New structures identified within our chromosomes

Since the beginning of this century, great technological advances at the molecular and genomic level, coupled with new approaches in very high resolution photonic microscopy, have revealed new chromosomal structures and new principles of organization of our genomes (the genome contains all the genetic information and therefore all the DNA sequences transmitted through cell divisions).

Frédéric Bantignies, University of Montpellier and Giacomo Cavalli, University of Montpellier

Left: chromosome DNA in a cell nucleus, seen through a fluorescent DNA intercalator and observation with super-resolution microscopy. Right: examples of TADs with several substructures, CNDs, seen thanks to specific fluorescent markers and observation in super-resolution microscopy. Quentin Szabo, Provided by the author

This knowledge is crucial for understanding how our cells function, but also for better understanding certain diseases and their evolution.

Deoxyribonucleic acid, or DNA, contains the genetic information specific to each living species. Chromosomes are the physical carriers of this information. The set of chromosomes is also called the genome and contains all the genes. Since the discovery in 1953 of the double helix structure of DNA, the question of its organization within the nuclei of our cells has been the subject of much research worldwide. In order to understand this question, we can specify here that man has 46 chromosomes, which contain 3 billion base pairs (bp) of DNA, which represents a molecular filament of about 2 m. This filament is capable of being organized into two or three parts. Now, this filament is capable of being contained in cell nuclei about 10 μm (one micrometer = one thousandth of a millimeter) in diameter. To better understand how the genome and our genes work, it was therefore essential to know how all this DNA is able to fold into a cell nucleus.

At the beginning of this organization, we have the nucleosome, whose structure was elucidated in 1997. The nucleosome is formed by very basic proteins called histones, which have a great affinity for the acidic DNA molecule. These histones will form a central body around which the DNA molecule will wrap, at a rate of 146 bp for a nucleosome, a kind of "macaroon" of 11 nm (one nanometer = one millionth of a millimeter) wide and 6 nm high. The nucleosome thus represents a first structural level of DNA organization in nuclei. Sign of its importance, this very particular structure is found in all organisms with nuclei (eukaryotes), whether they are unicellular or more complex like animals and plants.

For a long time, it was thought that the succession of nucleosomes (also called chromatin fiber), forming a kind of "pearl necklace", wound up in turn in a regular way to form a fiber of 30 nm in diameter. Spiral supercoils of this fiber would eventually lead to chromosomes. However, technical limitations, including the low resolution of microscopy techniques and the lack of other methods, made it impossible to demonstrate whether this hypothesis was correct.

On the other hand, it has been known since 1985 that the organization of chromosomes in cell nuclei is not random. Moreover, the well-known X-shape of chromosomes is not false, but represents only a very transitory stage of their organization. Indeed, this very condensed X-shape is conducive to their segregation (sharing) in the daughter cells during cell division. But the rest of the time, the shape of the chromosomes is quite different. Their visualization thanks to fluorescent molecules capable of interlocking specifically in the DNA double helix has shown that each chromosome occupies its own territory within the nucleus, thus avoiding too much entanglement with the other chromosomes. This "chromosome territory" property is also found in most species and seems to be very important, especially for species with a large number of chromosomes.

New technologies bring a new look at the organization of the genome

At the beginning of the 21st century, numerous researches around the world have led to a better understanding of the different levels of structural organization of the genome between the nucleosome and the chromosomal territories.

These advances were made possible by the development and use of entirely new technologies. First, there were new genomic techniques, including very high throughput DNA sequencing (next generation sequencing) and the ability to molecularly capture fine chromosome structures using the "Hi-C" method. In parallel, the arrival of super-resolution photonic microscopy, which uses fluorescent DNA markers, has made it possible to visualize these chromosomal structures directly in the nucleus of cells.

Representation similar to a necklace with beads spaced apart, some portions of the thread/beads intertwined with each other
Nucleosomes are organized in "clutches".
Frédéric Bantignies, Giacomo Cavalli, Provided by the author

So let's go back to our chromosome organization ladder. Its first level is the nucleosome. A second level of organization corresponds to clusters of a few nucleosomes, like small clusters called "nucleosome clutches" (they were so named by the authors of their discovery in analogy to the eggs found in brood nests). The nucleosomes are therefore not grouped in a regular way as previously thought, but rather in irregular clusters.

