Fundamental proteins that guide embryo development are co-opted by cancer

Wombs: a new parental battleground

Every organism gets two copies of its genes, one from each parent, but they don’t always have the same effect. Fathers primarily provide DNA. Having evolved solely to shuttle male DNA to an egg cell, mature sperm consist of little more than tightly compressed DNA, a cell membrane and a rapidly churning tail.

Eggs, on the other hand, contain DNA and other resources to help jumpstart the developmental process. Unsurprisingly, it’s not uncommon to find genes that cause problems when mom’s copies are defective, Malik said.

“It's very easy to imagine maternal defects being the failure for mom to package all the goodies in her oocyte [egg cell] that the zygote needs to survive,” he said.

Paternal-effect genes are rarer because during the earliest post-fertilization developmental phases, dad’s genes are still turned off while his DNA gets unpacked.

In mammals, it gets even more complex. Embryonic development occurs inside mothers’ bodies and draws on even more maternal resources. This evolutionary transition created a new battleground for conflict between the sexes, Malik said, as each parent’s goals for its offspring diverged a little more.

“If you're not in a monogamous species, dad’s basically saying, ‘Look, I have a limited opportunity to make these kids. I'm going to make sure that my kids sort of act like the aliens and extract maximum nutrition from mom,’” Malik said. “And mom’s like, ‘Hey, hang on, I'm planning to have a whole bunch of babies. You can't just pick up all of my resources, because then I'll have nothing left for the other babies.’”

Though Malik studies how genetic conflict shapes evolution, such as the genetic conflict between viruses and anti-viral proteins that block infection, genetic conflict in mammals was a new area for his lab.

“Ours is a fly lab,” he said. “I’ve never worked on mice, I’ve never trained in mouse genetics.”

As a Howard Hughes Medical Institute investigator with flexible funding, Malik was able to fund Molaro’s quest to understand these strange mammalian histones. But scientifically speaking, it was a little like sending him to an island and checking occasionally to make sure he had food and water, Malik recalled. At the beginning of the project, the island appeared to be waving distance from shore.

A new parental-effect gene

Mice have the H2A.B member of the short histone variant family of genes. When Molaro set up camp, the H2A.B protein was thought to be present only during sperm development, a process in which Molaro and Malik expected H2A.B to play an important role.

It didn’t.

Two and a half years after editing H2A.B out of mouse DNA, Molaro found himself with a lot of negative results and no idea how H2A.B might function in reproduction. His island was starting to feel more like a raft floating out to sea.

But the team had missed something important.

“The reason we were so fooled was that we never even considered the possibility that these genes might actually have a female role,” Malik said.

Molaro and Malik Lab research technician Anna Wood bred males lacking H2A.B to females that also lacked the gene — and with one experiment, his island became a peninsula.

Compared to litters bred from two normal parents, fewer pups were born from these crosses, and about four times as many embryos didn’t survive past implantation. Embryos in these litters were also about 30% smaller than normal. In Molaro’s previous experiments, the H2A.B from the mothers had masked the effect of losing paternal H2A.B, Malik said.

Further experiments strengthened the evidence that it was the parents’ histones that critically affected embryo development. Molaro was on solid ground at last.

“It was not just that these embryos [from parents lacking H2A.B] were less viable, but they were actually at a disadvantage to their siblings, depending on who their father was. They were also at a disadvantage compared to the siblings, depending on who their mother was,” Malik said.

H2A.B is one of the first parental-effect genes identified in mammals since the 1980s, Molaro said. He is now exploring several potential mechanisms by which parental H2A.B might affect embryo development in his own lab. He expects maternal and paternal H2A.B to act through different mechanisms.

Unusual histones give cancer a helping hand

As Molaro struggled to chart a course through the muddy waters of his H2A.B work, a collaboration with pediatric oncologist Sarthy and bioinformatician Chew helped form a lifeline. Sarthy was also studying histones, but in the context of cancer.

In cancer, classic histones can accumulate mutations that alter how they interact with DNA (a complex called the nucleosome). DNA often wraps more loosely around mutated histones, thereby creating less stable nucleosomes.

The short histone variants Molaro was studying also happen to wrap DNA more loosely, generating remarkably unstable nucleosomes. This makes it easier for sperm to remove their histones and replace them with even smaller proteins that facilitate the super-tight DNA packing they need in order to swim most efficiently. In cancer cells, relaxed DNA packing makes it easier to turn on genes that promote tumor development and progression.

“I started thinking, even though these histones that Antoine is working on are supposed to be in testes only, maybe they could be turned on in cancers. And if they were, they might have the same effect as these mutant histones,” Sarthy said.

Tumors often turn on genes that are usually turned on, or expressed, in specific tissues or during specific life stages.

Molaro was also interested in the potential connection between H2A.B and disease. With Chew, now a faculty member in the Cancer Institute of Singapore at the National University of Singapore, Sarthy and Molaro compared the natural genetic sequence of H2A.B genes and related short histone variants to the genetic sequences of cancer-associated mutated histones. They found that the natural histones had evolved the same changes seen in mutated cancer-associated histones.

The team also found that many types of tumors, particularly different kinds of B-cell lymphomas, turn on these short histone variants, suggesting that they also share unstable interactions between DNA and histones.

“It was kind of a big surprise that so many different cancers had unstable nucleosomes,” Sarthy said. “In some cancers, people had thought, ‘Oh, there are no histone mutations, so they don't have unstable nucleosomes.’ What we're finding is there's an entirely different way to get unstable nucleosomes.”

It means that even tumors with very few mutations may still be able to create the loose DNA-histone interactions they need to turn on cancer-promoting genes, just by turning on normal histones in the wrong spot, he said.

“We saw very little overlap between cancers with histone mutations or cancers that turn on H2A.B,” Sarthy said. It suggests that many cancers need this property, but either a mutated histone or a short histone variant is enough to achieve it.

“If you have both, the nucleosomes are too unstable,” he said. “There's a sweet spot that cancer’s trying to reach.”

It’s unlikely that H2A.B and related histones cause cancer by themselves. Instead, they work together with other genetic, molecular and cellular changes to promote cancer development and progression. In fact, Sarthy and his colleagues found that tumors that turn on short histone variants also have other changes in fundamental cell processes that could affect cancer development.

“It suggests there’s a functional consequence to this,” Sarthy said.

Sarthy is working to understand this phenomenon in lymphomas and exploring ways to potentially target tumors that have unstable DNA-histone interactions or that have turned on H2A.B.

One avenue he’s exploring, in collaboration with Hutch immunologist Dr. Marie Bleakley, is immunotherapy. The fact that H2A.B is never found outside sperm or eggs except in cancer makes it a great candidate target for selective cancer immunotherapy — that is, reprogramming a patient’s immune cells to recognize and kill cells carrying H2A.B.

If this finding leads to a new treatment, it would be the latest in a long line of discoveries about fundamental life processes that end up having profound impacts on human health.

“You could argue that histones are probably one of the most fundamental proteins we have,” Malik said. “And yet we have these variants. You could say, ‘Oh who cares about this weird evolution? It’s only expressed in the testes.’ But these aren’t siloed problems. [These histones] affect early development, they can be expressed outside of the testes, and they can cause disease.”