Dr. Edita Kriukienė: Goal of Research - to Help People Live Better
Hydroxymethylcytosine in DNA and proteins that interact with it
Research professor Dr. Edita Kriukienė and her team are developing methods that would help to better diagnose epigenetic disorders in human diseases, for example, when studying one of the deadliest children’s cancers: neuroblastoma. “There are a lot of achievements in the treatment of neuroblastoma, but children continue to die,” says the researcher at the Vilnius University Life Sciences Center.
You became interested in epigenetics quite early, in 2008. What prompted you to do this?
After defending my doctoral thesis, I worked in a completely different field, but Prof. Saulius Klimašauskas invited me to his leading Department of Biological DNA Modification. He offered to start a project on applying unique DNA labelling tools for epigenetic research.
At the time, I knew very little about epigenetics, but I soon realised that this interesting field covers all the kingdoms of life and the processes of every cell. I wanted to challenge myself in this new field, and I still don’t regret this decision. The more profound, the more interesting.
Keeping in mind the current knowledge of epigenetic regulation, epigenetics might be called the foundation of life, which continues to open up new layers and unexpected gene regulatory networks. It could be a lifetime’s work to explore this area.
How has your research changed since you started working in epigenetics? In addition, what news on epigenetics has most influenced your research?
As soon as I started my research in the epigenetics field, one of the most significant discoveries in this area took place. Nowadays, many have heard that DNA is made up of four bases - adenine, guanine, thymine, and cytosine and that DNA is the basis of genetic inheritance. Only one epigenetic DNA modification - methylcytosine - was known for many years. This cytosine modification has a defined function in mammals - it regulates when and which gene will be expressed or converted into an active protein in the cell.
However, in 2009, a new DNA modification, hydroxymethylcytosine, produced from methylcytosine, was discovered. In the following several years, two more new DNA modifications - formylcytosine and carboxycytosine - were detected, allowing the scientists to finalise the characterisation of the complete demethylation pathway of methylcytosine, which proceeds through several enzymatic steps.
A new technological challenge arose - it became clear that conventional methylcytosine assay methods could not detect these new modifications, and their presence remained hidden under the methylcytosine or unmodified cytosine signals.
Thus, after the discovery of the methylcytosine demethylation cycle, everything done in the field of epigenetics had to be re-evaluated.
It is now known that DNA methylation and demethylation occur every minute, in every cell, in every tissue. For example, extensive rearrangements of DNA modification occur during embryonic development. Even a slight disruption of the process can lead to disease. Therefore, studying each modification individually can facilitate a deeper understanding of the state of the cell and the mechanisms by which the cell regulates its functions. The study of several different states of modified cytosine required the development of new methods. In our work, we focus on the development of new methods that would enable the detection of all DNA modifications.
One interesting analogy used in popular science to describe epigenetics is the “gene closet.” Epigenetics determines whether our genes work harmoniously. Do you think this analogy is correct?
Yes, I agree with this analogy. All our tissues are different, and each has its own “closet” with its order.
However, random processes also occur in the cell, so the order in the “closet” is not ideal. The cell must be flexible because it needs to respond quickly to the changing environment. Flexible epigenetic variability in the regulation of gene expression can confer positive properties to the cell and facilitate survival. On the other hand, if the environmental impact becomes too strong, disorders appear.
For example, when a pregnant mother is under a lot of stress, the child may develop metabolic and heart disease or behavioural problems, all of which involve altered gene expression.
Epigenetics defines such processes: it investigates how the order of the cell’s “gene closet” is maintained or changed depending on the conditions.
Do environmental influences cause DNA mutations, or can they happen, for example, when a cell divides?
DNA mutations occur during cell division and due to unfavourable environmental exposures. However, this is just one way - at the genetic level – to influence gene expression.
The level of epigenetic mutations is much wider – it involves alterations in DNA modification and malfunctioning of various DNA-interacting proteins, histones, and non-coding regulatory RNAs.
And this is where highly complicated things begin. If the activity of the proteins that create necessary epigenetic labels in the genome is disturbed, abundant changes in the epigenome appear. Most importantly, the influence of some epi-mutations on the genome might be more challenging to detect than that of usually rarer genetic mutations. Therefore, the “communication” between the genome and the epigenome must be highly coordinated to avoid a risk of disease development.
What are the most significant epigenetic questions you want to answer?
Now, it has become increasingly popular to simultaneously analyse different epigenetic factors, for example, the formation of DNA or protein modifications and changes in DNA structure or the cumulative influence of these factors on gene expression. By identifying a single epigenetic factor, only limited information on regulatory processes can be obtained.
Recently, in our research projects, we started studying several factors simultaneously. In addition to the already mentioned DNA methylcytosine and hydroxymethylcytosine modifications, we analyse the structure of DNA maintained by various proteins and seek to determine how all these factors affect each other. This became possible due to our long-term experience in working with proteins isolated from bacteria, such as DNA methyltransferases, which we successfully engineered to introduce our desired chemical groups into the genome for the detection of the aforementioned epigenetic factors.
The process is somewhat like fishing - to catch a “fish”, for example, a modified cytosine in DNA, you need to cast a fishing rod with a “bait” - using DNA methyltransferases or glucosyltransferases to place labels in the genome, which are used as a “bait” to “pull out” the labelled regions of DNA.
