Exploring the universe of single cells using multi-omic approaches

In both unicellular and multicellular organisms, no individual cell is completely the same. The tissues of humans and other animals are composed of various cell types, each with a different function. Even within the population of a specific type of cell, all cells differ from one another on multiple molecular levels. This variation is constantly introduced in cells, making them more unique step by step. Variation can be introduced during cell division through for example DNA mutations or changes in the structure or the packaging of DNA. Diseases such as cancer can start in an individual cell when it quickly diverges from its predecessors through cell divisions and gives rise to a tumor. Chromosome loss in colorectal cancer The second chapter provides more insight into various modalities of individual cells. We use organoid models of colorectal cancer, which mimic the disease in tiny 3D structures grown in a dish. With NLAIII-seq for whole genome sequencing and viral barcoding for lineage tracing we perform and read out multiple measurements in individual cells to reconstruct the order in which chromosomal abnormalities occur in this type of cancer. Because of the integrated measurements, we are able to measure changes that occur in parallel in different cells within one population. Using this method, we found a recurring loss of chromosome 4 that only occurs after the loss of chromosome 18. These findings coincide with clinical observations in patients with colorectal cancer. Hematopoiesis In the third chapter we introduce a novel technique called sort-ChIC. This technique measures histone modifications in individual cells. Modifications in these molecules influence which parts of the DNA in that cell can be read and thereby influence the proteins that the cell produces. We apply sort-ChIC in two active (H3K4me1 and H3K4me3) and two repressive (H3K27me3 and H3K9me3) histone modifications on blood stem cells (HSPCs) and adult blood cells in the bone marrow of the mouse to gain insight into the histone modifications that occur during blood formation. Joint profiling of H3K4me1 and H3K9me3 demonstrates that cell types within the myeloid lineage have distinct active chromatin but share similar myeloid-specific heterochromatin repressed states. This suggests hierarchical chromatin regulation during hematopoiesis. State-of-the-art technology The fourth chapter introduces a new technique, scChIC-TAPS, that can measure various modalities – including histone marks and DNA structure – simultaneously in an individual cell. Our approach combines bisulfite-free conversion of methylated cytosines and targeted MNase digestion. With these integrated measurements we can resolve the local correlations of different histone modifications and DNA methylation states at base-pair resolution. We describe the validation of scChIC-TAPS and its application in the Fucci cell line, a system in which the position of every cell in the cell cycle can be measured precisely. We use this cell cycle information to integrate the data of multiple histone marks and compare their behavior throughout the cell cycle. Our data provides the first direct evidence that kinetics of replication-coupled methylation are influenced by the local chromatin environment

Exploring the universe of single cells using multi-omic approaches

In both unicellular and multicellular organisms, no individual cell is completely the same. The tissues of humans and other animals are composed of various cell types, each with a different function. Even within the population of a specific type of cell, all cells differ from one another on multiple molecular levels. This variation is constantly introduced in cells, making them more unique step by step. Variation can be introduced during cell division through for example DNA mutations or changes in the structure or the packaging of DNA. Diseases such as cancer can start in an individual cell when it quickly diverges from its predecessors through cell divisions and gives rise to a tumor. Chromosome loss in colorectal cancer The second chapter provides more insight into various modalities of individual cells. We use organoid models of colorectal cancer, which mimic the disease in tiny 3D structures grown in a dish. With NLAIII-seq for whole genome sequencing and viral barcoding for lineage tracing we perform and read out multiple measurements in individual cells to reconstruct the order in which chromosomal abnormalities occur in this type of cancer. Because of the integrated measurements, we are able to measure changes that occur in parallel in different cells within one population. Using this method, we found a recurring loss of chromosome 4 that only occurs after the loss of chromosome 18. These findings coincide with clinical observations in patients with colorectal cancer. Hematopoiesis In the third chapter we introduce a novel technique called sort-ChIC. This technique measures histone modifications in individual cells. Modifications in these molecules influence which parts of the DNA in that cell can be read and thereby influence the proteins that the cell produces. We apply sort-ChIC in two active (H3K4me1 and H3K4me3) and two repressive (H3K27me3 and H3K9me3) histone modifications on blood stem cells (HSPCs) and adult blood cells in the bone marrow of the mouse to gain insight into the histone modifications that occur during blood formation. Joint profiling of H3K4me1 and H3K9me3 demonstrates that cell types within the myeloid lineage have distinct active chromatin but share similar myeloid-specific heterochromatin repressed states. This suggests hierarchical chromatin regulation during hematopoiesis. State-of-the-art technology The fourth chapter introduces a new technique, scChIC-TAPS, that can measure various modalities – including histone marks and DNA structure – simultaneously in an individual cell. Our approach combines bisulfite-free conversion of methylated cytosines and targeted MNase digestion. With these integrated measurements we can resolve the local correlations of different histone modifications and DNA methylation states at base-pair resolution. We describe the validation of scChIC-TAPS and its application in the Fucci cell line, a system in which the position of every cell in the cell cycle can be measured precisely. We use this cell cycle information to integrate the data of multiple histone marks and compare their behavior throughout the cell cycle. Our data provides the first direct evidence that kinetics of replication-coupled methylation are influenced by the local chromatin environment

scChIX-seq infers dynamic relationships between histone modifications in single cells

Regulation of chromatin states involves the dynamic interplay between different histone modifications to control gene expression. Recent advances have enabled mapping of histone marks in single cells, but most methods are constrained to profile only one histone mark per cell. Here, we present an integrated experimental and computational framework, scChIX-seq (single-cell chromatin immunocleavage and unmixing sequencing), to map several histone marks in single cells. scChIX-seq multiplexes two histone marks together in single cells, then computationally deconvolves the signal using training data from respective histone mark profiles. This framework learns the cell-type-specific correlation structure between histone marks, and therefore does not require a priori assumptions of their genomic distributions. Using scChIX-seq, we demonstrate multimodal analysis of histone marks in single cells across a range of mark combinations. Modeling dynamics of in vitro macrophage differentiation enables integrated analysis of chromatin velocity. Overall, scChIX-seq unlocks systematic interrogation of the interplay between histone modifications in single cells

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