Hello from the dark genome

Within our genetic code lurk ghosts of ancient parasites, viral fragments and rogue elements that plagued our ancestors long ago. These DNA revenants stir in the shadows of a vast and mysterious realm known as the dark genome – a genetic underworld that is only now yielding its secrets.  

The 'light' genome, first illuminated a quarter of a century ago, comprises the 20,000 or so genes that build our cells – the known, the named, and the charted. The remaining 98 percent was initially dismissed as junk, yet within this shadowed script lie powerful fragments. Scientists investigating this dark domain are finding that, among the shattered genes and decayed DNA, there are viral fossils, inactive jumping genes and other shadowy elements that can shape our biology.  

George Kassiotis and Samra Turajlic in the lab. Credit: Dave Guttridge.

While the light side accounts for just 2% of our DNA, 5% of the dark genome is made up of endogenous retroviruses, which are the remnants of ancient infections. Remarkably, this percentage rises to almost half of our DNA if you include other virus-like elements. Some of these parasites still whisper instructions from the shadows, offering answers to many riddles of evolution, development and disease.

As I discovered during a tour of the Crick, the story of the dark genome shadows many key developments in genetics. More than seven decades ago, its extraordinary influence on evolution was glimpsed by a pioneering scientist, though was initially ignored by the establishment. Over the years, various Nobel Prizes have recognised discoveries that either directly or indirectly relate to the dark genome, which has been implicated in autoimmune disorders, neurological diseases and more besides.  

Though many of the ghosts in the dark genome have been tamed during our evolution, not all rest peacefully. The role of these crepuscular elements in cancer is being studied by teams lead by George Kassiotis and Samra Turajlic at the Crick, supported by the labs of Charlie Swanton and Julian Downward. With new insights and a battery of novel techniques, they hope to harness the dark genome to change the future of cancer medicine.

The genome’s hidden allure

“Your genome has more viral hitchhikers than it does genes,” says George Kassiotis. We are sitting in his office, which gives little away about his life or science, amplifying the sense of mystery. With its off-white walls and mottled dark grey carpet, the furnishings are stark, include ranks of empty box files (so the room is not so echoey) along with a computer. But George says he has everything he needs to explore the dark genome.

George Kassiotis, Principal Group Leader at the Crick. Credit: Dave Guttridge.

After he completed his PhD in immunology, George continued his research at the National Institute for Medical Research in Mill Hill, in northwest London, where he eventually set up his own lab to study the immune reaction of mice to a particular retroviral infection. Retroviruses use RNA as their genetic material and they insert a DNA copy of their RNA into the host cell’s genome. An infection of this kind stimulates an immune response including from a kind of white blood cell, called a T cell.  

Puzzlingly, the T cells of the infected mice George was studying behaved as if they had already encountered the retrovirus they had just been inoculated with. He subsequently discovered that they had mounted a response to proteins that were made by a remnant of another retrovirus that had integrated into the mouse genetic code: he had stumbled across an endogenous retrovirus, or ERV, part of the dark genome. “I was captivated - how do we recognise new retroviral infections, when we have so many endogenous ones, embedded in our DNA?”

George found that in immune-deficient mice unable to produce antibodies, the peaceful co-existence between the host and endogenous virus was disrupted – defective and dormant ERVs were ‘resurrected’, leading to retroviral infection and spread. “This suggested to us that immune responses to ERVs are not only possible to induce but must also be kept under control,” George tells me.  

Most ERVs in mice – and humans too - have accumulated mutations and deletions that mean the endogenous viruses have lost the ability to infect. They are also repressed by what are called epigenetic mechanisms, chemical modifications that turn genes on and off in cells. However, the hundreds of thousands of defective copies of ERVs that lurk in our genome have between them the necessary components to build a virus particle, with defective parts in one virus complemented by non-defective elements from another. “Particles made by ERVs are not infectious or even complete, but they may still be perceived as a threat by the immune system,” explains George. The immune system is tricked into believing that there is a genuine retroviral infection, so it responds by doing what it has evolved to do: get rid of the ‘infected’ cells.

