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In vitro assay for the detection of epigenetic effects during the differentiation
Project
Project code: BfR-CPS-08-1322-695
Contract period: 01.01.2018
- 31.12.2018
Purpose of research: Experimental development
Embryonic development is a highly complex process in which even the smallest disturbances can have drastic effects on the developing organism. These effects can affect a wide range of effects ranging from morphological malformations to cancer, impaired cognitive abilities or increased susceptibility to diseases. Yet, the respective toxicological focus for teratogenic substances still is the detection of apical morphological defects, mainly in lack of suitable testing alternatives. Meanwhile our understanding of the epigenetic changes during embryonic development has increased steadily. The permanently altered transcription of individual differentiated cells is stipulated by respective epigenetic modifications at the DNA, such as DNA-methylation. Catalysed by DNA methyltransferases and demethylases the latter targets predominantly cytosines within CG dinucleotide sequences. The palindromic nature of these sequences offers the inherent advantage of an easy transfer of methylation patterns during cell division. Routinely DNA methyltransferase 1 (Dnmt1) will recognize hemi-methylated DNA and modify the corresponding cytosine on the second strand. Methylation patterns are tissue specific and are usually permanent or even transgenerational. The only exception is a short time period immediately after fertilization at blastocyst stage, during which the paternal methylation patterns are completely removed. Tissue-specific methylation is subsequently reestablished by de novo DNA methyltransferases 3a and 3b. The only regions exempt from this are imprinted areas and of repetitive elements which maintain constant levels of methylation throughout embryonic development at 50 and 100 %, respectively. Unsurprisingly embryonic development hence is a particularly sensitive time window for adverse substance induced epigenetic changes. With clear mechanistic evidence still pending epidemiologic data do clearly indicate the potential adverse effects of altered DNA-methylation. One of the most prominent examples, although not substance induced, is the so called “Dutch hunger cohort”. This term subsumises Dutch nationals who, in their first trimester, were subjected to strong caloric restriction due to the Nazi-imposed famine in the Netherlands at the end of World War II. During this time daily rations fell below 1000 kcal and even 60 years onwards affected individuals feature reduced levels of DNA-methylation by up to 5 % while at the same time suffering from a significantly increased susceptibility for cardiovascular and other diseases. Similar changes in DNA-methylation observed for model teratogens such as valproic or retinoic acid. It thus appears that DNA-methylation is a sensitive marker for potential epigenetic toxicity. This is also confirmed by data from project 1322-593, which investigates substance induced effects on the epigenome. Building upon work performed last year we thus propose development of an methylation-sensitive in vitro assay. For this purpose, murine embryonic stem cells were differentiated into cardiomyocytes and sampled at different time points. The isolated DNA was examined by methylation-sensitive restriction and qPCR. This results in enrichment and monitoring of areas particularly sensitive to methylation. Firstly these will be genomic loci which feature constant levels of methylation and secondly levels with high variability. Proof of concept for the first category was established using the Pgk1 promoter, the imprinting control region of Igf2-H19, and the IAP promoter. The promoter of Oct4 was selected to represent category 2. The first gene, Pgk1, is a housekeeping gene that is highly expressed in all cells thus featuring an unmethylated promotor. The Igf2-H19 locus is an imprinted region and should therefore have around 50 % methylation. Contrastingly IAP (Intracisternal A Particle) is a silenced former retrovirus with around 100 % methylation, while Oct4 finally is a highly regulated pluripotency gene and as such subjected to strong hypermethylation in the course of embryonic development. Preliminary experiments were conducted with retinoic acid, valproic acid and bisphenol A. The latter was chosen as it shows methylation-sensitive effects in Agouti mice. In D3 mESCs bisphenol A likewise clearly affected methylation, particularly of the IAP-promoter. Interestingly this effect was concentration independent. Epigenetic effects of valproic and retinoic acid also correlated well with literature data. For example reduction of Igf2-H19-methylation by valproic acid fits reports of substance induced H19 expression. The assay shall now be further confirmed with complementary methods, a second cell line and other loci. Aim is the development of a multiplex assay for the detection of substance induced changes in DNA-methylation.
Epigenetic changes are an essential part of early embryonic development. There is sufficient evidence that external factors influence epigenetic mechanisms, which can be detected up to 60 years later and have an impact on the quality of life of those affected. Therefore, this project developed a new assay for detecting changes in the methylation status of individual selected regions and tested its transferability between various cell lines. The proof of concept study looked at varoius genomic areas known to be stable or subjected to changes during differentiation, respectively. Although first results were very promising the assay turned out to lack robustness in differing genomic backgrounds. Moreover, technical robustness appeared to be an issue too. Therefore the project will be terminated and the data be used for the ongoing work in other project and for commencing alternative detection systems.
Section overview
Subjects
- Biotechnology
- Genetic Resources
- Toxicology