These "nucleosome clutches" then cluster to form a structure called a Chromatin Nanodomain, or CND, which includes approximately 100,000 to 200,000 base pairs of DNA, forming large irregular clusters of nucleosomes 150 to 300 nm wide. These two levels were discovered recently (2015 and 2020, respectively), using super-resolution microscopy, which is capable of resolving 20-100 nm structures.

TADs structure the genome and regulate gene expression

The next level of this organization is called TADs, in English "Topologically Associating Domain", identified in 2012 with the Hi-C molecular method. TADs are composed of several CNDs, forming super clusters of nucleosomes about 500 nm wide. They thus comprise variable sizes of DNA, with an average of about 1 megabase (1 million base pairs). Our laboratory has contributed to the discovery of CNDs and TADs.

A large portion of the previous bead string is passed through a ring, forming a large loop
Nucleosome clutches are organized into CNDs, which in turn are organized into TADs. TADs are formed when the chromatin filament passes through a cohesin ring.
Frédéric Bantignies, Giacomo Cavalli, Provided by the author

TADs are quite heterogeneous structures, notably because of their dynamic formation mechanism. This mechanism involves the passage of the famous "string of pearls" (referring to the succession of nucleosomes) through the ring formed by cohesin. The chromatin fiber will continue to pass through the ring until it encounters nuclear factors called CTCFs at the borders of a TAD, a kind of "customs officer" that, when placed on the DNA, will block the progression of the fiber. As the fiber passes, the nucleosomes will organize themselves into clutches and CNDs. The TAD then represents the whole of this large chromatin loop that has passed through the cohesin ring.

Within chromosomes, gene activity is influenced by a whole host of regulatory sequences (switches) that can be tens of thousands of base pairs away from their gene. Thus, TADs will keep genes and their regulatory regions in the same molecular environment, which may be conducive to their expression (i.e. their reading to lead to the production of a protein) in a given cell type where their activity is required. Thanks to their boundaries, they will also allow to separate genes from each other, to avoid that active genes influence other inactive genes in a given cell type.

Recent studies have shown that chromosomal defects at the borders of TADs (such as inversion or deletion affecting the positioning of CTCF or rendering it inoperative) lead to defects in the isolation between genes, and therefore to erroneous activation of genes. In some cases, these rearrangements will cause the activation of genes called "proto-oncogenes", which can lead to the transformation of cells and the appearance of tumors.

Compartmentalization and territorialization of the genome

Representation resembling wool unrolled and spread on the ground; one half is red, the other blue
TADs are grouped into two compartments: mainly active regions in red, and mainly inactive regions in blue.
Frédéric Bantignies, Giacomo Cavalli, Provided by the author

Several TADs will then group into two distinct compartments, compartment "A" which contains mainly active genes and compartment "B", which includes mainly inactive genes. This grouping into compartments allows to reinforce the functions of the genome.

Sphere completely filled with what looks like bundles of wool, each bundle having its own color and not tangling with the other bundles
Each chromosome (1 chromosome = 1 color) occupies its own territory in the nucleus of cells.
Frédéric Bantignies, Giacomo Cavalli, Provided by the author

Finally, at the end of this scale of organization, we find the chromosome territories, which allow the chromosomes to be individualized from one another within the nuclei. This organization plays an important role in DNA replication (identical copies of genetic material) and cell division, where each duplicated chromosome is split into two in the daughter cells.

And the circle is complete! Well, almost: it is now a question of better understanding how this organization influences the various molecular processes inherent in the genome, which regulate, in particular, the expression of genes, the replication of the DNA molecule, its repair in the event of damage or stress, or its recombination, especially in reproductive cells. The field of investigation remains immense, but a better knowledge of the genome's organization will open the way to a better understanding of all these nuclear processes in normal cells, but of course also in cells with chromosomal defects that lead to diseases such as cancer.The Conversation

Frédéric Bantignies, Director of Research CNRS, University of Montpellier and Giacomo Cavalli, CNRS Research Director, University of Montpellier

This article is republished from The Conversation under a Creative Commons license. Read theoriginal article.