11 November 2016

Epigenetics with super resolution

MSU scientists investigated ways of packing DNA in the cell nucleus

MSU Press Service

Scientists of the A.N. Belozersky Research Institute of Physico-Chemical Biology of Lomonosov Moscow State University have created a method for marking working genes based on the differences between active and inactive chromosome sites during replication from DNA. The article was published in the highly rated journal Current Biology (Deng et al., Cytology of DNA Replication Reveals Dynamic Plasticity of Large-Scale Chromatin Fibers).

Comparison of conventional fluorescence microscopy of replicated chromatin
and microscopy with super resolution.

MSU scientists together with their colleagues from the USA investigated the ways of packing DNA in the cell nucleus and their changes in the process of its replicative synthesis. It is believed that the nature of DNA packaging has a significant impact on the work of genes and is one of the mechanisms of epigenetic control of gene expression.

"Epigenetic control of gene expression is expressed in the fact that, although all cells of the body have the same genetic information, not all genes work in this particular type of cell. It is the set of working genes that determines the fate of the cell. There are several cellular mechanisms that help the cell to remember which genes should work in it and which should not. This is epigenetic control, which is the memory of a cell about who it is, and thanks to the work of which genes it is," says Igor Kireev, one of the authors of the article, head of the electron microscopy department of the Belozersky Research Institute of Physico–Chemical Biology of Moscow State University, Doctor of Biological Sciences. "We were interested in the so–called higher levels of the structural organization of chromosomes, formed as a result of a series of successive stages of DNA strand packing."

DNA in the cell exists in the form of a complex with proteins – chromatin. The initial stages of chromatin compactification are quite well studied – these are nucleosomes, protein globules about 10 nm in size, consisting of eight molecules of histone proteins, on which DNA is wound. Then the chain of nucleosomes fits in an incomprehensible way into thicker fibrils, chromonemes, as a result of which a very high degree of compactification is achieved. Thus, the length of the maximally compacted mitotic chromosome is 20,000 times less than the length of the DNA laid in it.

Replication is the process of synthesizing a daughter DNA molecule on the matrix of the parent molecule, and transcription is the process of synthesizing RNA using DNA as a matrix. The DNA in the chromosomes is extremely densely and complexly packed, and it was traditionally believed that for the implementation of matrix synthesis processes (transcription and replication) this packaging interferes and should be disrupted on the scale of fairly large chromatin domains. It was quite difficult to identify these domains and analyze their structural organization with high spatial resolution without disturbing their natural structure.

"We have proposed a method for labeling working genes based on the differences between active and inactive chromosomal regions during replication from DNA. Thus, the active site, the so–called euchromatin, is replicated at the very beginning of the synthetic period of the cell cycle, and the "silent" – heterochromatin – in its second half, - comments Igor Kireev. – Our method made it possible to carry out non-destructive (non-destructive) chromatin studies. It is based on a combination of labeling of the newly synthesized (daughter) DNA by click-chemistry methods followed by detection of reaction products by correlation fluorescence microscopy with super-resolution and immunoelectronic microscopy. In other words, we can see the same molecule in a given cell both in an optical and an electron microscope."

Using this approach, the scientists made two unexpected observations. Firstly, "working" chromatin, contrary to traditional ideas, retains a very high degree of packaging, since it is represented by highly structured chromatin fibrils of the highest order – chromonems. Secondly, it turned out that the DNA in the composition of the chromosomes has a high structural plasticity, that is, it is able to "flow" from one part of the chromoneme to the neighboring one. At the same time, the overall dense structure of the chromoneme does not change. These observations do not fit into the framework of existing theories about the spatial organization of chromosomes, but at the same time allow us to express new hypotheses about the mechanisms of transmission of epigenetic information in the process of cell division.

"We made the assumption that in a new daughter cell, chromatin structures can move inside it, and not be fixed, interacting with the DNA that has not yet doubled and "remembers everything", and also contains the necessary molecular components to restore the lost epigenetic information," Igor Kireev continues.

Another conclusion, voiced in an article published in Current Biology, is that the structural organization of the genome is not a rigid hierarchy. Of course, there are some consistent levels of DNA organization. Previously, it was thought that there should be a clearly fixed system that unambiguously leads to the formation of chromosomes. Now it turns out that everything can be different: there are certain principles of construction, but within the given boundaries DNA has some freedom and plasticity.

"The further development of research consists, firstly, in the transition to the analysis of individual chromosomal loci, which we plan to mark (isolate) at the optical and electron microscopic levels using TALE technology, and secondly, in the development of even more native in vivo labeling methods compatible with such advanced technologies as cryoelectronic microscopy," Igor Kireev shares his plans for the future.

Scientists hope to come close to deciphering the principles of the spatial organization of DNA, using direct analysis methods using visualization of chromatin packaging methods with high resolution. In practical terms, the research will help to clarify the structural aspects of epigenetic control of gene expression and, possibly, suggest ways to regulate it, which will allow developing more effective approaches for therapeutic effects, for example, in the fight against cancer and aging – conditions in which the "epigenetic" component is very pronounced.

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