Chromosomes of an egg in 3D

Russian biologists "looked" into the nucleus of a fertilized egg

Daria Spasskaya, N+1

The laboratory staff of Sergey Razin from the Institute of Gene Biology of the Russian Academy of Sciences, as part of an international group of scientists, modified the method for determining the three-dimensional organization of chromosomes and applied it to study the spatial organization of DNA in mature eggs before and after fertilization. Article by Ilya M. Flyamer et al. Single-nucleus Hi-C reveals unique chromatin reorganization at oocyte-to-zygote transition published in the journal Nature.

The high level of complexity of multicellular organisms is determined by a wide variety of cell types that perform a variety of functions. This diversity appears during cell differentiation, which occurs from the earliest stages of embryonic development. The starting point for development and differentiation is the zygote, a cell that is formed after fertilization of an egg by a sperm. Despite the fact that the germ cells are strictly differentiated, the zygote formed as a result of their fusion has the property of totipotency, i.e. it is able to give rise to any type of cells in the body. In medical biotechnology, the possibility of reversing the processes of cellular differentiation and restoring the property of totipotence to cells is currently being actively investigated, however, despite some successes, the available knowledge is still insufficient for this.

It is known that in the process of "rebooting" cells from the differentiated state to the basic level, there is a change in the epigenetic status of DNA. Epigenetic changes include not only chemical modifications of DNA and related proteins, such as methylation, but also changes in the spatial organization of DNA inside the nucleus. The three-dimensional structure of the genome largely determines the work of individual genes, because in eukaryotes, the gene and its regulatory elements are often very far apart. More recently, this three-dimensional structure has been studied at the level of individual cells, but this was impossible for germ cells due to the extremely small amount of DNA available for analysis (recall that germ cells have a single set of chromosomes, i.e. twice as much DNA as somatic cells). In their work, Russian scientists proposed a modification of the currently main method for determining the three–dimensional structure of the genome - Hi-C, which made it possible to work not only with the DNA of individual oocytes, but also with the DNA of individual nuclei inside the zygote immediately after fertilization.

Drawing of the Institute of Molecular Biotechnology of the Austrian Academy of Sciences

Despite the fact that some of the most striking features of chromatin organization (DNA in combination with accompanying proteins and RNA) can be distinguished in a light or electron microscope, the active study of the three-dimensional organization of the genome began relatively recently with the invention in 2002 of the 3C method (Chromatin Conformation Capture, i.e. fixation of the chromatin structure). Its essence lies in the fact that the cells are treated with formaldehyde, which fixes DNA complexes with protein, then the DNA in the complexes is isolated, cut into small fragments with the help of enzymes and stitched again. As a result, the researcher has a library of hybrid DNA fragments in his hands, which include sequences that were located next to each other in the nucleus. Then everything depends on the methods of analysis of this library – the more productive methods are used, the more information can be obtained.

Initially, the 3C method assumed that it was possible to check the interaction of two specific sites in the genome ("one with one"). However, its inventors quickly began to increase the number of letters With, adding new stages that simplify the analysis of the library, which led to the emergence of technologies 4C ("circularized 3C", the study of interactions "one with all"), 5C ("3C carbon copy", "many with many") and finally Hi-C (from "high-throughput With", with which you can explore "all with all" interactions). Hi-C technology makes it possible to analyze the library completely thanks to the advent of high-performance DNA sequencing, although the basic principle remains the same as in 3C. Using Hi-C, it was possible to construct three-dimensional maps of human chromosomes, and to determine that the "active" chromatin, the genes in which are actively expressed, and the "inactive" form two separate compartments in the nucleus (A-B compartments). Another breakthrough in the field of studying the three-dimensional organization of the genome occurred in 2013, when with the advent of single-cell sequencing technology, it became possible to study the 3D structure of chromosomes in a single cell. However, this was still not enough for the study of oocytes.

The scheme of the Hi-C experiment – from the isolation of individual cores
before sequencing (from an article in Nature)

The standard Hi-C technique involves several stages of library enrichment using modified nucleotides, which are included in the composition of the obtained fragments. In the case when there is a lot of source material, these steps help to get rid of DNA, which for some reason has not been processed in the previous stages. However, if the initial amount of DNA disappearing is small, they only lead to the loss of material during the experiment. The authors modified the Hi-C protocol, discarding the stages leading to losses, and increased by an order of magnitude the sensitivity of the method, which they called single-nucleus Hi-C (Hi-C for a single core). In order to assess the changes occurring in the chromatin structure of oocytes, scientists compared the nuclei of immature and mature oocytes (eggs). As expected, with the maturation of the oocyte, chromatin became more compact and inactive.

The key experiment of the work was designed to provide answers to the questions of what happens to chromatin when the cell is "reset" after fertilization, whether the state of chromatin is inherited or formed anew, and also whether the state of chromatin in the maternal and paternal nuclei is different in the composition of the zygote formed after fertilization of the egg by the sperm (at the stage of the zygote, the nuclei coexist in the cell and do not merge).

It was found that the structures of the primary levels of organization – chromatin loops and so-called topologically associated domains - can be distinguished in the composition of both the paternal and maternal nuclei. Higher–order structures - A-B compartments of active and inactive chromatin in the paternal nucleus were poorly detected, however, to the surprise of researchers, they were not noticeable at all in the maternal nucleus. Thus, the authors of the work found that the zygote contains a nucleus that is in the interphase stage, i.e. non-dividing, but at the same time without signs of chromatin compartmentalization, which is a unique case among all mammalian tissues and cells. Based on these data, it is assumed that the formation of compartments in the nuclei occurs in different ways - in the maternal they are formed anew, and in the paternal they are inherited or formed earlier. In addition, based on the obtained structures and computer simulations, it can be assumed that the organization of chromatin in the maternal nucleus is closest to the organization of the metaphase chromosome, i.e. the maternal nucleus of the zygote is closer to the somatic cell than the differentiated oocyte or sperm. Finally, these data indicate that different levels of chromatin organization (namely loops and A-B compartments) are formed by different mechanisms.

I would like to note that this level of work has become feasible thanks to the explosive development of DNA and RNA analysis technologies literally over the past few years. Now the authors of the work want to supplement their work with the analysis of gene expression and DNA methylation at the level of a single cell, and we can only wonder and wait for what other opportunities progress will throw up to scientists today or tomorrow.

Portal "Eternal youth" http://vechnayamolodost.ru  30.03.2017