Immune responses against ERV particles or components are part of the body’s natural defences against cancer. As cells mutate, the escape of ERVs from the restraint of epigenetic controls can act as ‘red flags’ for the immune system, which, in its attempt to eliminate the newly sensed virus, also destroys the cancer cells.  

The dark genome runs up more flags in a cancer than just the ERV proteins. “The dysregulation of the dark matter in the transformed cells in a tumour produces a swathe of aberrant products – the result of mixed-up codes from virus and human – which can also be recognised and targeted as foreign by the immune response,” George says.

There are other ways that viral genetic ghosts influence cancer: they can interfere with genes related to immunity or the response of cancer cells. Some tumours hijack these viral sequences, using them to promote the uncontrolled cell growth that is the hallmark of all cancer. “For example, there is a cancer-causing form of the much-studied ALK kinase (a protein that controls cell growth) that is driven by one of these viral sequences in melanoma” says George. “There are many cases like that. In fact, ERVs were first discovered because they were causing cancer in mice and in chickens,” he adds.

To study how they sway the development of tumours, George works with cancer researchers around the world to study the trove of DNA data from cancer cells and how it is turned into the instructions to make proteins and other working molecules (the transcriptome). His collaborations include large-scale projects such as TRACERx (TRAcking Cancer Evolution through therapy/Rx), which tracks detailed changes in a cancer’s genetic makeup and behaviour. Using the Crick's computer cluster, which harnesses “sizeable computational power”, George’s team can interrogate the activity of dark elements in a few days, a job which would take decades on an office PC. From this he can learn how the dark genome helps a tumour outwit the body’s defences, establish a blood supply and spread. “Tumours use this adaptability for their own mini-evolution inside one human body,” says George.  

Tobias Plowman from the retroviral immunology laboratory. Credit: Dave Guttridge.

The rogue elements within

Aside from ERVs, another oft-cited example of how the dark genome can shape evolution was discovered almost a decade before the structure of DNA was even known. That glimpse came in the form of evidence that genomes contained mobile DNA elements, transposons or jumping genes, so named because these parasites can hop from one region of DNA to another, disrupting other genes in the process.  

While working in Cold Spring Harbor Laboratory in New York, Barbara McClintock studied maize, where transposons make up 85% of the genome, and first observed them in 1944. She published her findings in 1950, and called them ‘controlling elements’, acknowledging their important role. “The idea was that they were not only jumping, but they were also affecting the function of the gene near where they planted,” says George. “that’s actually how McClintock discovered them, because they changed the colour of a maize kernel.”

But this first hint of their regulatory role was dismissed by her peers. “Scientists of that time could not easily understand or accept the concept of a ‘controlling element’,” George says. “She faced criticism, to the point that she had to drop the term ‘controlling elements’, and rename these mobile genetic elements transposons.” McClintock would eventually win the 1983 Nobel prize for physiology or medicine for her discovery.

These movable pieces of genetic material now make up just 3.6% of the human genome but they’ve left a lasting impact. One example of how DNA transposons have shaped us can be traced back hundreds of millions of years, when a transposon was domesticated in a common forebear of humans and fish to create the ancestor of the modern-day RAG1 and RAG2 genes, crucial components of the recombinase enzyme found in the B and T cells of the immune system. RAG recombinase is essential for immune diversity, generating a varied repertoire of B and T cells that can detect and respond to the multitudes of different microbial invaders we encounter during our lives.

Hidden throughout our DNA are many other traces of ancient genetic parasites that have changed in influence over millions of years. These are remnants of our genetic history.

Rise of the genetic underworld

Long before humans walked Earth, a war raged in the genomes of ancient life. In plants like maize, mobile genetic elements had mastered the trick of transposition, moving themselves across the DNA within a single cell.

