At the start of its embryo-building career, the fertilized egg has packed within its nucleus DNA from mom and DNA from dad. How all this DNA is organized—and how it is reorganized in the earliest stages of life—has remained hidden from scientists, beyond the reach of the technology commonly used to assess genomic organization. This technology, called Hi-C, typically requires hundreds of thousands or even millions of cells, which simply aren’t available in embryonic studies.
A new form of Hi-C technology, developed by a team of scientists based at Lomonosov Moscow State University, can analyze cells at the single-cell level. With this new, improved Hi-C, the Lomonsov-led team established that global chromatin organization of zygote nuclei is fundamentally different from that of other interphase cells; that is, cells that are preparing to embark on cell division. The scientists also found that DNA of maternal origin and that of paternal origin are packaged differently within single-cell zygotes.
Detailed findings appeared March 29 in Nature, in an article entitled, “Single-nucleus Hi-C reveals unique chromatin reorganization at oocyte-to-zygote transition.” By improving our understanding of the zygotic chromatin “ground state,” the Lomonosov-led team, which included scientists from Austria and the U.S., could lead to more advanced stem cell technology. For example, stem cell scientists could discover how to reprogram cells to a state of totipotency.
“To study 3D chromatin organization in rare cell types, we developed a single-nucleus Hi-C (high-resolution chromosome conformation capture) protocol that provides greater than tenfold more contacts per cell than the previous method,” wrote the article’s authors. “Here, we show that chromatin architecture is uniquely reorganized during the oocyte-to-zygote transition in mice and is distinct in paternal and maternal nuclei within single-cell zygotes.”
The main novelty of the new 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.
“Whole-genome amplification makes possible direct operations with genomes of individual cells,” explained Sergey V. Ulianov, a coauthor the Nature paper. “We can sequence them and perform any other manipulations, including studies of the 3D 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 field of molecular biology.”
DNA molecules in nuclei are packed into chromosomes, which could be understood as complex but not randomly tangled tangles of genomic DNA associated with proteins and RNA. 3D chromatin organization is an important regulatory instrument, used by a cell to control gene expression. In the current study, the high-powered Hi-C technology revealed the 3D structures of chromatin at the earliest stages of embryogenesis—right after fertilization.
“Features of genomic organization including compartments, topologically associating domains (TADs), and loops are present in individual oocytes when averaged over the genome, but the presence of each feature at a locus varies between cells,” the authors of the Nature article elaborated. “At the sub-megabase level, we observed stochastic clusters of contacts that can occur across TAD boundaries but average into TADs. Notably, we found that TADs and loops, but not compartments, are present in zygotic maternal chromatin, suggesting that these are generated by different mechanisms.”
“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,” noted Ilya Flyamer, the first author of the Nature article. “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.”
“[The] Zygote is a totipotent cell, which gives rise to any type of cells in an organism,” Dr. Flyamer continued. “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 is possible by creation of induced pluripotent stem cells.”
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.