On June 16, 2001, it was announced that one of the most ambitious and significant scientific projects in human history had been completed: publication of a draft of the human genome in its entirety. Within a few years, we had moved from an almost complete lack of knowledge about our genes to being able to read the full text of the book of life. In many senses, that event could shape the progress of our species in the coming decade, century and millennium.
This past November, on “Black Friday,” biotechnology companies offered for sale on the web whole-genome sequencing services, for the affordable price of $200. It sounds good, but the benefit derivable from the abundance of genetic information now available is limited as long as we don’t understand how the system works overall – and that understanding requires research about the genome’s three-dimensional structure. Accordingly, one of the major challenges in genomic research in the years ahead will be to decipher that spatial structure. Research in this realm has only just begun, and in Israel there are but a handful of scientists involved in it. How, then, is this three-dimensional structure currently being studied?
Edited babies
Less than 20 years after genome sequencing became possible, the scientific progress in genetics is breathtaking, and sometimes scary. The cost of such sequencing is plunging, and today, for a relatively modest sum, each of us can receive an analysis of our genetic ancestry, as well as statistical information about our tendency to fall victim to illnesses with a hereditary component.
Every day too, scientists are discovering, by means of genome analysis, new details about the history of the human species, the causes and reasons for various illnesses as well as new ways to cope with them. We are moving ahead rapidly to a stage where every person will be able to receive personalized medical care based on his or her genome.
Today’s researchers are also capable of performing genome editing; in fact, a Chinese biophysicist stunned and shocked scientists everywhere last year by exploiting available tools to bring about the birth of genome-edited babies. The scientist, He Jiankui, of the Southern University of Science and Technology in Shenzhen, was inspired by a mutation that exists in a certain proportion of the world’s population (and in a higher proportion among Ashkenazi Jews). Its carriers are missing a gene that is responsible for creating a protein through which the HIV virus enters the cells, and as such are effectively immune from HIV infection. He Jiankui used the genetic editing tools available today to disable this gene in fertilized eggs of couples in which the father was HIV-positive. By these means he brought about the birth of babies who are supposed to be HIV-immune (though only one of them carries two copies of the engineered gene and actually acquired the immunization).
He Jiankui drew almost wall-to-wall criticism for breaching the ethics rules about performing experiments on human beings and about the use of insufficiently tested scientific tools. And then, a few weeks later, in a flabbergasting example of the complexity of the function of each gene in the body and of our limited understanding of that complexity, an American-Israeli study was published showing that the carriers of the mutation might possess certain cognitive abilities that are higher than average. In the wake of the publication of that research, the possibility arose that He Jiankui had been aware of this potential before choosing the gene for his procedure and that he may have been trying to bring about the birth of “enhanced” infants, possessing higher-than-average intelligence.
He Jiankui is currently under house arrest in China, his academic career apparently over. However, his name has already been recorded in the history books as the scientist who took the first step toward a post-human future, in which people will be able to choose the their offspring’s desirable traits. The implications of his deeds for the lives of Lulu and Nana, the twin girls born last November as a result of his experiment, and for the life of a third child, due to be born this summer, remain to be seen. But his actions also spotlight the current limits of understanding, despite all the progress, of the complexity of the information encoded in DNA.
The big challenge, which modern science is only now beginning to address, is that, in classical biology, genes are often studied individually. But the human body is a system, and in order to understand how it works, researchers need to understand how all the components interact and work together as a system. This realization has led to the emergence of a field of research known as “systems biology,” which is trying to bridge this knowledge gap. A major issue in the systemic study of the genome is that scientists often consider the DNA in only one dimension – its sequence. However, the organization of DNA in three dimensions (that is, spatially) plays a critical role in the genome’s activity, though the currently available tools offer only a glimpse of the vast complexity of the basic mechanism that is the foundation of all life.
In Israel of 2019, there are not many scientists who are coping with the challenge of trying to understand the three-dimensional structure of the genome as a whole. But in order to arrive at a deep understanding of how these chains of nucleic acids band together and write the book of life, it will be impossible to make do with its flattened version. According to Prof. Noam Kaplan, from the Faculty of Medicine of the Technion – Israel Institute of Technology, “We now possess the ability to edit DNA very precisely. We can read and write DNA easily. But what we’re lacking is a deep understanding of what we are reading and writing.”
