An Extended Culture System that Supports Human Primordial Germ Cell-like Cell Survival and Initiation of DNA Methylation Erasure.

The development of an in vitro system in which human primordial germ cell-like cells (hPGCLCs) are generated from human pluripotent stem cells (hPSCs) has been invaluable to further our understanding of human primordial germ cell (hPGC) specification. However, the means to evaluate the next fundamental steps in germ cell development have not been well established. In this study we describe a two dimensional extended culture system that promotes proliferation of specified hPGCLCs, without reversion to a pluripotent state. We demonstrate that hPGCLCs in extended culture undergo partial epigenetic reprogramming, mirroring events described in hPGCs in vivo, including a genome-wide reduction in DNA methylation and maintenance of depleted H3K9me2. This extended culture system provides a new approach for expanding the number of hPGCLCs for downstream technologies, including transplantation, molecular screening, or possibly the differentiation of hPGCLCs into gametes by in vitro gametogenesis

Strand-specific single-cell methylomics reveals distinct modes of DNA demethylation dynamics during early mammalian development

DNA methylation (5mC) is central to cellular identity. 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. While active and passive modes of demethylation have both been suggested to play a role in this process, the relative contribution of these two mechanisms to 5mC erasure remains unclear. Here, we report a single-cell method (scMspJI-seq) that enables strand-specific quantification of 5mC, allowing us to systematically probe the dynamics of global demethylation. When applied to mouse embryonic stem cells, we identified substantial cell-to-cell strand-specific 5mC heterogeneity, with a small group of cells displaying asymmetric levels of 5mCpG between the two DNA strands of a chromosome suggesting loss of maintenance methylation. Next, in preimplantation mouse embryos, we discovered that methylation maintenance is active till the 16-cell stage followed by passive demethylation in a fraction of cells within the early blastocyst at the 32-cell stage of development. Finally, human preimplantation embryos qualitatively show temporally delayed yet similar demethylation dynamics as mouse embryos. Collectively, these results demonstrate that scMspJI-seq is a sensitive and cost-effective method to map the strand-specific genome-wide patterns of 5mC in single cells. Erasure of DNA methylation from the parental genomes is critical to reset the methylome of differentiated gametes to pluripotent cells in the blastocyst. Here, the authors present a high-throughput single-cell method that enables strand-specific quantification of DNA methylation and identify distinct modes of DNA demethylation dynamics during early mammalian development

Developing single cell multiomics technologies to understand mammalian development

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

Control over single-cell distribution of G1 lengths by WNT governs pluripotency

The link between single-cell variation and population-level fate choices lacks a mechanistic explanation despite extensive observations of gene expression and epigenetic variation among individual cells. Here, we found that single human embryonic stem cells (hESCs) have different and biased differentiation potentials toward either neuroectoderm or mesendoderm depending on their G1 lengths before the onset of differentiation. Single-cell variation in G1 length operates in a dynamic equilibrium that establishes a G1 length probability distribution for a population of hESCs and predicts differentiation outcome toward neuroectoderm or mesendoderm lineages. Although sister stem cells generally share G1 lengths, a variable proportion of cells have asymmetric G1 lengths, which maintains the population dispersion. Environmental Wingless-INT (WNT) levels can control the G1 length distribution, apparently as a means of priming the fate of hESC populations once they undergo differentiation. As a downstream mechanism, global 5-hydroxymethylcytosine levels are regulated by G1 length and thereby link G1 length to differentiation outcomes of hESCs. Overall, our findings suggest that intrapopulation heterogeneity in G1 length underlies the pluripotent differentiation potential of stem cell populations.11Ysciescopu

Simultaneous quantification of protein-DNA contacts and transcriptomes in single cells.