Over generations, these early genetic parasites were reinforced by more insidious invaders. DNA transposons were joined by retrotransposons, stealthy agents that copy themselves into RNA, then, using an enzyme called reverse transcriptase, paste themselves back into a new location in the genetic code, occasionally causing havoc.  

Some transposons evolved to be even more ambitious. They hijacked proteins from their hosts, constructing viral envelopes that allowed them to slip between cells, like ghosts gliding from room to room. As they evolved, they developed keys to pick cellular locks – molecular receptors – that let them invade with eerie precision.  

Among them were the retroviruses, so named because they, like their sister retrotransposons, convert their RNA code into DNA, before inserting into the genome of a host cell to duplicate. When retroviruses infect cells that develop into egg or sperm cells, viral DNA can become a permanent fixture down the generations. It is estimated that around one in 100 human births acquires novel retroelement DNA and, over the eons, an uneasy cooperation has emerged between living things and their genetic phantoms: while cells provide retroelements with the means to multiply, retroelements arguably repay by churning the genetic code to help their hosts evolve.  

Today, thanks to the efforts of George and many others, we know more about the function of at least some of the around four million or so remnants of retroelements that are scattered across our DNA. Some researchers argue that the ability of ERVs and retrotransposons to reshuffle genetic material has sped up the evolutionary process, allowing our ancestors to adapt more rapidly to environmental challenges. “They are a force of evolution, a very powerful force,” he says.  

Humans are darkly complex

Towards the end of the 20th century, the regulatory role of the dark genome took on greater significance as molecular biologists found that humans seemed bewilderingly simple: water fleas, tomatoes and wheat, among other ‘simpler’ kinds of life, possess many more genes than we do.

It turns out that human complexity relies on how our 20,000 genes are used, for instance through alternative splicing, when parts of the same gene can be spliced together in different ways to create more proteins. In this way a person’s 20,000 genes can, when used in specific patterns, generate up to 400,000 proteins, by some estimates, to build an immune system, organ or whatever. Retrotransposons can become entangled in this process.  

“The coolest aspect of the dark genome is that it enabled humans to have a placenta,” says Rachael Thompson, a post-doc research assistant in George’s lab.

To create a placenta, mammals in effect stole genes that retroviruses use to bind with and enter a cell, fusing their membranes in the process. In this way, a once-harmful viral invader was captured and turned into a genetic architect. Quite different placental creatures have stolen equivalent genes but from different viruses, “which is pretty insane”, Rachael says. Successive retroviral invasions of the genome of our placental ancestors gave them the opportunity to use a better retroviral envelope than the previous one to make their placentas.  

This dark knowledge has shed light on pregnancy complications: a team at the Pasteur Institute found that, during serious infections, proteins induced by signalling molecules called interferons not only block infection but can interfere with the legacy viral machinery that today makes a placenta.

The dark genome also shapes how a baby develops. One ancient piece of viral DNA, called HERV-H, helps with looping, where DNA folds to bring distant parts of the genome together, like a long string bending to connect important sections. This looping helps turn genes on and off at just the right time, making sure the baby’s tissues grow and form correctly.  

“An ERV integration near one the amylase genes, which make enzymes that break down starch, drives amylase secretion in saliva, so bread tastes sweet,” says George “They’ve taken away our tail, due to a mutation of the responsible gene by a transposable element. They’ve changed our skin colour too, by affecting the production of the pigment melanin. And they’ve provided us with antiviral factors that helped fight of viral epidemics.”  

Other facets of the dark genome seem to play a role in neurodevelopment, potentially influencing conditions such as schizophrenia, addiction and autism. One theory suggests that viral sequences helped drive the evolution of complex brain functions, providing genetic material that enabled rapid advancements in cognition, memory and social behaviour; in this way, the dark genome may have helped to make us human.  

