Monitoring dynamics of DNA methylation in single cells

DNA methylation is considered a key epigenetic modification that shapes and maintains cellular identity. In recent years, advancements in sequencing technologies enabled the generation of detailed single-base resolution maps of DNA methylation in multiple cell types. However, current methods only provide a static "snapshot" of the methylation levels thus precluding the study of real-time epigenetic changes and limiting prospective mechanistic experiments. Here, we established a novel reporter system that allows region-specific dynamics of DNA methylation to be traced at single cell resolution.

HFSP Long-Term Fellow Yonatan Stelzer and colleagues
authored on Mon, 16 November 2015

While each individual cell in multicellular organisms shares the same DNA content, the overwhelming functional diversity and cellular specification is considered to be shaped and regulated by epigenetic modifications.  DNA methylation is the addition of methyl groups to the fifth carbon atom of cytosine nucleotides, and is among the best studied epigenetic modifications. Global DNA methylation patterns are highly dynamic during early embryogenesis while the zygotic signature is erased and the somatic signature is established in a tissue and cell-type specific manner. Nevertheless, it is still largely unknown how exactly DNA methylation patterns dictate cell type specific gene expression programs. In addition, misregulation of DNA methylation is associated with numerous human diseases and is frequently observed in cancer.

All current methods to study DNA methylation require the extraction of DNA and therefore provide only a snapshot measurement of the methylation landscape. Furthermore, the vast majority of sequencing methods are bulk, precluding the assessment of single-cell heterogeneity. Therefore, generating a traceable readout of endogenous DNA methylation changes is of great interest to the field. With that in mind, in the current study (Stelzer et al., 2015) we aimed to generate a reporter system that allows real-time regional DNA methylation dynamics to be traced at single cell resolution .

Figure: Region of interest DNA methylation state is affecting the Snrpn promoter activity resulting in differential fluorescent activity. Consequently, lack of DNA methylation (hypomethylation, open circles) is reflected by GFP positive cells, while methylated regions (filled circles) appear as GFP negative cells.

We show that a minimal imprinted gene promoter (Snrpn) can be used as a natural and neutral DNA methylation sensor for endogenous methylation state, thus allowing to transform epigenetic signals into transcription. In combination with fluorescent proteins, we utilized the Snrpn promoter to establish a dynamic on/off light-switch reporter. We further show that the reporter allows DNA methylation changes associated with both coding and non-coding regions to be traced at single cell resolution (Stelzer et al., 2015). The reporter system could be utilized to study DNA methylation dynamics during normal development and disease. Furthermore, combining the DNA methylation reporter with other conventional gene expression reporters, facilitates an attractive system to study the effects of epigenetic changes on gene expression programs in a tissue-dependent manner. 

Reference 

Tracing dynamic changes of DNA methylation at single cell resolution. Stelzer Y., Shivalila CS., Soldner F., Markoulaki S and Jaenisch R. Cell, 163(1), 218–229 (2015).              

Link to Cell article

Pubmed link