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The world’s only research to have unraveled the structure forming the regional centromere through observing atomic resolution

Hiroaki Tachiwana
Junior Researcher (Assistant Professor), Faculty of Science and Engineering, Waseda University

Entering the world of biology from an electrical engineering background

In writing this article, I am trying to unravel how chromatin, a complex consisting of DNA and protein, plays a decisive role in gene expression (Figure 1). All cells from a fertilized egg contain identical genetic information. Yet, some cells become part of eyes, some become part of a heart, and some others become part of a liver. Only necessary parts of the long threat of genetic information in a cell is unfolded and activated to form a particular part of the body. This mechanism is regulated by chromatin, and we believe that understanding the structure and properties of chromatin will unlock secrets behind genetic regulation of cellular differentiation.

The study of such gene expression’s regulation mechanism underlying activated genetic information is called epigenetics—which is the main subject of the laboratory of Professor Hitoshi Kurumizaka where I belong. We take a unique approach to artificially create chromatin as a biopolymeric material for conducting experiments. The basic structural unit of chromatin is a nucleosome comprising DNA and four types of core histone proteins. We use E. coli to produce each one of these DNA and histones to reconstitute nucleosomes.

Figure 1: Structure of chromatin

Waseda University reorganized its departments when I was about to start the fourth year of my undergraduate study. My department, the Department of Electrical, Electronics and Computer Engineering in the School of Science and Engineering, became the Department of Electrical Engineering and Bioscience and gave me the option to join a biology or life science laboratory. One of the faculty’s leading life science laboratories was headed by Professor Kurumizaka, who was in his mid-30s at that time. I visited his laboratory out of curiosity, but I was moved by his passion. By the end of the visit, I had already decided to apply to join his laboratory. In hindsight, that was the turning point of my life. I barely understood what DNA was when I joined the laboratory, but Professor Kurumizaka patiently guided me through the study.

             
Shedding light on the structural basis of testicular chromosomes

My first project was researching a testis-specific histone called H3T. We receive 23 chromosomes from each parent, totaling up to 46 chromosomes. When a child is born from a mother with 46 chromosomes and a father with 46 chromosomes, their sperm and eggs (i.e., germ cells) are formed to reduce the number of chromosomes to 23 through a division known as meiosis and avoid the child having 92 chromosomes. Due to this process, the next generation can maintain 46 chromosomes. Among numerous studies conducted in an attempt to clarify this mechanism specific to germ cells, we focused on H3T as a histone found only in testicles. Our analysis comparing normal nucleosomes with testicular nucleosomes with the H3T we produced in test tubes discovered that the latter chromosomes are incredibly brittle and vulnerable to immediate destruction.

The results from these experiments could explain actually observed phenomenon of spermatogenesis, during which almost 90 percent of nucleosomes are replaced by another protein called protamine. We hypothesized that the fragile property of nucleosomes with H3T plays a certain role. Our finding from this research was published in 2010 in Proceedings of the National Academy of Sciences (PNAS) in the United States. The report on the histone specific to spermatogenesis process by our researchers prompted other groups around the world to start paying closer attention to histones in spermatogenesis.

Identifying the determinant of the regional centromere

Figure 2: CENP-A nucleosome and canonical H3 nucleosome

Later as a postdoctoral researcher, I began to focus on the regional centromere formed on genomic DNA. Identical genetic information contained in DNA is inherited during the cell division process when the DNA is accurately copied and distributed evenly into divided cells. The centromere plays a vital role in ensuring such equal distribution. As the research on the determinant of the regional centromere took place worldwide, CENP-A, a type of histone H3, was identified as an important determinant. However, nobody knew how an extremely small protein likes CENP-A could fulfill such an important function.

We made an attempt to produce CENP-A nucleosomes and determine their structure, which is quite a challenging task. Their crystallization for X-ray crystal structural analysis is even harder. We single-mindedly kept producing numerous crystallized samples and brought every last one of them to SPring-8 (a large synchrotron radiation facility) for X-ray radiation. Luckily, our efforts were rewarded by the discovery of the structure.

