Single-cell studies point to the role of DNA methylation in cell fate decisions

Research from the Reik lab at the Barbraham Institute advances our understanding of
the role of DNA methylation in the early stages of development.
Technological advances in this field, which provide the ability to collect parallel data types from individual cells, as well as the presence of cell atlas references and synthetic data sets, are revolutionizing our understanding
of the processes that determine cell fate.
Rules that elucidate how different cell types are formed have applications
in regenerative medicine as well as in understanding developmental disorders and diseases.

When the tissues and organs of the embryo are formed, the elimination and reintroduction of DNA methylation is essential
to establish the properties of the cell.
In some cases, the absence of key methylases in mice can lead to severe developmental defects and embryonic death
.
Despite the importance of DNA methylation during development, little
is known about the underlying mechanisms by which it is achieved.
This is due to the limited information previously gathered by researchers to understand the effects of changes in the normal process of DNA methylation during development, limited to analysis of developmental defects, sample imaging, and limited genome-wide analysis
using bulk samples.
These methods are not sufficient to address the effects
at the different cell type levels.

Using mice where key methylases have been deleted, researchers from the institute's epigenetics project, Reik, performed single-cell gene expression analysis
at the beginning of organ development (day 8.
5 after fertilization).
Harnessing the power of the single-cell approach, the researchers were able to track which types of cells were affected, proposing the mechanisms behind the effects seen at the
entire organism scale in terms of their inability to form in mouse embryos.

The study used genetically engineered mice in which two key groups of methylases were deleted: knockout mouse lines of DNA methyltransferases (DNMT 1, 3a, and 3b) that introduced and maintained DNA methylation, respectively, and a system to study the effects of
the combined deletion of all three TETase (ten-11 translocation (TET) methylcytosine dioxygenase 1/2/3) that cause demethylation.

At the time of the study, Dr.
Stephen Clark, a senior researcher in Reik's lab, said: "The use of single-cell methods has indeed provided us with the resolution
needed to study the mechanisms of DNA methylation during development.
" The images we were able to build confirm the inhibitory effect of DNA methylation at this developmental time point, first, that maintaining proper DNA methylation requires inhibiting past and other cell type identities, and secondly, that DNA methylation needs to be removed from parts of the genome to allow for the formation
of specific cell types.
”

A technique called single-cell RNA sequencing is used to measure gene expression
in the genome of each mouse.
Comparing these expression profiles with the reference dataset can identify all cell types of
the embryo.
After this step, the effect of methylation perturbations on cell fate can be assessed by comparing the composition of knocked out embryos (where methylases are removed) with wild-type embryos at the same developmental stage to highlight differences in cell type ratios
.

The researchers were able to link the effects of day 8.
5 of development on cell type formation, which matched the phenotype observed, and analyze cell type-specific gene expression changes
that may be associated with defects in cell fate commitment.

Dr.
Ricard Argelaguet, a former postdoctoral researcher in the Institute's Reik Lab and co-first author of the paper, commented: "Being able to observe changes in cell type and gene expression from both a holistic perspective and changes in cell type and gene expression at a granularity provides us with the ability to comb through the role of DNA methylation and demethylation in developing embryos at this particular point in time, creating new insights
.
" 。 "It would be equally interesting
to apply this method to a later point in time to learn more about the role of DNA methylation during development.
"

The study has created an interactive data platform that provides single-cell-level gene expression readings from Dnmt and Tet-mutant mouse embryos
.

Professor Wolf Reik, Director of the Cambridge Institute of Science in Altos, who led the study and is also the team leader of the Barberaham Institute's Epigenetics Project, commented: "This study provides a wealth of resources
for exploring the link between DNA methylation and cell fate.
This study benefits from published datasets and reference atlases, and we hope that our work will in turn be helpful
to other researchers in the field of developmental and epigenetics.
" ”