When the stadium is DNA, swapping one spectator may affect the results

The human genome consists of more than three billion pairs of specific nitrogen bases called nucleotides. Surprisingly, within this pool, genes -- the DNA fragments encoding proteins -- constitute less than 2%. Geneticists have long been intrigued with the following riddle: since such a large part of the genetic code is disabled, why replacing just a single nucleotide within it can sometimes lead to the development of this or some other disease? The answer has been found by the team of Prof. Yijun Ruan from The Jackson Laboratory for Genomic Medicine (JLGM) in Farmington, USA. A significant contribution to this study was made by a group of scientists from the Nencki Institute of Experimental Biology of the Polish Academy of Sciences, in Warsaw. The results of the study have been recently published in the scientific journal Cell.

The DNA in cells is wrapped around proteins known as histones. Such an arrangement produces chromatin fibers that are responsible for the packaging of the genetic material into chromosomes, with their characteristic X-shaped structure.

"Based on data obtained with the use of cutting-edge methods of DNA sequencing, our American colleagues came to some very interesting conclusions on the three-dimensional structure of chromatin in the nucleus of human cells. Our task was to verify these results in another way: by using sophisticated morphological techniques, i.e. fluorescence microscopy combined with advanced computational imaging," says Dr. Grzegorz M. Wilczyński, a Professor of the Nencki Institute.

The Warsaw group, inspired by Prof. Wilczyński, involved Dr. Adriana Magalska, Paweł Trzaskoma, Prof. Jakub Włodarczyk and Dr. Błażej Ruszczycki.

Microscopic confirmation of the results of the US team required the Polish scientists to record images of superior resolution. An obstacle, however, was the same physics: the diffraction making it impossible to focus the light to a point, precluding to see details smaller than half the wavelength of the light projected on the sample. In the case of classical optical microscopy, the best resolution is therefore approx. 200 nanometers. Thus, the Nencki Institute's scientists resorted to one of the varieties of superresolution microscopy the invention of which was awarded the 2014 Nobel Prize. The essence of the method used by the researchers from the Nencki consisted of applying a light source through a special diffraction grating. Digital processing of a series of images taken at different positions on the grid allows doubling the resolution of microscopic images.

The second way of imaging, used by the group from the Nencki Institute, was a microscopy based on the phenomenon of resonance energy transfer (FRET, Förster Resonance Energy Transfer), imaging of fluorescence decay time (FLIM, Fluorescence-Lifetime Imaging Microscopy). The technique of FRET tested energy transfer between the molecules occurring without emission of photons. A characteristic feature of such transfers is their very short range: they decrease until the sixth power of the distance between the molecules, allowing them to be used to support the observations made using superresolution microscopy.

The stacks of microscopic images were then refined, analyzed and converted into the three-dimensional visualization, using the proprietary software written by Dr. Błażej Ruszczycki (a co-author of the study).

Polish measurements and analysis made it possible to confirm the existence of an interesting relationship between one of the chromatin proteins, CTCF, and the enzyme RNA polymerase II. According to previous views, CTCF protein was found in parts of inactive chromatin, where they serve, among others, the similar function to a fastener: they can bind to one another strongly and stably. Following the binding of CTCF protein, the chromatin fibers form stable loops. On the other hand the RNA polymerase II enzyme is responsible for the transcription of the gene, i.e. the rewriting of a DNA strand to a new RNA strand. Later, in the process of translation, these RNA threads are used to make protein molecules encoded by the gene.

Microscopic examination performed in the Nencki Institute has demonstrated the validity of the measurements made by the American side: at sites where the 'velcro' protein CTCF makes loops of chromatin, one can also find the RNA polymerase II. What is more, the enzyme not only occurs in the vicinity of a 'plywood' of chromatin fibers, but it also works there, i.e. performing the active transcription of genes; this finding changes the established views on the architecture of chromatin. This is a critical observation, reaching beyond the previously considered paradigm of the three-dimensional structure of the genome.

"Our observation also explains why the replacement of a single nucleotide for another in the inactive fragment of DNA can cause a person to become susceptible to a disease," says Prof. Wilczyński and explains: "If a change occurs, for example, within the CTCF protein binding site, it may not be able to serve as a velcro. As a result, the proper chromatin loop cannot form, leading to the changes in the geometric conditions in which RNA polymerase II transcribes genes."

The geometry of the environment affects the expression of genes, as it may facilitate or impede the access of the other molecules to a gene. This situation resembles the experience of people trying to read a book in a crowded bus during the trip. The crowd in the middle of the vehicle, where everyone pushes against one another or tries to go somewhere, is not the place of a pleasant reading. The comfort increases significantly when the passerger can lean against a wall. Thus the geometry decides on the efficiency of reading. Similar principles operate in the interiors of cells and decide about activating or deactivating one or another gene.