Dr. Edita Kriukienė
Have you already tried this method? Maybe you want to share the results?
The development process of any new method usually involves its optimisation and testing phases and application in a biological context in order to evaluate its utility. We applied our method to study the development of mouse stem cells into neural cells and demonstrated the importance of hydroxymethylcytosine in this process.
The results of this study were recently published in the journal Cell Chemical Biology. We also described the importance and epigenetic roles of hydroxymethylcytosine in an invited review published in the Chemical Society Reviews journal earlier this year.
You were running a project on neuroblastoma research funded by the Research Council of Lithuania. Why did you choose this particular cancer?
Neuroblastoma is one of the deadliest children’s diseases. Currently, there are many advanced treatments for this disease, but children continue to die. Since neuroblastoma is a disease of the impaired development of the nervous system, it usually appears relatively early - in children who are only a few months old or newborns.
The development of the child’s nervous system takes place before and after birth. If these processes are disturbed, tumours can be formed and spread throughout the body. Neuroblastomas have few genetic mutations but display abundant epi-mutations that drive the disease. We want to better understand epigenomic changes in various neuroblastoma cells after their exposure to drugs. We are currently studying the effect of epigenetic drugs on the cells of both extremely malignant cancers and mild forms, in which the tumour can even disappear without severe treatment.
We believe our new cost-efficient methods might be applied for treatment monitoring and evaluation of tumour state in personalised therapy. I think identifying every patient’s epigenome and selecting the right treatment strategy is a promising future.
Since you mentioned personalised medicine, how can personalised therapy help treat cancer?
Clinical trials usually take a very long time, and a drug is approved if it shows a significant effect in most cases studied, often involving thousands of patients. Unfortunately, there are always cases when cancer resists treatment. Why? What global changes are taking place in that “stubborn” tumour? Such patient-oriented questions should be the focus of epigenetics. As a researcher, I want scientific discoveries to help people live better.
By the way, there are already cases in the world when clinics create tests that can be applied immediately during surgery. For example, during surgery, a tumour sample is taken and analysed quickly to determine the size of an incision to remove the cancerous cells. In the case of a brain tumour, the surgeon knows which part of the affected tissue should be removed to protect the person’s brain. I see such an example as a beautiful alignment of science and treatment practice.
How else have you applied your new methods?
We have applied our methods in a research project on prenatal diagnostics in cooperation with colleagues from the universities of Tartu and Helsinki.
Do you mean NIPT tests?
Yes, that’s right. Our studies have shown that testing for hydroxymethylcytosine can more sensitively detect fetal genetic disorders early in pregnancy. As you know, the mother’s blood carries the DNA of a child. The smaller the fetus, the lower amount of its DNA can be detected in the mother’s blood, which complicates prenatal testing.
The cost of available NIPT (non-invasive prenatal testing) tests is high, and many women cannot afford it. These tests usually detect fetal Down syndrome, which shows changes in the 21st chromosome, and several other syndromes. Our goal was to make this test highly effective and widely available. We are cooperating with a foreign company that wants to continue our research.
Does it mean that the method you developed will be used for fetal genetic testing of pregnant women in the future?
It may be, but at the same time, it raises ethical questions. For example, is it necessary to expand the market to all women, especially if the possibility of fetal abnormalities is lower due to the young age of a woman? After all, there are also false positive cases because these tests show the probability and not the diagnosis.
The diagnosis can be confirmed by invasive tests, such as amniocentesis, which are known to increase the risk of miscarriage. And maybe many women don’t even want to know the diagnosis.
Your work was nominated for the National Science Prize (NSP). Tell us more about this work.
We presented this work to the NSP competition with my colleague Dr. Giedrius Vilkaitis. It combines my group’s research on the development and application of epigenetics tools and the methods created by my colleague’s group, which are devoted to analysing regulatory small RNAs and studies of DNA methyltransferase functioning in living cells. We presented a set of unique tools - mTAG-seq, TOP-seq, hmTOP-seq, caCLEAR, Mx-TOP, Dnmt-TOP-seq, and mDOT-seq, demonstrating their innovation and showing a successful combination of sophisticated multidisciplinary research to study various complex epigenetic diseases, such as cancer, cardiovascular diseases, lactose intolerance, etc.
What are your plans for the future? What new goals are you establishing for yourself?
Since my main research subject is epigenetic technologies, I continue to think about other technological possibilities that would improve the accuracy of epigenetic research and fill in existing niches.
In my opinion, the future of epigenetics in disease treatment is related to spatial or single-cell biology. It is essential to know that each cell in a cancerous tissue can respond differently to drugs. Epigenetic processes in individual cells are a huge separate area of research. When analysing tumours, it is crucial to know their cellular composition and the impact of tumour cell communication with each other and surrounding healthy cells. Spatial biology can identify not only the different types of cells in a tumour, for example, malignant cells or stem cells that cause cancer recurrence, or the presence of various immune cells that help fight against that tumour, but also their location in the tumour, and the connections between cells. Many different pieces of information have to be collected to assess a tumour’s state and envisage its response to treatment. This is especially relevant for personalised treatment. I would very much like to contribute to this in some way.
Interviewed by Goda Raibytė-Aleksa