As early as the 19th century, scientists saw via microscopy that DNA is organized in particular structures within the cell nucleus and that this form of organization changes in the course of such biological processes as cell division. The tremendous breakthrough by James Watson and Francis Crick (with the underappreciated aid of the chemist Rosalind Franklin), published in 1953, dealt with understanding the structure of DNA molecules and with how the “ladder” of sugar and phosphate forms the double helix, providing insight into DNA’s encoding and replication of information. However, not until a few years ago did scientists have a practical way to understand how the whole genome is arranged in the space of living cells, how that structure connects with the sequence of letters of DNA, and how this three-dimensional structure affects the cell’s function.
In principle, each of our cells contains the same DNA sequence, with the same 20,000 or so protein-encoding genes. This raises the question of how the same DNA sequence exists in cells as different in form and function as neurons and white blood cells. The answer is that in each cell of the body a different set of genes is activated at any given moment. Some genes are active all the time, others only when the cell is required to perform a certain action or only in certain types of cells. If DNA is a recipe book, when we go into the kitchen we don’t want to prepare all the recipes together, only those that are appropriate for that particular meal.
One of the 20th century’s great discoveries was of how DNA’s double-helix form enables it to function. In order to produce protein, the cell needs to unravel a portion of the double-stranded DNA. In order to replicate, the cell must separate the strands completely. What, then, decides which genes will function in which situation? According to Prof. Kaplan, the physical structure can control activity: For example, if some genes are densely packed, their information is simply not accessible. Returning to the metaphor of the genome as the book of life, it can be compared to a situation in which some of the pages in the book are glued together and therefore are unreadable.
In addition, protein-encoding genes possess “regulatory sequences,” to which other proteins bind before the gene is activated. In some cases, Kaplan notes, the regulatory sequences are adjacent to the gene itself, but there are other cases in which the regulatory sequence can be situated hundreds of thousands of letters away from the gene itself, so it becomes difficult to understand which regulatory sequence affects which gene, and why. One mechanism of regulation in the genome is the folding of the DNA chain so that the regulatory sequence draws physically closer to the gene. Thus, says Kaplan, two cells bearing an identical DNA sequence but whose DNA is folded differently, can behave in different ways.
Kaplan likens this regulatory system to the cell’s “brain,” which decides, based on the cell’s situation and its surroundings, which genes to activate. In fact, one of the standard methods currently used to define cell types is analysis of the genes that are active in them. With the progress made in reading the genome, scientists found that there is no necessary connection between the number of letters in an animal’s “book of life,” and its complexity. Often, “the complexity of living beings may depend on the complexity of the regulatory system that decides which genes are active,” explains Kaplan.
Understanding the three-dimensional structure of the genome and the way it affects gene regulation is also related to another field that has been gathering momentum during the past 20 years. Epigenetics deals with the processes that influence the manner in which genes are expressed and inherited, without changes in the DNA sequence itself. A key principle in Darwinian evolution is that acquired traits – those brought into being by the influence of the environment in our lifetime – are not passed on to the next generation. According to this view, evolutionary changes are caused only as a consequence of genetic mutations, which occur randomly and change the organism according to the principles of natural selection. Studies in recent years, however, suggest a more complicated picture. It turns out that parents can pass on modifications to their children even without changes in the DNA sequence. One example that has drawn considerable attention is the way in which trauma influences epigenetics and how its effects are inherited by future generations.
There are genes whose function is relatively clear, and genes for which scientists have been able to identify a direct link between a mutation in them and a disease (such as cystic fibrosis). Still, a large number of hereditary diseases, and most of the traits that make us human, each depend on the activity of dozens if not hundreds of different genes. Accordingly, many of the discoveries concerning the activity of a particular gene suggest only a certain statistical connection, limited in its power, between the gene and the trait. “To achieve deep understanding of how genes cause complex phenomena, we have to understand how genes interact and how they are regulated. To understand that, we need to understand the three-dimensional structure of DNA,” suggests Kaplan.