Shedding light on kidney cancer

While George Kassiotis explores viral ghosts and immune sentinels within the dark genome, I visit his colleague Samra Turajlic, who is studying renal cell carcinoma, the main kind of kidney cancer. “My interest in the dark genome came about because of kidney cancer, which is a fascinating disease for many reasons,” says Samra, who is also a consultant medical oncologist and Director of the Cancer Research UK Manchester Institute. “Intriguingly, one aspect that has really come into focus over the last decade is the paradox of why immunotherapy works in kidney cancer.”

Samra Turajlic, a Group Leader at the Crick. Credit: Dave Guttridge.

The immunotherapy in question inhibits immune checkpoint proteins – molecules that evolved to prevent excessive immune responses to pathogens but are frequently co-opted by cancer cells, which may appear ‘foreign’ to the immune system, to protect themselves from immune attack. Checkpoint inhibitor drugs take the brakes off the immune system and have been successfully used to treat melanoma, a form of skin cancer caused when the ultraviolet in sunlight leaves a trail of mutant proteins (‘neoantigens’) that the immune system can spot like foreign flags. But kidney cancer harbours far fewer such mutations, so should not be easily targeted by immunotherapy. That mystery led Samra to suspect the answer lay in the genome’s shadowy vaults.

She began to suspect that immunotherapy worked because it wasn’t targeting conventional mutations at all but proteins linked to the dark genome. To test this ideas, she approached George Kassiotis for help.  

Samra focused on the cellular machinery that monitors whether cells have enough oxygen to thrive, which is disrupted by cancer. Normally, when there’s low oxygen, cells turn on emergency signals. Thanks to work by the 2019 Nobelists Bill Kaelin, Peter Ratcliffe (another Crick colleague), and Gregg Semenza, and others, it’s clear that a mix-up happens where cancer cells think they have no oxygen – even when they do. This ‘false low-oxygen alarm’ (called pseudo-hypoxia) flips on the dark genome, activates blood vessel growth and more, and helps cancer cells to multiply and spread.  

Using data from the TRACERx study, Samra’s team found that an important milestone foreshadowing the evolution of kidney cancer is damage during adolescence to a key part of a cell’s oxygen sensor. Called the VHL gene, it is the recipe for a protein that that helps turn off the hypoxia response.  

Fortunately, all normal cells have two copies of the VHL gene, but the second copy can be damaged, usually many decades after the first VHL gene copy is lost – and this induces pseudo-hypoxia. Samra, along with Bill Kaelin, discovered that pseudo-hypoxia triggers the manufacture of certain endogenous retroviral elements, which are pervasive in kidney cancer. Another reason that upregulation of part of the dark genome occurs is that cancer affects how the DNA in cells is packaged in a form called chromatin, which organises the DNA ‘recipe book’. But in cancer the chromatin is loose, so it is easier to flip to a page, allowing more of the genome, both light and dark, to be read.  

“The biggest change in the transcription of endogenous retroelements happens very early on in cancer evolution and then remains quite steady,” says Samra. To pick out the dark genome proteins presented to the immune system, which she likens to hunting a needle in a haystack, Samra uses immunopeptidomics, in which mass spectrometry, which ‘weighs’ molecules to identify them, is used to study immune-peptide complexes, the molecules 'shown' by a tumour cell to mark it out for clearance by the immune system. “We are going to have to understand which ones are good for the immune system and which ones are good for the tumour,” she says.

The implications of solving this ‘big puzzle’ go much wider than kidney cancer. “If we can unlock this kidney cancer conundrum, we can unlock it for other types of cancer as well,” says Samra. That is why the Cancer Grand Challenge programme, run jointly by the US government’s National Institutes of Health and Cancer Research UK, has announced that it intends to funnel £20m into ‘dark proteome’ research

To complement her detailed work on kidney cancer, Samra is leading a four year study of thousands of patients, called MANIFEST, to understand immunotherapy responses and its side effects more generally, funded by the Medical Research Council, Office for Life Sciences, and industry. “We are conducting a deep study into many elements of patients’ coding genome, the dark genome immune system, the blood, the gut microbiome and the tumour environment,” says Samra. “It's a kitchen sink approach because these interactions hold the key – we are taking the dark genome and contextualising it.” 
 

