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I want to unravel the “black box of chromosomes” described in textbooks

　Chromosomes are intracellular structures that carry genetic information to daughter cells during cell division (Fig. 1). As a postdoctoral fellow studying chromosomes at University of California, San Francisco (UCSF), Tatsuya Hirano discovered a new group of proteins (now known as SMC proteins) that were completely unknown at the time. He then went on to head his own laboratory at Cold Spring Harbor Laboratory (CSHL) in the suburbs of New York City, where he continued to develop his findings. This period (from the mid-1990s to the early 2000s) was an exciting period in which the field of chromosome research not only made great advances, but also found links to the cell cycle and human hereditary diseases and cancer. Hirano’s discovery of the multiprotein complex “condensin” could be described as “one of the last great discoveries in the last unexplored field.” This article traces the footsteps of Hirano, who has focused on analyzing the cellular functions and molecular mechanisms of SMC proteins involved in a wide range of chromosomal functions.

　When I began my research career as a Ph.D. student at Kyoto University Graduate School of Science in the early 1980s, recombinant DNA technology was just beginning to be applied to elucidate basic biological phenomena. However, genome sequence information was scarce, and it was a time when genes had to be isolated and analyzed one by one. Research on DNA replication and transcription was very popular at the time, but I focused my attention on the fundamental question of how long DNA molecules are folded in the cell. Although the structure of the nucleosome, the first stage of DNA folding, had been elucidated in the 1970s, the higher stages of folding observed during cell division (i.e., mitotic chromosome assembly) remained a complete black box. Since Prof. Mitsuhiro Yanagida was one of the few researchers working on this problem, I knocked on the door of his laboratory.

　My laboratory now uses a biochemical approach, but Yanagida’s laboratory used a genetic approach. Looking back, I used yeast genetics in his laboratory to search for a protein component that later became known as condensin. In fact, a junior student of mine later found that some of the yeast mutants I had isolated were actually mutant strains of condensin.

　Let’s compare the difference between the two approaches to a car. In the biochemical approach, we take the car apart and try to understand the function of each component. We make speculations such as, “The accelerator pedal is connected to the engine, so it must be involved in its control,” and “The brake pedal probably helps stop the rotation of the tires.” Ultimately, when we understand the functions of each part and can reassemble a car from all its parts (this process is called “reconstitution”), we will feel that we understand how a car works. On the other hand, the genetic approach tries to understand the function of each part by randomly destroying the parts one by one and observing how the car works (or does not work). In many cases, we reach a comprehensive understanding of a biological phenomenon when the biochemical and genetic approaches converge. My research on condensin is an excellent example of the convergence of these two approaches.

Hirano’s research did not bear fruit in his first year abroad. A “course correction” led to a great discovery.

　After completing his Ph.D. at Kyoto University Graduate School of Science, Hirano moved to the U.S. as a postdoctoral fellow. At that time, there was no postdoctoral training system in Japan, which made it difficult for researchers to find employment after receiving their Ph.D. Hirano also recognized the limitations of genetic approaches and sought a new biochemical approach. So, he decided to start his postdoctoral work abroad at UCSF in 1989. At the time, UCSF was considered a mecca for cell biology. He was amazed by the low barriers between laboratories and the rapid exchange of information and materials.

　At UCSF, I learned a new experimental method called “frog egg biochemistry”. In my first project, I tried to make kinetochores (special structures on chromosomes) in vitro. Although I spent more than a year on this project, it bore no fruit. So I decided to redirect my research from a new angle: “To make the special kinetochore structure on chromosomes, it would be necessary to first make the whole chromosome itself as a foundation.” Since this decision eventually led to the discovery of the SMC protein, I truly feel that nothing can be wasted in the research process.

　My experiments were performed as follows. First, I obtain a large number of Xenopus eggs, crush them in a centrifuge tube, and collect an extract (a highly concentrated soup containing all the contents of the eggs). One advantage of this experimental system is that we can make chromosomes in a test tube by simply adding sperm nuclei to this soup. Using such an “in vitro” experimental system makes it much easier to perform various experiments compared to isolating and analyzing chromosomes assembled inside the cell. For example, we can easily identify the protein components bound to the assembled chromosomes. We can test whether the protein of interest plays an essential role in the chromosome assembly process by inhibiting its activity in the test tube. Through these experiments, I was able to discover a pair of proteins, “SMC2–SMC4,” as a key factor in mitotic chromosome assembly.

　Despite these great achievements, there were few opportunities at that time for researchers who had completed their postdoctoral studies abroad to work independently in Japan. The only, but undesirable, option was to work as a research assistant to a professor at a Japanese university and wait for an opportunity to be promoted. Since my postdoctoral work was highly rated in the U.S., I decided to stay there and work independently by setting up my own laboratory. Fortunately, I was offered a position at CSHL. In 1995, I started my own laboratory at CSHL, looking forward to complete independence and a top-notch research environment. While many Japanese researchers now have their own laboratories abroad, this was rare in the 1990s.

