by the particular course of study he followed in Marburg. It was human biology, which had been established there in the 1970s. With a curriculum that relates biological processes between health and disease to humans, he also benefited from Marburg’s excellence in the field of infection biology. After his undergraduate degree, he conducted as many laboratory internships abroad as possible. That took him to the Karolinska Institute in Stockholm for a summer. “I would very much like to go to America, too, but as an unknown student from Germany I probably don’t have a chance,” he confided to a young researcher there. “Apply, just try it out, you’ll be surprised,” the latter replied. “I took that to heart and emailed Bob Weinberg to ask if I could do an internship with him.” Against all expectations, he received a positive response. Robert A. Weinberg is a luminary in cancer research. In the early 1980s, he had isolated the first human oncogene and the first tumor suppressor gene and co-founded the legendary Whitehead Institute for Biomedical Research. Lucas Jae liked the internship in Weinberg’s lab in Cambridge in the U.S. so much that he returned to the Charles River for his diploma thesis. While he thus was working on tumor research, he met the Dutch researcher Thijn Brummelkamp on the same floor. This one had just set out to solve a challenging problem: Making forward genetics possible in humans. Until then, this method had mainly been applicable in model organisms such as yeasts or fruit flies. What he learned from Brummelkamp fascinated Jae. When Brummelkamp moved to the Netherlands Cancer Research Center in Amsterdam to set up his own laboratory, he followed him as a doctoral student.
The problem of undirected mutagenesis
The problem addressed by Brummelkamp explains as follows: The first step in forward genetics is to manipulate a phenotype to be studied in an organism. To do this, mutations must be generated in its genetic information. However, except for germ cells, this genetic information is present in duplicate on two chromosomes each in almost all multicellular organisms. They are diploid. The mutation of the gene on one chromosome is therefore often masked by an unmutated version on its counterpart. Mutant model organisms such as the fruit fly are therefore crossed with each other until homozygous variants are produced in which both chromosomes carry the same mutation and show the characteristic phenotype. Of course, it is not possible to experiment with humans in this way – but neither is it possible to experiment with human cells. This is because human cells reproduce asexually by dividing and producing identical diploid copies of themselves, in which the unmutated phenotype usually prevails. In cultures of haploid cells, however, forward genetics should also be possible in humans, Jae’s doctoral supervisor thought, and resorted to so-called HAP1 cells to verify this idea. These haploid human cells were derived from tumor cells discovered at Tufts University in Boston in patients with chronic myeloid leukemia. “Because these cells no longer have a back-up mechanism, you can do unbiased genomic research with them,” Jae explains. Although “beautiful genetics” can also be done in human cell cultures these days thanks to RNA interference or genome editing, says Lucas Jae, these methods rely on specific preliminary considerations. “In contrast, the method invented by my Ph.D. advisor allows for undirected mutagenesis.” In other words, he says, you don’t know in advance which mutations you’re going to introduce, as was once the case with the experiments of future Nobel laureates Christiane Nüsslein-Volhard and Eric Wieschaus, who fed flies a mutagen that produced random mutations, from which they made their groundbreaking discoveries. “We are, in a sense, asking the cell to tell us how it works through mutations. The whole genome is our playground in this.”
The tracing of genetic wiring maps
From now on, Jae’s goal was to explore this playground, and accordingly the title of his dissertation was “Genetic Expeditions with Haploid Human Cells,” which he completed in 2015. In it, he further developed the method for phenotypic forward screens introduced by Brummelkamp. In the simplest case, he said, one infects a cell culture with a virus that kills cells, for example, then generates cells that are resistant to the virus through random mutations, and eventually finds out which genes mediate that resistance. “We were the first to be able to do it that way.” The vast majority of cellular phenotypes involved in human disease, however, did not follow this simple pattern of life or death. These had to be looked at in a more differentiated way, he said, and their genetic wiring maps had to be traced for this purpose. In 2017, Jae and Brummelkamp showed how this can be done without complicated assays in a high-profile Naturepublication. [1]
The invention of an ingenious process
They subjected HAP1 cells, which exhibited various phenotypes, to random mutagenesis by gene trapping. These are prefabricated DNA sequences that destroy the function of each gene into which they nest along with the retroviruses by which they are transported. Jae and Brummelkamp chose specific protein states of the cells as phenotypes. For the proof-of-concept of their concept, they generated such a phenotype by stimulating the formation of the interferon-regulatory factor IRF 1, whose signaling cascades are well known, in a culture of 100 million previously mutagenized HAP1 cells by adding interferon-gamma. They stained this factor with fluorescent antibodies and then grouped all cells in a fluorescence-based flow cytometry according to the intensity of their color signal. In almost every one of these cells, the retrovirus had created a different mutation in the genome. Then they selected 15 million cells each with particularly weak and with particularly strong color signal. In the first, mutations of those genes had accumulated that act as positive regulators – i.e. agonists – in the IRF-1 signaling pathways. These mutations had weakened the production of IRF-1 and thus the color signal. Conversely, mutations of inhibitory genes occurred particularly frequently in the group with a strong color signal.
