17 June

New method to study gene regulation and expression simultaneously in single cells

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Researchers from the group of Jop Kind have developed a new method to study both the output and the regulation of genes in the same single cell. This method may be used to increase our understanding of how a single cell can develop into an organism that consists of many cells with different functions, or to study how cancer can develop from previously healthy tissue. The new method was published in the scientific journal Nature Biotechnology on the 17thof June.

One genetic code, many different cell types
All cells in our body contain the same genetic information (DNA), yet consist of many different cell types with very diverse functions. The identity of a cell, for instance being a skin cell or a muscle cell, is determined by the regions of the DNA (genes) that are actually being used in each cell. For a gene to be used by the cell, it has to be accessible. Therefore, the level of accessibility determines to a large extend the activity of genes and thereby the identity of a cell. So far, we understand little about how the accessibility and activity of genes are regulated. The researchers in the group of Jop Kind have now developed a new method with which both the activity and the proteins that determine the accessibility of genes can be analyzed in the same cell. This method will lead to a better understanding of the regulation gene activity, and will teach us for instance how a complex organism with many cells of diverse functions can form from a single fertilized oocyte.

Accessibility and activity
Active genes in the cell are copied into intermediate templates, called mRNAs. The identity and function of a cell can therefore be determined by analyzing the mRNAs that are made by that cell. For a gene to be active, it needs to be accessible: the cell needs to be able to “read” the gene. This accessibility depends on how extensively the DNA is wrapped in a complex protein structure called chromatin. The key to understanding how different cell types arise, lies in determining the mechanisms that control the packaging of genes into chromatin. The opening and closing of chromatin is generally achieved by special proteins. By simultaneously studying the location of these special proteins on the DNA and the activity of genes, we can gain insight into how the packaging works. However, until now there was no technique available that can measure both aspects in the same cell.

a very powerful new technique with which the mechanism and the output of genes can be measured simultaneously with high sensitivity in the same single cell

New method
The researchers found a way to combine two techniques that previously seemed impossible to combine in the same cell, to study both proteins that regulate the packaging of the DNA and the activity of genes at the same time. DamID, a method to ‘stamp’ the DNA in those locations where a certain protein is present on the DNA, was used to stamp the DNA in those places where the specialized proteins that open and close the chromatin were present. By looking at all the places in the DNA that have this stamp, we can reconstruct where the protein has been. At the same time the activity of genes was measured by studying the mRNA molecules made by the cell. This resulted in a very powerful new technique with which the mechanism and the output of genes can be measured simultaneously with high sensitivity in the same single cell.

The body exists of many different cell types that each have their own specialized function. Although they differ a lot in structure and function, they each have the same genetic blueprint: the DNA. This diversity is achieved by using different parts of the DNA to generate mRNA, for example the purple region in the neuron and the red region in the heart cell. Which regions are used or not is determined by specialized proteins that can tightly package or open up the DNA. This combination of protein and DNA is called chromatin. The method we developed, scDam&T-seq, combines two techniques to get a dual readout from a single cell: DamID to uncover which parts of the DNA are regulated by a protein of interest, for instance those proteins that regulate the packaging of the DNA, and CELseq to detect which genes are active in the cell.

Applying the method
The new method will provide a valuable tool for future studies. It may for instance be applied to study how a single cell develops in an organism that consists of many cells with different functions during embryonic development. Another application may be to study how cancer cells can arise from a previously healthy tissue.

Simultaneous quantification of protein–DNA contacts and transcriptomes in single cells. Koos Rooijers, Corina M. Markodimitraki, Franka J. Rang, Sandra S. de Vries, Alex Chialastri, Kim L. de Luca, Dylan Mooijman, Siddharth S. Dey and Jop Kind. Nature Biotechnology 2019.



Jop Kind is group leader at the Hubrecht Institute and Oncode Investigator.