Architects of discovery

In the southeast corner of the fourth floor of the Crick, I visit some of the twenty plus members of Samra’s cancer dynamics team. Most are busy working at ranks of desks with computers though a few are in neighbouring laboratory spaces, where their studies focus on both kidney cancer and melanoma.

Mihaela Chisca from the retroviral immunology laboratory. Credit: Dave Guttridge.

One of them, Taja Barber, one of the Crick’s laboratory research scientists, tells me how she has to use barcodes, computers and more to keep track of the many samples from cancer patients that flow through Samra’s laboratory, from storage in liquid nitrogen to analysis. Scattered around her is the usual molecular biology lab paraphernalia: centrifuges, Rainin pipettes (ergonomic, to prevent repetitive strain industry, says Taja), PCR (to multiply DNA) and so on. These can be found in greyish boxes near biosafety cabinets to handle and grow cells, both animal and human, in phials suspended in reddish nutrient media.  

Taja shows me one impressive box of tricks that is used to shed light on the dark genome: the Phenocycler, a concoction of white and clear blue plastic that contains a powerful microscope to highlight cellular details inside tissue, or a tumour, not just the cancer itself but the immune cells that invade it. Running the Phenocycler takes two technicians, Steph Hepworth and Anne-Laure Cattin. They add glowing tags (made of a fluorescent molecule linked to an antibody that can target a particular molecule) to different parts of the sample, take pictures, then erase the tags and repeat the process.

Two floors below, in the northwest corner of the Crick, George shows me millions of pounds worth of instruments, called flow analysers, which are also expertly tended by technicians. These powerful instruments can study thousands of cells per second, identifying the parts of the immune system that dark elements activate using antibodies honed to dock with specific molecules, each carrying a fluorescent tag that glows under lasers of different wavelengths.

In one room, a confection of black tubes, silver drums and yellow laser warnings make up an analyser that can not only detect labelled cells but divert a single cell into a well for further study, or to be multiplied. This is one of many of the Crick’s ‘science technology platforms’, each operated by a dedicated team – in this case, 12 technicians, led by Andy Riddell. "We are very proud of this facility," says George, "It is one of the largest in the country.” Using these instruments, the Crick teams have gathered evidence that targeting the dark genome can provide new avenues for cancer treatment.

From the shadows, next generation medicine

When it comes to cancer, the good news is that the immune system can recognise dark viral elements when they are reactivated, so that these endogenous elements can, in the words of George, “act as a kind of alarm system.” Efforts are under way to detect them in blood, providing early warning of cancer, though he emphasises that proven, reliable, practical tests remain some way off.

Various companies have also been set up to make potential cancer vaccines that target the proteins made by dark retrotransposable elements. Among them is one that George has helped to set up, called Enara Bio, though again he is the first to caution that it can take decades to turn basic scientific insights into practical treatments.

As we uncover more details of the dark genome, expect more shockwaves across medicine. Scientists are already exploring ways to silence harmful ERVs, potentially offering new treatments for other conditions such as autoimmune diseases. Others are investigating whether beneficial viral sequences could be harnessed for gene therapy, using ancient viruses as tools to repair faulty genes.

“They have been linked to age-related inflammation,” adds George. “even to the point that some have suggested we should be on antiretroviral therapy to slow down ageing and age-related inflammation.”

Recent technological advances, and studies by a global network of researchers, have transformed our understanding of these parasitic dark elements, says George. As they uncover the dark genome’s secrets, scientists will reshape our understanding of evolution, development and health. Rather than fearing what elements lurks in the shadows of our genome, we should recognise them for what they are: not just the architects of our past and present but also of our future.