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Background
DNA methylation impacts many cellular processes including cell differentiation, genomic imprinting, DNA replication, X chromosome inactivation, and suppression of unneeded transcription from oncogenes.
For example, this is CopyCat:
CopyCat is the first cloned pet, born in December 2001 (Shin, Taeyoung, et al. (2002)), and this is Rainbow:
Rainbow is CopyCat’s genetic donor; note that they have totally different fur patterns – this is due to their epigenetic differences, particularly a phenomenon called X-Chromosome inactivation. This is a dosage compensation mechanism for females wherein one X chromosome in each cell is randomly selected for inactivation by DNA methylation and other modifications.
DNA methylation also has another important effect in mammalian cells; methylation of CpG sites can function to repress gene expression, as shown where methylation prevents binding of DNA polymerase upstream of a promoter, which is then silenced.
Clearly, DNA methylation is one of nature’s most powerful mechanisms for transcriptional regulation, yet synthetic biologists don’t talk about it. Penn iGEM is adding synthetic biology as a tool for epigenetics researchers; we are providing researchers with the tools they need to safely and precisely engineer the epigenome.
Targeted Methylation: the Possibilities
If the tools existed to allow researchers to target specific DNA sequences for selective methylation, they could be used to inactivate strong promoters (permanent repression without gene knockout), repress overactive oncogenes, inactivate excess chromosomes, correct hypomethylation in imprinting disorders and even control differentiation in stem cells.
Our team aimed to address this need by creating a target methylation toolkit which includes a modular plasmid that enables fast, cheap, and simple screening of targeted methyltransferases.
MaGellin: A one plasmid system for screening targeted methyltransferases
MaGellin has several key features:
- DNA binding domain/methyltransferase can be swapped out
- Easy digest assays both components: methylation and targeting
- Inducible system for controlled expression of protein
- Verified working bisulfite sequencing primers
Restriction digest of miniprep screens targeted methyltransferase efficacy
This assay gives Results 3 hours after miniprep. Bisulfite sequencing would take 5 days and much more money.
The following table compares bisulfite sequencing (the currently used standard for measuring DNA methylation) to Magellin.
Bisulfite Sequencing |
Modular Plasmid |
|
Time after Sample Collection |
5 days |
2 hours |
Cost |
$$$ |
$ |
Reports On-Target and Off-Target Effects |
Requires separate assays |
Can be assayed simultaneously |
Quantitative Measurement of Methylation |
Yes |
No |
Ease of Designing Sequencing Primers |
Difficult |
Primers already designed |
Ease of Cloning |
Must either co-transform or design cloning plan for single-plasmid system |
Backbone with target is ready for cloning |
Our team used MaGellin to Characterize Existing and Novel Targeted Methyltransferases:
