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Establishment of diverse cell lines for the detection of epigenetic changes in single cell resolution

Project


Project code: BfR-CPS-08-1322-696
Contract period: 01.01.2018 - 31.12.2019
Purpose of research: Experimental development

Epigenetic mechanisms are particularly crucial during early embryonic development. However, although our understanding of the underlying molecular biology grows we are still way from sufficiently understanding the consequences of dysregulation. It is not the least this reason, why toxicology still lacks a reliable test system for epigenetic noxae, be it in vivo or in vitro. There is even no concept of what would constitute a sufficiently universal and reliable biomarker, for this matter. Toxicologically the consequence of epigenetic dysregulation often is severe, though. Therefore the aim of this project is to develop fluorescent indicator cell lines which allow studying epigenetic regulation in situ. This not only will foster a deeper molecular understanding but crucially also allows for the identification and verification of potential biomarkers. Importantly, the anticipated system is designed not only to record the dynamics of epigenetics but to work on single cell level. This is crucial as the two best-studied epigenetic mechanisms include DNA methylation and histone modifications. In both cases, the analysis of these is very laborious and destructive, only allowing recording of molecular stills. While this already abandons essential information about the dynamics of epigenetics, interpretation of the corresponding data is complicated further by the fact that they integrate signals from complex cellular mixtures. This inherently limits the sensitivity and specificity of nearly all epigenetic analysis tools currently available. Two recent publications in Cell reported on the use of methylation-sensitive protein expression for studying the endogenous methylation state of the corresponding regions in mice in vivo (Stelzer et al. 2015 and 2017). The method is contemplated to work in embryonic stem cells also and hence was adapted accordingly. Last year’s work saw the generation of the respective reporter constructs and their initial testing in embryonic stem cells. The constructs designed and used for the initial proof of concept rely on two 400- to 500 bp-long promoter fragments, that is the constitutively active promoter of the gene Pgk1 and the strong but normally inactive promoter of the intracisternal A particle (IAP), respectively. However, the IAP-construct proved to deliver unreliable results, most likely due to the use of an incomplete promotor-construct (Lienert et al. 2011). The construct is currently redesigned appropriately and will be re-tested next year. A second approach relies on the visualization of specific histone modifications affecting DNA-accessibility in situ. In the nucleus DNA is wrapped around histones. These are globular proteins that have long N-terminal ends which can undergo various protein modifications, the most prominent examples being phosphorylation, methylation, and acetylation. Functionally these modifications usually exert either a transcriptionally activating or repressive effect on the proximal genes. Classic representatives are, for example, histone H3K4me3, which is found mainly in the promoter region of active genes or H3K27me3, which acts as a repressor. Interestingly, in all but embryonic stem cells these modifications are mutually exclusive. In embryonic stem, however, there is a third category of promotor regulation that results in so-called bivalent promoters. These carry both histone modifications and are in an open and readily accessible state. Yet, they remain transcriptionally inactive. The functionally complex context of different histone modifications is also called the histone code. Cells possess many different reader proteins that can recognize specific histone modifications. The project used this as a first starting point, creating fusion proteins that link a specific reader protein (or a protein domain) fluorescent mCherry. The idea was to construct a fluorescent reporter system that specifically reacts to the histone modification set out by the reader’s activity. First proof of concept studies with parallel antibody staining show the system to work and indicate a high specificity. However, the H3K4me3-reporter still needs optimization with regard to fluorescent background reduction. Work on this construct will thus continue in the following year. The constructs will now be analyzed for their long-term stability and possible interference with cell viability. Also, the suitability for recording signals over longer periods of time in vivo shall be established. Finally, mCherry is to be replaced by bipartite fluorescence proteins as tol restrict signaling to spatial proximity of the modifications in question. The ultimate goal is the generation of corresponding stable cell lines as a tool to study the effect of potential epigenetic noxae on histone modifications and identify suitable biomarkers, respectively.

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BMEL - research cluster

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