Sequence switching
In light of all this, how are scientists currently investigating the three-dimensional structure? The problem, says Prof. Yuval Garini, a biophysicist at Bar-Ilan University, is that there are no tools today with the appropriate resolution for directly viewing the spatial structure of DNA. Optical microscopes, even the most sophisticated, allow each chromosome to be seen in its entirety, but not to decipher all its details. And electron microscopes first require the sample first to be dried out and fixed, ruling out the possibility of studying a living cell in this way.
Garini likens the DNA in the cell’s nucleus to a plate of spaghetti (though emphasizing that this comparison appears in older textbooks and is now known to be inaccurate). If the optical microscope allows scientists to see the whole serving of pasta but without being able to distinguish the details of the spaghetti’s folds, with the electron microscope, scientists obtain the structure of a small slice of a noodle, but even that information is not very informative about the genome’s spatial structure.
In that case, how can the structure be deciphered? One method that is drawing much attention does not, according to its developers – who are considered potential candidates for a Nobel Prize – even try to view the genome directly, but rather seeks to learn about its structure by resequencing the DNA following various manipulations. The basic principle of this method was originally developed by the Dutch-American scientist Job Dekker in 2002, but its full potential was only realized after technological advances made it possible to sequence vast quantities of DNA at very low cost. The updated method, published by Dekker and colleagues in 2009 and known as “Hi-C,” established an entire field of research.
The idea behind Hi-C technology is that of copy, cut and paste. Because DNA is densely packed in the cell’s nucleus, there are regions in the chain that are spatially close to one another. In the first step, the scientists add a chemical to the cells that causes the DNA segments that are spatially adjacent to become attached to each other. An enzyme is then added that cuts the DNA into fragments, resulting in a collection of short DNA fragments that are stuck together according to their original spatial proximity. In the third step, attached pairs of DNA fragments are linked together to form a new DNA sequence, resulting in a fusion of the sequences of the two fragments that were proximal in space. In the end, hundreds of millions of these fused sequences are read by sequencing machines, and by means of comparison to the original genome sequence and the use of advanced algorithms, researchers are able to identify the points in the genome where proximity originally existed between two different parts of the chain.
This method is being applied by Noam Kaplan (who previously was in Job Dekker’s research group), in his new laboratory at the Technion. The method is not yet able to create precise models of the genome’s spatial structure, Kaplan notes, but it does allow researchers to produce maps depicting the mutual proximity of different DNA regions. The maps have led to important breakthroughs in understanding the function of the genome. For example, he says, this method led to the discovery that a previously unknown internal division exists in DNA – “neighborhoods,” as it were, of regulatory sequences. Through this division, genes are influenced only by regulatory sequences in their “neighborhoods.”
Moreover, relates Kaplan, a group of researchers from Germany, led by Prof. Stefan Mundlos of the Max Planck Institute, has used the insight about “neighborhoods” of regulatory sequences to decipher the mechanism underlying a group of genetic diseases that cause disfiguration in the fingers. Surprisingly, no disruption was found either in the relevant gene sequences or in the control sequences that dominate them. The new method showed that a disruption in the DNA of these patients modified its spatial structure. As a result of the structural change, a gene was influenced by the regulatory sequences from a foreign “neighborhood” and was activated at the wrong time during development – thus causing the disease. In cancer, too, examples have been found recently in which changes in the organization of the genome led to the activation of the wrong genes and thus to the disease.
Mice and the atomic microscope
Noam Kaplan’s laboratory, which was established in 2016, was recently involved in measuring for the first time the spatial organization of the genome of sperm cells during their development. “Sperm cells are biologically very much unique,” Kaplan says, explaining his interest in the subject. “Biological mechanisms are usually based on quantity in order to ensure success. However, in the case of a human sperm cell, successful reproduction ultimaely depends on one cell carrying out its mission flawlessly. It is thus of supreme importance for the sperm cell to be created in an orderly, regulated process.”
