Tracing histone modification dynamics in single cells during differentiation and early development

Epigenetics is derived from the Greek prefix -epi that means “around” and the word genetics which is the study of genes. Therefore, epigenetics is the study of the effects that occur around the DNA helix that have an impact in the expression or repression of genes without changing the sequence per se. There are many types of epigenetic factors, but the one studied in detail in this thesis are histone modifications (Chapter 1). Histones are a group of 8 protein units that enable the almost 2 metre stretch of DNA to be correctly compacted and contained within each cell. These histones have a protruding tail that is susceptible to post-translational modifications. The type and amount of chemical modifications added to these tails changes how tightly compacted the DNA is, creating a more “loose” and accessible DNA conformation or a more “closed” and restricted one.To be able to study these modifications at a single-cell level, we have developed a new technique termed “sortChIC” (Chapter 2). First, we use a combination of surface markers on an heterogeneous sample to identify and sort out the cell types of interest. Second, we add an antibody that will specifically recognise the histone modification we are measuring. Afterwards, we add a protein fused with an enzyme that will recognize the antibody. Next, we activate the enzyme so that it will start cutting the DNA around where the histone mark is present. In this way we are able to retrieve these fragments produced by the enzyme, ligate adapters to differentiate them from one another, amplify the material, and finally sequence and perform analysis. We have applied this novel technique during the blood cell differentiation process in mice, called hematopoiesis (Chapter 3). This study showed that depending on the cell type, histone modifications are deposited on different parts of the genome, establishing specific regulatory patterns of gene expression. Moreover, we have improved upon this technique to allow for the parallel measurement of another modality, the transcriptome. This technique is called T-ChIC, and it enables the detection of not only the histone modifications but also the snapshot of the genes being transcribed at that specific moment. This allows us to distinguish specific cell types from one another. We then used T-ChIC to study the impact of histone modifications in the specification of cell types and tissues in early embryonic development of the zebrafish (Danio rerio) (Chapter 4). From our data we concluded that there is a spatiotemporal accumulation of a repressive mark responsible for the silencing of expression in early embryonic development. The aim of our research is to be able to understand in detail how histone modifications influence the expression of genes in tissue development and organism development. For this, we contributed two novel techniques that can be applied to many animal models and systems to further advance our understanding of epigenetics (Chapter 5)

Single-cell sortChIC identifies hierarchical chromatin dynamics during hematopoiesis

Post-translational histone modifications modulate chromatin activity to affect gene expression. How chromatin states underlie lineage choice in single cells is relatively unexplored. We develop sort-assisted single-cell chromatin immunocleavage (sortChIC) and map active (H3K4me1 and H3K4me3) and repressive (H3K27me3 and H3K9me3) histone modifications in the mouse bone marrow. During differentiation, hematopoietic stem and progenitor cells (HSPCs) acquire active chromatin states mediated by cell-type-specifying transcription factors, which are unique for each lineage. By contrast, most alterations in repressive marks during differentiation occur independent of the final cell type. Chromatin trajectory analysis shows that lineage choice at the chromatin level occurs at the progenitor stage. Joint profiling of H3K4me1 and H3K9me3 demonstrates that cell types within the myeloid lineage have distinct active chromatin but share similar myeloid-specific heterochromatin states. This implies a hierarchical regulation of chromatin during hematopoiesis: heterochromatin dynamics distinguish differentiation trajectories and lineages, while euchromatin dynamics reflect cell types within lineages