Protein-DNA interactions are critical to the regulation of gene expression, but it remains challenging to define how cell-to-cell heterogeneity in protein-DNA binding influences gene expression variability. Here we report a method for the simultaneous quantification of protein-DNA contacts by combining single-cell DNA adenine methyltransferase identification (DamID) with messenger RNA sequencing of the same cell (scDam&T-seq). We apply scDam&T-seq to reveal how genome-lamina contacts or chromatin accessibility correlate with gene expression in individual cells. Furthermore, we provide single-cell genome-wide interaction data on a polycomb-group protein, RING1B, and the associated transcriptome. Our results show that scDam&T-seq is sensitive enough to distinguish mouse embryonic stem cells cultured under different conditions and their different chromatin landscapes. Our method will enable the analysis of protein-mediated mechanisms that regulate cell-type-specific transcriptional programs in heterogeneous tissues

Simultaneous quantification of protein-DNA interactions and transcriptomes in single cells with scDam&T-seq.

Protein-DNA interactions are essential for establishing cell type-specific chromatin architecture and gene expression. We recently developed scDam&T-seq, a multi-omics method that can simultaneously quantify protein-DNA interactions and the transcriptome in single cells. The method effectively combines two existing methods: DNA adenine methyltransferase identification (DamID) and CEL-Seq2. DamID works through the tethering of a protein of interest (POI) to the Escherichia coli DNA adenine methyltransferase (Dam). Upon expression of this fusion protein, DNA in proximity to the POI is methylated by Dam and can be selectively digested and amplified. CEL-Seq2, in contrast, makes use of poly-dT primers to reverse transcribe mRNA, followed by linear amplification through in vitro transcription. scDam&T-seq is the first technique capable of providing a combined readout of protein-DNA contact and transcription from single-cell samples. Once suitable cell lines have been established, the protocol can be completed in 5 d, with a throughput of hundreds to thousands of cells. The processing of raw sequencing data takes an additional 1-2 d. Our method can be used to understand the transcriptional changes a cell undergoes upon the DNA binding of a POI. It can be performed in any laboratory with access to FACS, robotic and high-throughput-sequencing facilities

An Extended Culture System that Supports Human Primordial Germ Cell-like Cell Survival and Initiation of DNA Methylation Erasure.

The development of an in vitro system in which human primordial germ cell-like cells (hPGCLCs) are generated from human pluripotent stem cells (hPSCs) has been invaluable to further our understanding of human primordial germ cell (hPGC) specification. However, the means to evaluate the next fundamental steps in germ cell development have not been well established. In this study we describe a two dimensional extended culture system that promotes proliferation of specified hPGCLCs, without reversion to a pluripotent state. We demonstrate that hPGCLCs in extended culture undergo partial epigenetic reprogramming, mirroring events described in hPGCs in vivo, including a genome-wide reduction in DNA methylation and maintenance of depleted H3K9me2. This extended culture system provides a new approach for expanding the number of hPGCLCs for downstream technologies, including transplantation, molecular screening, or possibly the differentiation of hPGCLCs into gametes by in vitro gametogenesis

Simultaneous quantification of protein-DNA interactions and transcriptomes in single cells with scDam&T-seq

Protein-DNA interactions are essential for establishing cell type-specific chromatin architecture and gene expression. We recently developed scDam&T-seq, a multi-omics method that can simultaneously quantify protein-DNA interactions and the transcriptome in single cells. The method effectively combines two existing methods: DNA adenine methyltransferase identification (DamID) and CEL-Seq2. DamID works through the tethering of a protein of interest (POI) to the Escherichia coli DNA adenine methyltransferase (Dam). Upon expression of this fusion protein, DNA in proximity to the POI is methylated by Dam and can be selectively digested and amplified. CEL-Seq2, in contrast, makes use of poly-dT primers to reverse transcribe mRNA, followed by linear amplification through in vitro transcription. scDam&T-seq is the first technique capable of providing a combined readout of protein-DNA contact and transcription from single-cell samples. Once suitable cell lines have been established, the protocol can be completed in 5 d, with a throughput of hundreds to thousands of cells. The processing of raw sequencing data takes an additional 1-2 d. Our method can be used to understand the transcriptional changes a cell undergoes upon the DNA binding of a POI. It can be performed in any laboratory with access to FACS, robotic and high-throughput-sequencing facilities