Advances in single-cell technologies have not only made it possible to analyze multiple molecular families in individual cells, they have also enabled scientists to parse through limitations in data analysis arising from the intercellular diversity of measured parameters. Although single-cell sequencing is now widely available, techniques to detect epigenetic markers and transcription at single-cell resolution in a combined protocol has been unavailable until now.

Scientists at the Hubrecht Institute for developmental biology and stem cell research in the Netherlands have now developed a new experimental technique to measure genome-wide transcription and DNA packaging in a consolidated assay in individual cells.

When DNA is not actively expressed, it is tightly coiled around histone proteins forming a dense chromatin that prevents the decoding machinery from accessing it. When expressed on the other hand, histones are chemically modified (post-translational modifications, PTMs), which loosens the dense chromatin and allows RNA polymerase and its molecular entourage to access regions of the chromatin and transcribe it into proteins. Histone PTMs and packaging of DNA therefore constitute epigenetic states (heritable changes that do not involve changes in the DNA sequence) that regulate which genes are expressed.

EpiDamID is a single-cell application that provides a combined epigenetic and transcriptomic readout [Rang FJ, et al, Molecular Cell, 2022].
The new method called EpiDamID locates modified histones. It improves upon the DamID (DNA adenine methyltransferase identification) toolkit that maps binding sites of proteins on DNA- and chromatin in a large group of cells. EpiDamID can be used in a variety of systems and workflows, including the simultaneous single-cell measurement of epigenetic state and transcription during embryonic development. By leveraging the binding specificities of single-chain variable fragment antibodies, engineered chromatin reader domains, and endogenous chromatin-binding proteins, EpiDamID is equipped to target different types of chromatin.

The technique was developed by Franka Rang and Kim de Luca, PhD students in the laboratory of Jop Kind, PhD, who is an Oncode Investigator at the Hubrecht Institute.

The findings were published on April 1, 2022, in the journal Molecular Cell, in an article titled “Single-cell profiling of transcriptome and histone modifications with EpiDamID.” Single-cell analysis using EpiDamID improves upon earlier protocols that provided average measures of DNA packaging in cell clusters.

“EpiDamID is a new addition to a vast toolbox to study chromatin states during dynamic cellular processes,” the authors note.

EpiDamID can be used to determine the location of specific PTMs on histone proteins in single cells. The method requires only a limited amount of material to generate enough data since it analyzes single cells. Furthermore, EpiDamID can be combined with other methods, such as microscopy, to study the regulation of gene expression using multipronged approaches.

Zebrafish notochord nuclei at 15-somite stage showing DAPI-stained nuclear DNA in gray and histone H3K9me3 stained in red (Phong Nguyen, Franka Rang & Kim de Luca / Hubrecht Institute).

To test the application of their new method, the investigators use EpiDamID to study the epigenetic modification called Polycomb (H3K27me3) and its relationship to transcription in mouse embryoid bodies. They identified several hierarchical transcription factor networks that are Polycomb dependent and independent. The team also mapped another epigenetic modification (H3K9me3) in early zebrafish embryonic development, and detected densely packed inaccessible regions of the chromatin that are specific to the notochord, a cylindrical cartilaginous structure that supports the embryonic body in all chordates.

In future studies, the Kind group will focus on investigating the role of PTMs in individual cells during embryonic development.