UC Santa Barbara

Next generation sequencing has been key in unlocking the ability to detect thousands of features at single base resolution in thousands of single cells simultaneously in a single experiment. In addition to the DNA bases present, DNA sequencing libraries can contain information on RNA transcripts as well as epigenetic features central to cellular identity like DNA methylation (5mC), DNA hydroxymethylation (5hmC), and DNA accessibility. This dissertation develops multiple single cell sequencing methodologies to explore these epigenetic features simultaneously from the same cell. We first developed scMspJI-seq to detect 5mC from single cells. To investigate 5mC, DNA accessibility, and the transcriptome from the same cell we built upon scMspJI-seq to create scMAT-seq. Then by incorporating 5hmC detection into this measurement, we gained the ability to detection of all 4 features (scMATH-seq) or a subset of them (scMTH-seq). Finally, by combining these technologies with more traditional techniques we developed scDyad&T-seq to detect the transcriptome and the presence of 5mC on both strands of the same piece of DNA. Using these techniques, 4 key areas of human development were investigated: 1. pre-implantation development, 2. gastrulation and primordial germ cell (PGC) specification, 3. PGC maturation, and 4. stem cell pluripotency.Pre-implantation development: The global erasure of 5mC from the parental genomes during preimplantation mammalian development is critical to reset the methylome of gametes to the cells in the blastocyst, but how this process occurs remains unclear. By applying scMspJI-seq, we discover that methylation maintenance is active till the 16-cell stage followed by passive demethylation in a fraction of cells within the early mouse blastocyst. In human embryos we find slightly delayed but similar demethylation dynamics as was found in mice. Gastrulation and primordial germ cell specification: Human gastrulation is marked by dynamic changes in cell states that are difficult to isolate at high purity, thereby making it challenging to map how epigenetic reprogramming impacts gene expression and cellular phenotypes. Applying scMAT-seq to 3D human gastruloids, we characterized the epigenetic landscape of major cell types corresponding to the germ layers and human primordial germ cell-like cells (hPGCLC). Here we find hPGCLCs are specified from progenitors which emerge from epiblast cells and show transient characteristics of both amniotic- and mesoderm-like cells. Finally, we find that during gastrulation DNA accessibility is tightly correlated to both upregulated and downregulated genes, while reorganization of gene body DNA methylation is strongly related to only genes that get downregulated. Primordial germ cell maturation: PGC maturation is marked by global erasure of 5mC followed by transient high levels of 5hmC. Extended culture systems can achieve passive demethylation in a subset of hPGCLCs, but what initiates this heterogenous process is unknown. By applying scMTH-seq to hPGCLCs in extended culture we observe that DND1 and SOX15 likely play a role in the initial phase of passive demethylation experienced by hPGCLCs. Additionally, we find that the hPGCLCs in this system stall in their maturation and do not accumulate high levels of 5hmC. Stem cell pluripotency: Genome wide erasure of 5mC is associated with the acquisition of pluripotency. By applying scDyad&T-seq to different time points of mouse embryonic stem cells transitioning from a primed to a naïve state of pluripotency, we observe extreme demethylation dominated by passive processes and discover this process is highly heterogenous and delayed in some cells. By connecting RNA expression from the same cells, we detect a small set of genes directly linked to 5mC levels during this transition. Finally, we determine that regions of the genome which escape 5mC reprograming do so by retaining high levels of 5mC maintenance and are associated with specific histone modifications.