For the first time, scientists from the Faculty of Biology of the Lomonosov Moscow State University in collaboration with their colleagues from Austria and the USA have built up detailed maps of three-dimensional genome organization in individual cells and studied the characteristics of three-dimensional organization of maternal and paternal genomes in mouse zygotes. The obtained results have been published in the Nature journal.
The international research team including the biologists from the Lomonosov Moscow State University, have proved an earlier proposed model, arguing that chromatin fibril packaging could significantly vary in individual cells. Achievement of these results became possible due to the development of a new experimental approach for studying the three-dimensional genome organization in nuclei of individual cells.
DNA molecules in the cell nuclei are packed into special structures, chromosomes, which could be understood as complex but not randomly tangled tangles of genomic DNA associated with proteins and RNA. The biologists have developed a new technique, allowed to study how this DNA-protein complex called chromatin is packed a nucleus of a living cell. This technique is an advanced version of the classical approach for studies of three-dimensional genome organization, called Hi-C (high-throughput chromosome conformation capture).
Sergey Ulianov, a member of the research team, explained: "Let's take three arbitrary DNA fragments: A, B, C. The first two, being neighbors, are located one by one in the genome, and the third one - let's say - is located over a distance of several million base pairs from the previous two. However, a chromosome could be packed in such a way that fragment C will be close in space to A or B. We can identify this fact (not for these three arbitrary DNA fragments but simultaneously for the whole genome) and use this information for building up maps of three-dimensional chromatin organization in a living cell. This is how the Hi-C method works."
The standard Hi-C method requires several hundreds of thousands and even millions of cells in order to performone experiment. However, the new technique allows researches to work with single cell and to drawits individual map of three-dimensional chromosome organization. The main novelty of this technique is the selection of single nuclei at the final stage of the Hi-C experiment with subsequent whole-genome amplification. This process provides a possibility to get dozens of thousands of DNA copies from a single nucleus by using a special enzyme.
Sergey Ulianov says: "This is the key stepin our experiment. Whole-genome amplification makes possible direct operations with genomes of individual cells. We can sequence them and perform any other manipulations, including studies of the three-dimensional chromatin organization in a single cell. So-called single-cell technologies, namely studies of single cells and manipulations with them, now belong to a rapidly developing fieldof molecular biology."
This new method allowed scientists to study separately maternal and paternal nuclei from mouse zygotes and examine how the three-dimensional organization of maternal genome differs from the same one of paternal genome.
Ilya Flyamer, the first author of the article, comments: "We conducted the analysis of three-dimensional genome organization in mouse zygotes. It turned out that both maternal and paternal nuclei co-existing in one cell, in a zygote, considerably differed in the way of the genome packaging. In the paternal pronucleus, formed from a sperm nucleus, active parts of the genome are separated in space from inactive ones. Surprisingly, we don`t see that in the maternal pronucleus. So, the maternal pronucleus is the first mammalian nuclei type where active and repressed chromatin compartments are not spatially segregated from each other."
Thus, the new method allows one to study the earliest stages of embryogenesis - right after fertilization.
Ilya Flyamer adds: "Zygote is a totipotent cell, which gives rise to any type of cells in an organism. That's why the obtained results could probably help to understand the nature of totipotency and provide an opportunity to approach a more complete reprogramming of somatic cells, than it's possible by creation of induced pluripotent stem cells."
Three-dimensional chromatin organization is an important regulatory instrument, used by a cell to control gene expression. There are more and more reports in scientific literature that disclose a connection between the abnormal DNA packaging in the cell nucleus and development of various human diseases, including some cancers. In the future, the single-cell Hi-C technology will allow scientists to study certain subpopulations of tumor cells, including rare ones. Moreover, it will probably move us closer to understanding mechanisms of malignant tumors emergence.
The project has been done in cooperation with the Institute of Gene Biology of the Russian Academy of Sciences and colleagues from Austria and the USA.