Ghost in the genome

It turns out genetics is not as complex as we thought. It’s even more complex. Associate Professor Kevin Morris has recently arrived from the US and is working on uncovering the startling complexity of gene regulation.

While the genome revolution has been underway for many years now, another one has been taking place in its shadow, one that has the potential to explode our understanding of the manifold mechanisms that manage the genes themselves. This is the study of the regulation of our gene expression, the systems that can effectively switch our genes on and off (or ramp their expression up or down), sometimes in lasting ways that can pass between generations.

Gene regulation can occur at multiple junctures, such as at the transcriptional level via DNA methylation or the post translational modification of histones. Another mechanism, discovered in the late 1990s, occurs post transcriptionally during gene expression and is mediated by a growing family of RNAs.

It’s this latter approach that is of particular interest to Associate Professor Kevin Morris, who last year made the journey from the Scripps Institute in California to the University of New South Wales. This rising star of the RNA world was lured here by Australia’s pedigree in noncoding DNA, in the form of individuals such as the Garvan’s Professor John Mattick, Diamantina’s Dr Marcel Dinger and University of Queensland’s Dr Ryan Taft, as well as his passion for surfing. Clearly, he’s come to the right place.

At UNSW he is embarking on a programme to understand the processes behind the RNA-directed epigenetic regulation of gene expression, with a particular eye towards some enticing therapeutic applications. He will be speaking at the Lorne Genome conference about his work and what he sees as the tremendous potential of both transcriptional gene silencing and activation.

Make hay

Far from being ‘junk’, it appears the roughly 98% of our genome that is constituted by noncoding DNA is indeed treasure of sorts. It performs the essential role of regulating the transcription of our genes to produce and assemble the protein building blocks of life. While some outspoken researchers, such as John Mattick and Malcolm Simons, had been waving the flag that this noncoding DNA must serve some essential function, the general consensus over the past decade or two had been one of overwhelming scepticism.

Yet Morris and his team at Scripps were convinced something interesting was going on in the noncoding regions of the genome, and they showed as such in their lab. “Initially, we found that some RNAs can control transcription,” says Morris. “In subsequent follow-up work, we showed how it was working mechanistically, how the RNAs interacted with the chromatin, and that in some cases there are higher ordered RNA-RNA interaction that occur.

“We first did this using synthetic small interfering RNAs, because that’s back when siRNAs were really hot, but we didn’t know what the mechanism was. We just threw stuff at the cell and we could see the silencing effect. But we did know this form of silencing was occurring through epigenetics because we could use certain cancer drugs, which are used to turn tumour suppressor genes back on, to inhibit the process.”

It was around 2007 that Morris became sure that in human cells it wasn’t small RNAs that were responsible for the gene epigenetic-based regulation but longer forms of noncoding RNA. Yet, even after some publications in prestigious journals stating as much, the reception from the wider genetics community was still unenthusiastic.

Morris remembers discussing his situation with Professor John Mattick in a bar following a day at a conference in Brussels. “I was really bummed out that we’d been working our butts off, struggling to get funding, and nobody was believing us. I’d go to give talks and everybody was just sceptical. I told him how frustrating it was, and he said, ‘Kevin, listen, make hay while the sun is shining, because this is our time.’ And he was so right about that. Now I look at it and everybody’s doing it.”

The turning point in popular sentiment was the publishing of the Encyclopedia of DNA Elements, or ENCODE, which reported in September of 2012. It finally gave some concrete evidence to quash the ‘junk’ conjecture once and for all, finding that at least 80% of the genome serves some kind of biochemical function. That’s far more than the 2-3% that had been widely believed to be functional only a few years ago. Morris felt vindicated.

Layers

At Scripps, Morris and his team went some way towards understanding the mechanism that underlies this process of gene regulation. “What we’ve learned is that we can make small RNAs that can turn genes off in a very stable manner that are also heritable,” he says.

“The way many RNAs are controlling transcription in human cells is epigenetically based. What that means is that it changes the chromatin code around the DNA, making it compacted or opening it up. So when we target a particular region with RNA, we can compact the DNA, and if we do that to a promoter, if we hit it for a long period of time, we can get DNA methylation. Then all these epigenetic changes in the DNA that make it resistant to being transcribed are heritable and they can be passed on to other cells.”