　At CSHL, I enjoyed working with young scientists from different countries around the world. I feel that I was able to grow as a person by “getting jostled” in an active and stimulating environment both inside and outside the laboratory. Although it was not easy to secure research funding, once I did, I was able to concentrate on my research because I had few other responsibilities. Also, an atmosphere where you were fairly valued as long as you did a good job suited my nature. On the other hand, of course, the competition was fierce. I continued my research in such an excellent environment at CSHL, and in 1997, I was able to report the discovery of condensin, a new multiprotein complex containing SMC2-SMC4 that plays a central role in mitotic chromosome assembly. We were surprised to find that condensin had a very peculiar conformation that no one had ever seen before (Fig. 2).

A research field that illuminates not only basic biology but also the future of humanity

　Condensin, discovered by Hirano, was a completely new type of protein machine, so his efforts to understand its molecular mechanisms are still ongoing. After expanding his research program at CSHL for a little over a decade, Hirano returned to Japan in 2007 and established Chromosome Dynamics Laboratory at RIKEN Discovery Research Institute (currently RIKEN Pioneering Research Institute). In 2015, he published a landmark paper on in vitro chromosome reconstitution.

　Although it was generally accepted that condensin plays a central role in mitotic chromosome assembly, it was not known how many other proteins were required for this process. One day, a member of my laboratory asked me the following question: “Is it possible to assemble a chromosome using a limited number of purified proteins?” This question led us to develop a new experimental system. Based on the information we had gathered up to that point, we first prepared three structural proteins, including condensin, and two other proteins that assist in their functions (collectively called chaperones). We tried to reconstitute chromosomes in a test tube by mixing these five proteins with sperm nuclei, but it did not work. After many attempts, we finally identified another essential chaperone protein. Since the sperm nuclei were successfully converted into mitotic chromosomes when this protein was added to the previous cocktail of five proteins, we concluded that chromosomes could be assembled using only six proteins. This simple conclusion surprised chromosome researchers around the world, but in fact, the logic behind the success was actually in the SMC2-SMC4 paper that I had published in 1994. In other words, it was the result of 20 years of research. The establishment of this chromosome reconstitution assay was hailed as a milestone that rewrote the biology textbooks.

　Ever since I became a researcher, I have been working toward one major goal: understanding mitotic chromosome assembly. I have expanded my research program by incorporating new approaches depending on the backgrounds of the people who gather in my laboratory. The chromosome reconstitution mentioned earlier is just one example of our many accomplishments. In addition to biochemistry and cell biology, I have also introduced structural biology, single-molecule biophysics, mouse genetics, and non-model organisms. Currently, my research focuses on fundamental questions, such as “How do the chromosome components work at the mechanistic level?” and “How do the multiple components work together?” In particular, I consider understanding the molecular mechanisms and regulation of condensin to be my life’s work.

　Our laboratory’s website lists three research subjects: “Molecular mechanisms of action of SMC protein complexes,” “Regulation of SMC protein complexes,” and “SMC protein complexes and human diseases.” While I plan to continue basic research that contributes to the fundamental understanding of biology, in the long term I also hope to contribute to the field of clinical medicine by addressing questions such as how chromosomal aberrations are associated with diseases including cancer and genetic disorders.

Be original or die – Swim in your own blue ocean!

　Hirano has continued his research to gain a comprehensive understanding of chromosome assembly and dynamics. During his career, he has witnessed the historical developments in the field of chromosome biology: the first wave (mid-1990s to early 2000s), when the field made a series of groundbreaking discoveries, and the second wave (from the late 2010s), when the field embraced new technologies such as single-molecule analysis and Hi-C, as well as new concepts from polymer physics. What is Hirano most proud of in his long research career?

　Scientists are often perplexed when asked the following question: “What good will your research do?” But no one would ask athletes, “What good will your sport do?” No one would ask writers, “What good will your novel do?” Just like sports and novels, the first principle of science is not to create short-term economic value or immediate social impact. I see science as a kind of cultural activity that generates new knowledge that will eventually become a treasure for all humanity. I am proud to be a part of such an activity. I am also proud to have been one of the leaders in my research field for nearly 30 years, even though I am “almost out of breath.” SMC proteins are now being actively studied all over the world. This field has grown so much that we had the opportunity to hold an international conference on SMC proteins in Japan in 2017, of which I myself was one of the organizers (Fig. 3).

　Because my research is basic, continuity is very important. While a major discovery may put you in the spotlight, you cannot end up as a one-hit wonder. It is important to maintain a broad range of knowledge and flexible thinking skills in order to continue research over the long term. The motto in my laboratory is therefore “discuss, discuss, and discuss.” Based on careful and constructive discussions, we set up our own questions, work out our own strategies, and take our own approaches. And we should always be aware of how we can differentiate ourselves from our competitors. These considerations are essential for a researcher to survive in the competitive field of modern science. “A scientist should be the president of a start-up company!” “Do not aim for a red ocean. Find your own blue ocean and swim as hard as you can!” I have a duty to pass on this spirit to the next generation of scientists.

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