Identification and characterization of methyl-deoxyadenosine in the eukaryotic genome.

The aim of our proposal is to study a novel, currently unreported modification that can directly modify a part of the genetic material, and to determine how this affects an organism.

DNA encodes the genetic instructions used in the development and functioning of all living organisms. It is composed of 4 molecules. About 30 years ago, it was discovered that one of these DNA molecules could be directly modified on a molecular level. This modification, occurring on the molecule called dC, has been studied extensively ever since, revealing a fundamental role in different biological pathways and a better understanding of many human diseases such as cancer. Ultimately, research in this field has led to new drug therapies and improved human health.

Since the discovery that dC can be modified, no other DNA molecule has been known to undergo direct molecular alterations. However, just recently we have discovered the presence of a modification on another DNA molecule, namely affecting the DNA molecule dA. This novel finding has so far not been reported and is still unknown to the research community.

Hence, the aim of our proposal is to study this novel and direct DNA modification and determine how it affects an organism. Since this novel modification directly alters the DNA, the building blocks of 'life', it has the potential to generate a similar fundamental impact on biology and human health as the discovery of the modification affecting dC had and still continues to do. Hence, we would like to investigate this novel modification.

Every cell of an organism has the same DNA. However, molecular modifications ensure that even though the DNA is the same in every cell type, the cells still differ. For example, a liver cell is different and has other functions from a skin cell. We have recently established that the dA modification varies between different cell types. This suggests that this novel modification could be responsible for the differences observed between different cell types.

To get a better understanding of the novel dA modification, we will describe and compare the location of the dA modification in different cell types. By studying this modification during the early development of an organism, we will get a better understanding how this novel modification is established and regulated on a molecular level.

Due to ethical concerns, experiments on human development cannot be performed. However, to understand the underlying molecular events, researchers use animal model organisms that have similar and conserved molecular mechanisms. In our case, we use the frog, which is a commonly used model organism to study the development of an organism and the differences between different cell types.

Ultimately, we expect that our research will lead to the better understanding why different cell types are distinct despite having the same genetic instruction. In future, this information could be used for medical purposes and the generation of organs for tissue replacement therapies. Bearing in mind that any errors in the modification affecting dC can lead to various diseases such as cancer, it is likely that the misregulations of the novel modification dA could also cause health defects. Hence, it is vital to study this novel dA modification, as this has the potential to open up the knowledge of currently unknown causes of disease. This knowledge is essential to path the way for novel and better future drug therapies, ultimately benefiting human health.

More than 30 years ago, it was discovered that some cytosine deoxynucleotides are methylated in genomic DNA. This "fifth element" of DNA has been studied extensively ever since, revealing a major genetically heritable function in regulating gene transcription. Up till now, no modifications affecting other deoxynucleotides have been reported.

We have recently discovered that adenosine deoxynucleotides can also be methylated in eukaryotic genomes. Our preliminary analysis of this novel mark reveals that its location is distinct between different cell types, suggesting that this methylation mark may have cell-type related function(s) and is actively regulated. Further, this novel base seems to be encountered across the genome, but seems to be depleted from exons.

The aim of this proposal is to further explore the distribution and dynamics of methylated deoxyadenosines in the eukaryotic genome and to elucidate their function: First, we will extend our identification and genome wide description of the novel mark in the context of the developing and differentiating X. laevis embryo. Second, we will elucidate the function of deoxyadenosine methylation by identifying proteins that bind to and/or alter the deoxyadenosine methylation mark. We will then test their function with overexpression and depletion experiments, and evaluate their impact on the level of deoxyadensoine methylation, development and gene expression.

We believe that this research proposal will allow us to elucidate a novel, currently unknown modification that directly targets the DNA. We will shed light on the distribution and function of the deoxyadenosine methylation mark in eukaryotes, which will lead to a better understanding of fundamental biological pathways. In future, this work has the potential to generate a similar impact on biology and human health as the discovery of m5C has done and still continues to do.

About 30 years ago, it was discovered that eukaryotic DNA could be directly modified on a molecular level. This modification, affecting the molecule called dC, has been studied extensively ever since, revealing a fundamental role in different biological pathways and a better understanding of many human diseases such as cancer and ageing. Ultimately, research in this field led to new drug therapies and improved human health. Since the major discovery that dC can be modified, no other DNA molecule has been known to undergo direct molecular alterations in eukaryotes.

Recently, we have discovered the presence of a modification on another DNA component in the eukaryotic genome, namely affecting the DNA molecule dA. Since this newly discovered modification directly alters the DNA, the building blocks of 'life', and is genetically heritable, elucidation of this modification has the potential to generate the same fundamental impact on biology, human health and ageing as has the discovery of the modification affecting dC, as it still continues to do.

Hence, the overall aim of our proposal is to study this novel and direct DNA modification. Our preliminary data indicates that the dA modification varies between different cell types, suggesting that this novel modification could also be responsible for the differences observed between distinct cell types. Also, it is likely that the m6A mark is genetically heritable, and that changes arise during the development of an organism, when cells begin to differentiate. This we also intend to elucidate. Studying the modification of dA in eukaryotes will reveal fundamental biological regulatory pathways that might have consequences on differentiation and even ageing, as was the case with methylated dC. For example, the study of methylated dC gave rise not only to a better understanding of various diseases such as cancer, but also shed light on ageing and contributed to the development of various drugs and cures.

The immediate beneficiaries of the results of our proposed work will be the scientific community. Our data will contribute towards greater knowledge, which will be shared and can be used as a resource around the world. Our work may reveal a fundamental regulatory mechanism in biology, which will not only attract further research investments into the academic field, but also into pharmaceutical companies. Hence, our proposal has an excellent 'value for money' ratio.

On the long run, we believe that this study will benefit the economy and ultimately enhance the quality of life: Since this novel modification directly alters the DNA and might be genetically heritable unlike all other modifications except affecting dC molecules, any missregulation might have fundamental effects on human health. Hence, studying this novel modification in eukaryotes might not only help to better understand the causes of many human diseases, but ultimately lead to future drug therapies. Also, the missregulation of various biological pathways that are important for early development are known to induce cancer. Hence, studying modifications at these stages will also ultimately contribute to a better understanding of cancer. This project will also discover novel molecules that could represent potential targets for cancer drug therapies, ultimately leading to therapeutic cures. Overall, this is expected to boost not only more academic research, but also the pharmaceutical industry, which in turn will help the economy and ultimately improve the quality of life not only on the health level, but also financially. In addition, research into development has unraveled how unspecialized cells differentiate into specific tissues and organs. Therapeutic cell replacement of this kind could therefore give an improved quality of life while decreasing the cost of care to the National Health budget. This would result in a considerable economic benefit for government spending.