Gene mutation research calls for rethink on cancer gene analysis

Johns Hopkins researchers report that the deletion of any single gene in yeast cells puts pressure on the organism’s genome to compensate, leading to a mutation in another gene. Their discovery, which is likely applicable to human genetics because of the way DNA is conserved across species, could have significant consequences for the way genetic analysis is done in cancer and other areas of research, they say.

Summarized in a report published November 21, 2013 in Molecular Cell, the team’s results add new evidence that genomes, the sum total of species’ genes, are like supremely intricate machines, in that the removal of a single, tiny part stresses the whole mechanism and might cause another part to warp elsewhere to fill in for the missing part.

“The deletion of any given gene usually results in one, or sometimes two, specific genes being ‘warped’ in response,” says J. Marie Hardwick, Ph.D., the David Bodian Professor of Molecular Microbiology and Immunology at the Johns Hopkins Bloomberg School of Public Health and a professor of pharmacology and molecular sciences at the school of medicine. “Pairing the originally deleted gene with the gene that was secondarily mutated gave us a list of gene interactions that were largely unknown before.”

Hardwick says the findings call researchers to greater scrutiny in their genetic analyses because they could unwittingly attribute a phenomenon to a gene they mutated, when it is actually due to a secondary mutation.

“This work has the potential to transform the field of cancer genetics,” Hardwick says. “We had been thinking of cancer as progressing from an initial mutation in a tumour-suppressor gene, followed by additional mutations that help the cancer thrive. Our work provides hard evidence that a single one of those ‘additional mutations’ might come first and actively provoke the mutations seen in tumoursuppressor genes. We hope that our findings in yeast will help to identify these ‘first’ mutations in tumours.”

The beauty of working with yeast, Hardwick says, is that it is easy to delete, or “knock out,” any given gene. Her team started with a readily available collection of thousands of different yeast strains, each with a different gene knockout. At their preferred temperature, each of these strains of yeast grows robustly even though they each have a different gene missing. Hardwick’s team first asked a fundamental question: Within a given strain of yeast, does each cell have the same genetic sequence as the other cells, as had generally been presumed?

“We know, for example, that within a given tumour, different cells have different mutations or versions of a gene,” explains Hardwick. “So it seemed plausible that other cell populations would exhibit a similar genetic diversity.” To test this idea, her team randomly chose 250 single-knockout strains from the thousands of strains in the collection. For each strain, they generated six substrains, each derived from a single yeast cell from the “parental batch.”

They then put each sub-strain through a “stress test” designed to detect sub-strains with behaviours that varied from the behaviour of the parental batch. All of the sub-strains grew indistinguishably without stress, but when the temperature was gradually raised for only a few minutes, some sub-strains died because they could not handle the stress. When the Hardwick team examined their genes, they found that, in addition to the originally knocked-out gene, each of the sub-strains that faltered also had a mutation in another gene, leading the team to conclude that the cells in each strain of the single-gene knockouts do not all share the same genetic sequence.

They then tested all 5,000 of the original single-gene knockout strains to find sub-strains that could overgrow when given low-nutrient food – a trait that tumour cells often possess. This was another stress test designed to detect differences between the individual cells taken from the parental batches.

They identified 749 such knockout strains and showed that their growth differences were often due to secondary mutations. In total, the team’s evidence indicates that 77% of all the knockout strains have acquired one or two additional mutations that affect cell survival and/or excessive growth when food is scarce. Hardwick believes that stressing yeast in other ways may lead to an even higher percentage of double-mutant strains. In fact, she said she believes that “essentially any gene, when mutated, has the power to alter other genes in the genome”.

Deleting the first gene seems to cause a biological imbalance that is sufficient to provoke additional adaptive genetic changes, she explains. Furthermore, in all of the strains that they examined, they found that the secondary mutations that appeared after a given knockout were always in the same one or two genes as in their earlier observations.

