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Whitehead Team Reports Method for Real-Time, Single-Cell Tracking of DNA Methylation

NEW YORK (GenomeWeb) – A Whitehead Institute team this week reported a new technique for tracking changes in DNA methylation in individual live cells in real time, allowing for the dynamic monitoring of a process that has previously only been viewed in static images.

According to the researchers, the method — dubbed reporter of genomic methylation (RGM — is expected to not only help gain a better understanding of methylation but also enable efforts to develop drugs that target the DNA-modification process.

"Pharmaceutical companies have been interested in manipulating methylation in disease," Yonatan Stelzer, who led the Whitehead team, said in a statement. "Now that we have a reporter for methylation, they can screen for small molecules or genes that can change a cell's phenotype."

DNA methylation is an epigenetic cellular process in which methyl groups are added to DNA —often at CpG sites — to suppress gene transcription. Methylation is key to proper cell development, and its dysfunction has been implicated in various diseases including cancer.

While the availability of new technologies have allowed for the creation of methylation maps for multiple human and mouse cell types, DNA methylation is dynamic, and existing tools only provide static snapshots of the process during cell-state transitions.

"The difficulty in translating real-time epigenetic changes into a traceable readout is, to date, a limiting factor in our ability to follow the dynamics of DNA methylation," the Whitehead researchers wrote in a paper published today in Cell. "Therefore, a key challenge in the field is to generate tools that allow tracing changes in DNA methylation over time," particularly at single-cell resolution.

To tackle this issue, the scientists developed a reporter system based on two key premises: that CpG sites can serve as cis-acting signals, affecting the methylation state of adjacent CpGs; and that a methylation-sensitive promoter, when introduced near a target CpG region, may be used to report on methylation changes of the adjacent sequences.

RGM is specifically based on a minimal imprinted gene promoter that drives a fluorescent protein. Because the promoter is not subjected to methylation changes by itself, expression of the fluorescent protein is dependent on the methylation state of surrounding sequences.

In their paper, the Whitehead group showed that when RGM is inserted next to an unmethylated region, it matches this state and allows the fluorescent protein to be expressed. When it is placed next to a methylated region, fluorescence is suppressed.

The researchers also used the system to study demethylation events acquired during cellular reprogramming by generating reporter cell lines for the pluripotency-specific super enhancers of Sox2 and microRNA-290, which are demethylated and activated during the reprogramming of somatic cells into induced pluripotent stem cells.

With RGM, the team was able to visualized loci-specific DNA methylation changes and their relationship with transcription during cell-state transition following differentiation of mouse embryonic cells, as well as during reprogramming of somatic cells to pluripotency.

"This reporter is a very important tool," Rudolf Jaenisch, senior author of the Cell paper, said in the statement. "We believe it will allow us to look in a very detailed way at issues like imprinting during development and screening for the activation of genes silenced in diseases like cancer."