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New method for analysing epigenetic modifications - in the service of medical progress

It is not just genetic factors that influence developmental processes and diseases; it is becoming increasingly evident that epigenetic changes play a major role too. Thanks to a new method developed at the University of Stuttgart, epigenetic processes can now be investigated in living cells.

Over the past decades, much has come to light about the role of genes and gene mutations in the development and progression of diseases. However, the DNA sequence of genes is not the whole story. For many years, researchers have been exploring an aspect that has more impact on gene activity than was originally thought, i.e. the fact that DNA bases can undergo chemical modification without leading to changes in the DNA sequence and thus the blueprint of proteins. Methylation, i.e. the addition of a methyl group to the cytosine base in a DNA strand, is the most common type of epigenetic modification.

Prof. Dr. Albert Jeltsch has been the director of the Department of Biochemistry at the University of Stuttgart since 2011. © A. Jeltsch, IBTB, Uni Stuttgart

Such superordinate, epigenetic changes play a substantial role in determining whether and to what extent genes are activated or repressed. Some DNA sections are literally peppered with methyl groups, which lead to characteristic patterns. The methylation process is dynamic; many factors including diseases can influence the methylation patterns. And to complicate matters, histones, the proteins that package DNA in chromosomes, can also be methylated. The methylation of histones is a regulator of gene expression, marking genes in DNA to be or not be transcribed. 

Investigating epigenetic changes and influences is therefore an important research activity, especially with regard to diseases such as cancer that have a genetic as well as an environmental component. The expectation is that this research will identify new therapeutic approaches that can be used to specifically interfere with epigenetic modifications. Up until now, however, it has not been possible to detect epigenetic signals live – i.e. in the living cell, for the simple reason that suitable methods were not available.

Prof. Dr. Albert Jeltsch, who heads up the Department of Biochemistry at the Institute of Biochemistry and Technical Biochemistry at the University of Stuttgart, and his team have delivered a real milestone in the study of epigenetic modifications. Using a sophisticated fluorescence labelling strategy, the researchers can track epigenetic processes over a period of several days or weeks.

Live: watching the flow of epigenetic processes

The BiAD sensor detects methylated DNA regions (yellow) in the nucleus of a human cell. The sensor components are shown schematically in the top left-hand corner. The anchor protein (zinc finger protein) is marked blue, the detector protein (MBD2 protein) red; both proteins are coupled to fluorophore components that can assemble into an active fluorophore (green). © A. Jeltsch, IBTB, Uni Stuttgart

"We can use this new method to investigate dynamic epigenetic processes in living cells, either as the cell develops or as pathological changes manifest themselves. This has previously not been possible. “Standard methods for analysing DNA methylation and chromatin modifications are discontinuous methods which provide us with a heterogeneous mixture of cells rather than the profile of an individual cell. This heterogeneous mixture of cells has different methylation states that cannot be correlated with cellular phenotypes,” explains Jeltsch. The labelling of cells, including living ones, with fluorescent proteins is nothing new. In fact, fluorescent labelling of cells is part of researchers’ standard laboratory repertoire. However, fluorescence-labelled proteins could not previously be used for analysing epigenetic processes at individual gene loci.

"CRISPR/Cas has led to a major breakthrough in making site-specific genome changes. We can use CRISPR/Cas to bring our BiAD sensor to new gene loci and analyse methylation patterns,” said Jeltsch. BiAD stands for “bimolecular anchor detector” and is a rather sophisticated two-component sensor system. The gene that encodes an anchor protein with two important characteristics is introduced into the cell: it consists of a non-fluorescent fluorophore fragment and is able to bind to a specific gene locus. On its own, this fluorophore fragment cannot emit a fluorescent signal; this is only possible when it joins a complementary fluorophore fragment.

The second, complementary fluorophore fragment is bound to a detector protein that recognises and binds to typical methylated sites on the DNA sequence. Only if the detector protein and anchor protein come to be located in immediate vicinity to one another, i.e. when the gene to be examined is methylated, can the fragments fuse to form the whole fluorescent protein. The fluorescence signal can then be detected under a fluorescence microscope. "The sensor system is specific, robust and modular, meaning that it can be combined with different detector domains, and it basically works with all cell types," says Jeltsch, summing up the key benefits of the BiAD sensor system.

Schematic showing BiAD sensors prior to binding to their target structures. The schematic shows the anchor domain (orange), which can bind to specific DNA sequences, and a detector domain (purple), which can bind to a chromatin modification. BiAD sensors consist of an anchor domain (orange), which recognises a specific DNA locus, and a detector domain (purple), which recognises a target epigenetic modification. Both proteins are fused to the non-fluorescent parts (grey) of a fluorophore. The right-hand side of the schematic shows the bound sensor components, and a complete fluorophore (green) called "Venus" after fusion of the two components. The complete, functional fluorophore emits a signal that is symbolised by a green corona around the molecule. 

When the targeted DNA sequence is methylated, the two domains bind in close spatial proximity, leading to the formation of an active fluorophore (green).
BiAD sensors consist of an anchor domain (orange), which recognises a specific DNA locus, and a detector domain (purple), which recognises a target epigenetic modification. Both proteins are fused to the non-fluorescent parts (grey) of a fluorescent protein. When the targeted DNA sequence is methylated, the two domains bind in close spatial proximity, leading to the formation of an active fluorophore (green). © A. Jeltsch, IBTB, Uni Stuttgart

Epigenetic modifications could be the key to new therapies

Research involving BiAD is still in its infancy. However, some highly interesting applications are already emerging. In diseases such as cancer, repetitive DNA elements frequently become hypomethylated, while tumour suppressor genes often gain methylation (hypermethylation), thus becoming ineffective. Different fluorescent proteins could be used for shedding light on these epigenetic abnormalities. Epigenetic modifications that lead to gene repression could be visualised with green fluorescence, and gene activation could be visualised using red fluorescence. However, the BiAD system can be applied to more than just DNA methylation. It can also be adapted to other epigenetic changes, for example, the acetylation of chromatin structures. Once we start looking at the different possibilities, many moves are possible.

However, despite all the euphoria, the effects of the method on living cells still need to be explored in greater detail. Jeltsch comments: "The basic requirement to ensure a quality analysis is that the investigation does not affect the cell too much. For example, we still have to study the effect of the method on chromatin structure in greater detail.” At the same time, Jeltsch and his team are working on using fluorophores with even greater luminous power. At present, the detection limit is 15 to 25 fluorophores per gene segment. This is why the team focuses mainly on repetitive DNA elements. So there is quite a lot of research needed before it becomes possible to detect individual fluorophore signals. "In cooperation with a team of researchers headed up by Prof. Dr. Monilola Olayioye from the Institute of Cell Biology here in Stuttgart, we now want to advance the method and make it suitable for single-spot detection," says Jeltsch.

In the long term, the researchers’ vision is to be able to use BiAD for deciphering the development- or disease-related reprogramming of epigenetic patterns in living organisms. Clinically, the new analysis method could be used in tumour models, for example, in order to clarify how and when abnormal epigenetic patterns develop in tumor tissues. And to take it even further, such findings would then provide starting points for new therapies that could specifically prevent or promote methylations and other epigenetic chromatin changes.

Website address: https://www.gesundheitsindustrie-bw.de/en/article/news/new-method-for-analysing-epigenetic-modifications-in-the-service-of-medical-progress