Decoding Developmental Epigenetics: Innovation and implementation of single-cell techniques to study epigenetic regulation in embryonic development

The DNA is often referred to as the blueprint of the cell. However, nearly all cells in the body contain the same DNA, while displaying a vast array of phenotypes and functions. To achieve this phenotypic diversity, different types of cells activate distinct gene sets through various mechanisms, including epigenetic regulation and spatial organization of the genome. Histone post-translational modifications (PTMs) are an important form of epigenetic regulation and strongly influence chromatin density and gene expression. An example of the spatial organization of the genome is the association of chromatin with the nuclear lamina (NL), a filamentous meshwork lining the inner nuclear membrane. Regions of the DNA that are in contact with the NL are called lamina-associated domains (LADs). LADs are typically gene poor and form a gene-repressive environment. While histone PTMs and LADs have been extensively studied, a lot remains unclear about their role in regulating gene expression, especially during complex biological processes such as embryonic development. To better understand these regulatory mechanisms, it is important to measure both protein-DNA interactions and gene expression. Ideally, these measurements are taken in single cells so they can be directly related.

In my dissertation, I describe the innovations and studies that we performed during my PhD to gain more insight in epigenetic and spatial regulation. First, we developed scDam&T-seq, a technique to simultaneously measure protein-DNA interactions and gene expression in single-cells. Next, we developed EpiDamID, an extension of scDam&T-seq that enables its application to histone PTMs. We apply both these techniques to study histone PTMs and LADs during the very early stages of mouse development. During these early stages (from the moment of fertilization until implantation), the epigenome and spatial organization of the DNA are extensively rewired. The patterns inherited from the parental gametes are largely reset to enable the embryo to give rise to all cell types of the organism. In our research, we find that LADs are extremely variable in the 2-cell mouse embryo: There are large differences in which regions of the genome are LADs, both between embryos and between cells of the same embryo. We demonstrate that this variability is the result of broad domains of the histone PTM H3K27me3, which are uniquely present in the early embryo. These domains cover regions of the DNA with a strong inherent affinity for the NL, but effectively work as a repellent for NL association. These opposing forces result in the strong LAD variability and removal of H3K27me3 stabilizes LADs. These findings provide insight in the interactions between different regulatory layers of the genome and emphasize the power of single-cell technologies.