Bilateral BBSRC-SFI: Deciphering the function of the human Dihydrofolate reductase 2 gene

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Folates are a type of B vitamin that consists of a group of small molecules that are needed in nearly every cell in the body to fulfil a number of essential functions including synthesis of DNA for cell division. Like all vitamins, we need to ensure that we consume a sufficient amount of folate to maintain our health. Inadequate folate is implicated in birth defects, inborn errors of metabolism, neurological problems, autism, fatty liver disease, age-related and cognitive impairment and many cancers. We need to understand the pathway that uses folate, known as folate metabolism, and how it is regulated in different tissues and stages of development before and after birth.The individual steps of folate metabolism are mediated by a specialised set of proteins, called enzymes, one of the most important being Dihydrofolate Reductase (DHFR). Lack of DHFR function suppresses folate metabolism and prevents cells dividing - this is very harmful in normal tissue but can be exploited as the basis of action of some drugs for the treatment of cancer. DHFR is also the route of entry into folate metabolism of folic acid, which is included in fortified foods and vitamin supplements for prevention of birth defects such as spina bifida. Despite public health messages, many women still become pregnant with inadequate folate status. Further research on folate and human health is essential to inform the public health discussion on the introduction of mandatory fortification policies across Europe.Notably we found that humans and other primates have acquired a second DHFR gene, DHFR2, during evolution whereas other mammals have only one. Little is known about the function of DHFR2 but we find that the gene is active in many tissues and genetic studies show that alteration of DHFR2 may be linked to increased risk of a group of severe birth defects termed neural tube defects (NTDs), in which the early events of brain and spinal cord development fail. These findings suggest that DHFR2 may play a key role during development. To gain a better understanding of human folate metabolism it is important to investigate the role of DHFR2 and to ask whether it has similar or distinct functions to DHFR. We will study how DHFR2 protein abundance and location changes as stem cells differentiate and become more specialised cell types such as neurons. We will use genetic tools to remove DHFR2 from cells in culture and investigate the consequences for cellular properties such as proliferation and differentiation, as well as activity of folate metabolism. In parallel, we will ask how the relative levels and location of DHFR and DHFR2 differ in varying conditions. This knowledge will all help to understand what it does. The next step will be to investigate the role of DHFR2 during development and we will find out where DHFR2 is expressed in human embryos at differing stages. In order to move this work to living embryos we will generate new mouse strains in which the mouse DHFR gene is replaced with human DHFR or DHFR2 or both. These mouse models will allow us to ask detailed questions about the function of each protein. Can human DHFR or DHFR2 substitute for the mouse protein? Is the presence of only DHFR2 sufficient for normal development or do these embryos show changes in growth, neural development and/or folate metabolism? In this way, the study of 'humanised' mice that express only DHFR or DHFR2 will tell us about the individual functions of the enzymes that is difficult to address in human cells that have both.Having established the functions of DHFR2 in human cells and mouse models, the final part of the project will examine the regulation of the protein in more detail. We will test whether it is present in different structural forms, whether it is modified at particular sites and how its production is regulated. Overall, this project will give new insight into a fundamental metabolic pathway that is crucial for human health.