Abstract
Spatiotemporal gene regulation guides proper embryonic development, by governing lineage specification and cell fate decisions. This is achieved through the complex interplay between gene-regulatory mechanisms, such as various modes of epigenetic gene regulation and chromatin organization. To fully understand these mechanisms, their individual aspects as well as their interconnectivities need
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to be carefully dissected. In this dissertation, we implement and develop single-cell multimodal sequencing technologies to integrate multiple features of gene regulation, in order to ultimately obtain a complete interpretation of this phenomenon. In chapter 1, different mechanisms underlying epigenetic gene regulation are introduced alongside a brief overview of the state-of-the-art and single-cell methodology to measure these processes. Next, in chapter 2, we review and discuss one prominent feature of spatial genome organization, the lamina-associated domains (LADs). These large genomic regions reside in spatial proximity to the lamina meshwork at the nuclear periphery and form a repressed chromatin state. We specifically focus on the role of LADs in gene regulation during development and disease. In chapter 3, we provide an extensive protocol for scDam&T-seq, a technology that is based on the DamID approach. DamID is one of the primary methods to study LADs, by generating measurements of protein-DNA interactions. The combination of DamID with transcriptomic measurements at single-cell resolution, as obtained with scDam&T-seq, allows for a direct comparison of LAD dynamics with changes in gene expression. We apply the scDam&T-seq technology in chapter 4, in which we jointly study changes in LADs and gene expression during neural development. By performing in utero electroporation of DamID constructs in the developing mouse embryonic cortex, we measure genome-lamina association in combination with transcription during neurogenesis. In parallel, we implement an in vitro neural differentiation system to study the global rewiring of LADs throughout the trajectory at a high temporal resolution. In chapter 5, we present the MAbID technology, a method for combined profiling of multiple histone modifications and chromatin-binding proteins in one sample. After first validating the approach on a wide range of chromatin types, we establish the potential of MAbID in single cells and study changes in combined epigenetic signatures upon X-chromosome inactivation. Moreover, we demonstrate the applicability of the technology in primary tissues by obtaining multifactorial single-cell profiles of mouse bone marrow cells. To integrate single-cell sequencing technology with live-cell imaging, we introduce Waldo as a novel cell-barcoding approach in chapter 6. We technically validate the Waldo barcoding strategy and demonstrate that we can uniquely barcode individual cells to intersect measurements of live-cell imaging with sequencing. Furthermore, we establish that the Waldo approach can be implemented for lineage tracing assays and can generate combined measurements with whole-genome karyotyping and DamID. Finally, in chapter 7, the research in this dissertation is discussed, including the potential underlying molecular mechanisms, opportunities and limitations for the presented technologies and overall future perspectives. Collectively, this research has revolved around the implementation and development of single-cell multimodal sequencing technology, to benefit researchers in fully unravelling the principles governing gene regulation during development and disease.
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