It is easy to determine which genes are enriched or depleted in this way. “We do this by sequencing from the gene trap into the adjacent genome section and aligning the result with annotation databases to identify which gene is affected.” In the 2017 paper, he and Brummelkamp wired eight different protein-defined phenotypes with their genetic origins as if in a wiring map. Using one of these screens, the authors were able to demonstrate how elegantly this can expand our knowledge of biological signaling pathways. The screen focused on the phosphorylation of a specific amino acid of protein kinase B and led to the discovery of a previously unknown off-switch-molecule of the extremely important signal transduction via G protein-coupled receptors.
The discovery of a mitochondrial distress signal
Jae is now successfully applying the method he co-developed to research into the molecular mechanisms of mitochondrial stress response. To the stress caused by infections or by an excess of misfolded proteins, for example. With its own genes, the mitochondrion cannot produce a protein to defend itself against it. Rather, it must send stress signal to the cytosol, which activates a transcription factor there that sends the message to the nucleus: Shut down general protein production and focus on producing those proteins that help others fold properly! This emergency signal is a protein with such a short half-life that it has remained largely invisible to classical biochemistry. The gene that codes for this protein, on the other hand, is naturally stable. Therefore, Jae was able to find it in haploid human cells using his random mutagenesis approach. He manipulated these cells by genome editing of the known transcription factor so that they lit up green when it mediated a stress response. By exposing the mitochondria to various stressors and then separately examining the mutagenized cells with particularly weak and particularly strong color signals, he discovered the gene whose product DELE1 sends the mitochondrial emergency call into the cytosol so that it can be relayed from there to the cell nucleus. [2]
New starting points for pharmacological interventions
The discovery of DELE1 and some associated molecules opens up many new questions about the involvement of mitochondrial disorders in the development of neurodegenerative diseases as well as in the progression of aging processes. SOLID – Suppression of Organelle Defects in Human Disease is the topic of the ERC Starting Grant project in which Lucas Jae and his group have been investigating these questions, among others, since 2019. Above all, however, in this project they are interested in finding previously unknown starting points for drug interventions in such disorders. “These are often rare disorders characterized by the mutation of a gene,” says Lucas Jae. In principle, he says, gene therapies can be developed against them. Direct pharmacological intervention, on the other hand, is often not possible because the target protein is missing or crippled due to the mutation, he says. However, this could possibly be changed if a second gene were found that interacts genetically with the mutated one. “For example, if the mutation of one gene turns on a signaling pathway that leads to the disease, perhaps a second gene exists that turns off that signaling pathway again, restoring the healthy state if you inhibit it,” Jae explains. And that’s where his method comes in again. With this method, it is possible to first find all the genes that determine a signaling pathway in a discovery screen. Genome editing can then be used to “rebuild the patient’s condition” in order to investigate in a modifier screen whether there are genes in it that could repair the diseased phenotype. Their protein products would then be potential targets for therapeutic intervention. Such treatment of rare diseases is still a long way off in most cases, Lucas Jae cautions. “We are doing the basic work for this.” At the Gene Center of LMU Munich, he finds the best conditions for this, spurred on by the Life Sciences Bridge Award of the Aventis Foundation.
Author: Joachim Pietzsch, Wissenswort
Photos: © Uwe Dettmar
[1] Brockmann M*, Blomen VA*, Nieuwenhuis J, Stickel E, Raaben M, Bleijerveld OB, Altelaar AFM, Jae LT, Brummelkamp TR. Genetic wiring maps of single-cell protein states reveal an off-switch for GPCR signalling. Nature. 2017 Jun 8;546(7657):307-311.
[2] Fessler E, Eckl EM, Schmitt S, Mancilla IA, Meyer-Bender MF, Hanf M, Philippou-Massier J, Krebs S, Zischka H, and Jae LT. A pathway coordinated by DELE1 relays mitochondrial stress to the cytosol. Nature. 2020 Mar 4;579(7799):433-437.