Unexpectedly, Hardwick said, the altered growth of the sub-strains was usually due to the secondary mutations, not the original knockout, and many of those secondary mutations were in genes that are known to be cancer-causing in humans. l doi: 10.1016/j.molcel.2013.09.026



Two human proteins found to affect “jumping gene”

Using a new method to catch elusive “jumping genes” in the act, researchers have found two human proteins that are used by one type of DNA to replicate itself and move from place to place.

The discovery, described in Molecular Cell in November 2013, breaks new ground, they say, in understanding the arms race between a jumping gene driven to colonize new areas of the human genome and cells working to limit the risk posed by such volatile bits of DNA.

Jumping genes, more formally known as transposons or transposable elements, are DNA segments with the blueprints for proteins that help to either copy the segment or remove it, then insert it into a new place in the genome. Human genomes are littered with the remnants of ancient jumping genes, but because cells have an interest in limiting such trespasses, they have evolved ways to regulate them.

Most jumping genes have mutated and can no longer move, but these “rusting hulks” are still passed down from generation to generation. One exception is a jumping gene called L1, which has been so successful that copies of it make up about 20% of human DNA. While many of these copies are now mutated and dormant, others are still active and thus the subject of much interest by geneticists.

“Human cells have evolved ways of limiting jumping genes’ activity, since the more frequently they move, the more likely they are to disrupt an important gene and cause serious damage,” says Lixin Dai, Ph.D., a post-doctoral associate at the Johns Hopkins Institute for Basic Biomedical Sciences, who led the study.

To find out more about how cells control L1 and what tricks the jumping gene uses to get around these defences, Dai and others in the laboratory of Jef Boeke, Ph.D., first induced lab-grown human cells to make large amounts of the proteins for which L1 contains the blueprints. As expected, the two types of L1 protein joined with human proteins and genetic material called RNA to form so-called ribonucleoprotein particle complexes, which L1 uses to “jump”.

To find out which human proteins interact with ribonucleoproteins – and are therefore likely to have a role in either tamping down its activity or helping it along – Boeke’s team collaborated with researchers at The Rockefeller University who had developed a technique for fastfreezing yeast with liquid nitrogen, then grinding it up for analysis with steel balls and very rapidly pulling out the ribonucleoproteins with tiny magnetic particles. “It’s a good way of preserving the interactions,” Dai says.

Adapting this powerful technique to human cells, the team found 37 proteins that appear to interact with the ribonucleoprotein, and they selected two for further analysis. One of these, UPF1, is known for its role in quality control; it monitors the RNA transcripts that carry instructions from DNA to the cell’s protein-making machinery and destroys those with mistakes.

In this case, Dai says, UPF1 binds to the L1 ribonucleoprotein, probably because L1 RNA contains instructions for two proteins rather than one – a red flag for UPF1. When the researchers disabled the UPF1 gene, cells produced more L1 RNA and protein.

But they still haven’t figured out exactly how UPF1 interacts with the ribonucleoprotein, Dai says. The other human protein, PCNA, helps to copy DNA strands before a cell divides into two. The researchers found that PCNA interacts with a critical segment of one of the ribonucleoprotein’s L1 proteins; when they tried altering that section, L1 could no longer jump.

In contrast to UPF1’s role in suppressing L1 activity, Dai says PCNA seems to have been co-opted into helping the jumping gene, perhaps by repairing gaps left in human DNA after L1 splices itself into a new spot. Dai notes that these discoveries would not have been possible without two methods pioneered in this study: growing large quantities of human cells and inducing them to make ribonucleoprotein, and adapting the fast-freezing technique to study interactions in human cells.

He expects that these methods will enable biologists to greatly increase their understanding of L1, a jumping gene that has played a key role in the evolution of the human genome and whose activity has been implicated in some cancers.

“Our study shows how the jumping gene tries to be smart and get around the host cell’s control mechanisms, and how the host tries to minimize its activity,” Dai says. “We’re looking forward to learning more about this arms race.”



 

                                   
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