It turned out that CENP-A nucleosomes have unique structure not seen in canonical nucleosomes. For the first time in the world, we revealed that this unique structure of CENP-A (Figure 2) holds the key to the formation of the centromere, as we reported in Nature in 2011.

         
Enjoying research in Paris

After spending approximately 10 years of research at the Kurumizaka laboratory, I began thinking about studying abroad. Yet, choosing the right laboratory was a difficult task for me. As I confessed to the professor that I no longer knew what I wanted to do, he told me, “What more do you expect than enjoying your research? To do so, the first thing is to enjoy your life.” His statement made complete sense.

Photo: Dr. Tachiwana (front row, third person to the left from the center) and Dr. Almouzni (front row in the center) at the Jardin du Luxembourg near Institut Curie with laboratory members

I had spent my entire life in Tokyo, so I couldn’t imagine myself on a grocery run to a large shopping mall by car. I narrowed my choices down to four locations: London, Paris, New York, and California. I did not base these choices on laboratories but rather on cities where everything was at my disposal. Ultimately, thanks to an introduction by Professor Kurumizaka, I was recruited by the laboratory led by Doctor Geneviève Almouzni at the Institut Curie in Paris (who would later become the director of the institute). During my stay in Paris of two years and three months, I published two research papers. On a private note, I met my future wife there—a very fulfilling moment of my life indeed.

The Curie Institute mainly focuses on cell biology, so I investigated the formation of the centromere from a cellular biological perspective. The centromere is a crucial region for cells which explains its constant presence. For this reason, the regional centromere is hardly shaped from scratch, but it does happen in rare cases. In order to capture this irregular phenomenon through experiments, I hypothesized that the centromere is created entirely when CENP-A is intentionally dislocated.

To replicate this intentional dislocation is quite difficult. I applied a method employing an E.coli protein as reported in some papers at the time to make LacI, a protein binding specifically to a DNA sequence. By expressing a fusion protein between CENP-A and LacI as a cultivated human cell and arranging LacO on the desired destination of the genomic DNA, CENP-A followed the LacI to the desired destination as well, duplicating a mechanism for forming a new regional centromere (Figure 3). The outcome from further analysis with this experiment system was reported in Cell Reports in January 2015.

Figure 3: Method for reproducing the formation mechanism of the regional centromere

Back in Japan, I received the 2015 Waseda University Research Award (High-Impact Publication) as my research to date was favorably evaluated. I could not have released my research results as often without the strong support of Professor Kurumizaka, the laboratory staff, and other fellow researchers. The number of members of Kurumizaka laboratory has currently grown to 40, and I am one of those researchers who have been encouraged by our powerful boss Professor Kurumizaka and his exemplary leadership in the laboratory to write an astounding number of papers. More than 10 members of the laboratory have already obtained doctorates, and the number is soon to reach 20. As a member since the founding of this laboratory, I would also like to be a good example and perhaps an inspiration to others. I aspire to lead my own laboratory in the near future, and I feel excited already just imagining myself advancing the field of science in collaboration with the junior members in their own laboratories.

Hiroaki Tachiwana
Junior Researcher (Assistant Professor), Faculty of Science and Engineering, Waseda University

Professor Tachiwana graduated in 2004 from the Department of Electrical, Electronics and Computer Engineering in the School of Advanced Science and Engineering, Waseda University. In 2009, he completed the doctorate program at the Graduate School of Advanced Science and Engineering in the Department of Electrical Engineering and Bioscience. He has held positions as a special research fellow of JSPS (DC2), Research Associate at Waseda Research Institute for Science and Engineering, Junior Researcher at the Graduate School of Advanced Science and Engineering, Faculty of Science and Engineering, Waseda University, and a postdoctoral fellow at the Curie Institute (special overseas research fellow of JSPS) prior to the appointment of Junior Researcher at the Graduate School of Advanced Science and Engineering, Faculty of Science and Engineering, Waseda University in September 2014. He won the JBS Award for Excellent Presentation in 2009, the Koichi Suzuki Memorial Award in 2011, the 28th Inoue Research Award in 2012, and the Waseda University Research Award for High-Impact Publication in 2015.