Hundreds of millions of sperm cells are created in the human body every day. A critical stage in the development of sperm cells is meiosis, at the end of which only half the amount of the original DNA remains. Meiosis also entails the reorganization of the DNA in which the chromosomes are condensed and exchange different segments between them, thus increasing genetic diversity and preventing in practice the possibility that two identical offspring will be born. In collaboration with a group led by Prof. Satoshi Namekawa, from the Cincinnati Children’s Hospital, Kaplan and a student in his group, Haia Khoury, were able to monitor the meiosis of sperm cells in mice.
“We wanted to see how the organization of DNA changes during this process, and how, despite the condensation, some of the genes continue to be active,” explains Kaplan. The study was published in February in the journal Nature Structural & Molecular Biology. The researchers propose that the three-dimensional organization of sperm cells is important for genes that provide germ cells with he ability to divide into specialized cells following fertilization.
The organization of DNA is critical not only for humans but also in beings of very distant species. In a study published recently by an international group of scientists in the journal Nature, the researchers, led by Prof. Nicolai Siegel, from the University of Munich, used Hi-C technology to decipher the spatial organization of Trypanosoma brucei, a parasite that causes African sleeping sickness. “With the aid of a special spatial organization of the genome, the parasite activates genes that modify its outer surface, and thus allow it to evade the immune system,” explains Prof. Kaplan, who was also involved in the study.
Another approach to understanding the three-dimensional structure of DNA is to try to discover what holds it in place. This is the path that has been pursued by Yuval Garini in his laboratory since 2007. One of the basic tools in molecular biology is the ability to color parts of the genome (there are different ways to add fluorescent material that acts as a “lantern” when it is viewed through the microscope). When the chromosomes are fully colored, relates Garini, it’s apparent that each one has its own “territory” in which it remains within the cell nucleus. That territory can be different in each cell; and even though there are no barriers within the nucleus separating the chromosomes, each of them remains in its own territory.
In his study, the Bar-Ilan University scientist notes, “lanterns” were attached to the ends of the chromosomes (called telomeres), with the result that 92 dots of color appeared across the cell nucleus. The next stage was to monitor the movement of the glowing dots. Prof. Garini draws a comparison with the Waze navigation application, which by monitoring travelers is able to trace the roads they used and also learn about other regions on the map. The monitoring of the telomeres showed that the ends of the chromosomes also hardly move.
After years of research, says Garini, he and his colleagues have succeeded in identifying the protein that is responsible for this fixedness of the DNA strands. He likens the function of the protein, called lamin A, to the way that clothespins can fix a twisted rope in a relatively stable condition. The inner lining of the cell nucleus is also made of the same protein, he adds, and thus the “clothespins” connect to it and stabilize the chromosomes in their location within the cell. Researchers are also capable of coloring the lamin A “clothespins,” which also enables them to obtain additional information about the points of connection between the various twists in the DNA chain.
Scientists use additional tools to decipher the structure of the genome. One of them is known as the atomic-force microscope. Prof. Garini compares it to a needle whose point has a diameter of one atom. That needle can be shifted with nanometric movements across surfaces, thereby enabling their texture to be deciphered at the atomic level. This tool, he says, offers scientists a precise picture of the way in which the genome is connected by various proteins, but without the ability to obtain similar information about its internal structure. Beyond this, the field of microscopy is itself undergoing significant revolutions, with more anticipated in the years ahead, providing researchers with a more accurate look at the genome’s twists within the cell nucleus.
Optimistically, Yuval Garini estimates that 10 years down the line, we will already be able to get a direct glimpse of the spatial structure of the code that shapes us. Noam Kaplan is more cautious – he believes decades will be needed – but he too is optimistic. Even if we don’t succeed in constructing the exact three-dimensional model of all DNA in the near future, he says, the insights we are getting about its organization are leading and will lead to many scientific breakthroughs. Noam Kaplan sums up: “Ultimately, we want to understand genome function, and therefore a precise description of every detail of the structure at very high resolution is not necessarily the final goal. The question is, what can we learn from that about the function of the genome.”