Effectively, Morris figured out how to target specific genes and ramp them up or suppress them in a lasting way, all without affecting the underlying genetic code. The therapeutic potential is enticing.

“So we can hit something and turn it off, and remove our signal that turned it off, and it stays off. But the even more amazing thing is this: if you target a particular gene - let’s say in a case of cystic fibrosis - there are two noncoding RNAs we’ve discovered that are regulating CFTR [cystic fibrosis transmembrane conductance regulator] gene expression, and if you knock those down or turn them off, you can increase CFTR expression four-fold.

“If you do that, you’re going to end up increasing some level of the defective CFTR receptor on the surface of the cell, which helps with the chloride ion transport even though it’s defective. If you take a cystic fibrosis patient and give them a four-fold increase in the receptor, it might be beneficial.

“What gets even more fascinating about this is, in the case of cancer, you have tumour suppressor genes that control the cell cycle and cell division. What happens over time in a lot of cancers is that they get turned off, presumably by noncoding RNAs. These noncoding RNAs that are regulating them are getting out of control and turning them off, and when they’re off, the cell divides uncontrollably. So almost all the tumour suppressor genes have these noncoding RNAs, and if you know which ones are turned down in a particular cancer, you can target them and turn them back up.”

He has also been exploring the role of noncoding RNAs in viruses like HIV. “HIV has its own RNAs that it uses to control itself in viral latency. So if you target that noncoding RNA that regulates HIV, and you find a way to stably target it, it might be that the virus can’t go into a latent state and it’s always replicating. Then you can use drugs to inhibit it from spreading.”

In principle, the mechanisms for RNA-mediated gene expression that Morris is investigating could be used to hone in on problem genes and flip their expression in lasting ways. But there’s a hitch.

“The big elephant in the room is targeting,” he says. “We can turn genes on and off willy nilly - pick your gene and we can do it - but the problem is getting it to the right cells that need it.”

Targets and inputs

Half of Morris’s lab, still based at Scripps, is working on this problem of developing targeted therapeutic approaches to delivering RNAs that can regulate gene expression, helped by a $14 million NIH grant. The other half, which he is establishing at UNSW, will focus on uncovering the basic mechanisms at work, particularly in human and primate cells, and exploring the complexity of RNA gene regulation, as well as its implications on evolution.

After all, if 80% of our genome appears to regulate the 2-3% of protein coding regions, and these regulatory patterns are conserved through generations, then this seems to be a ripe candidate to undergo evolution. John Mattick has already made claims that in order to understand the evolution of startlingly complex organisms like ourselves, we need to look not only at protein coding regions but the noncoding regions alike. Morris agrees.

One of the giveaways that noncoding RNAs have some link to the evolution of complexity is the fact that the number of protein coding genes has only a loose correspondence to the complexity of an organism, yet the proportion of noncoding regions in the genome increases with complexity.

As Morris points out, gene regulation gives a whole new level of plasticity and responsiveness to the environment, allowing organisms to adapt to changing conditions over generations without requiring significant changes in the protein coding regions of the genome. “So you can have different exposure to the environment causing different transcriptional paradigms, and that leads to adaptability,” he says.

The question that remains to be answered is how the noncoding regions sense changes in the environment and respond accordingly. “So now we have the connection that the RNA can control transcription, but the missing connection is how the outside stimulus is causing the activation of particular RNAs that lead to the different transcriptional changes. Input is the missing thing, but we’re working on that.”

There can be no question that it’s exciting times for genomics, but particularly for epigenomics. If Morris’s suspicions about the role and function of noncoding RNAs are correct, then they have the potential to radically shake up our understanding of our genome and could pave the way to a new era of personalised medicine. We’ve long known the genome is complex. But it’s only beginning to dawn just how complex it might truly be.

Associate Professor Kevin Morris received his PhD in comparative pathology, with a particular interest in HIV, at the University of California Davis, and continued working there in the years following his PhD. In 2005 he shifted over to the Scripps Research Institute in California looking at antisense noncoding RNAs to regulate transcription of HIV-1 and genes involved in cancer. In 2012 he also took up a position at the School of Biotechnology and Biomolecular Sciences at the University of New South Wales. He has received several awards throughout his career including the 2010 Outstanding Young Investigator Award from the American Society of Gene and Cell Therapy.