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.

A crucial step towards engineering biological systems is the ability to precisely tune the genetic response to environmental stimuli. In the case of Escherichia coli inducible promoters, our incomplete understanding of the relationship between sequence composition and gene expression hinders our ability to predictably control transcriptional responses. Here, we profile the expression dynamics of 8269 rationally designed, IPTG-inducible promoters that collectively explore the individual and combinatorial effects of RNA polymerase and LacI repressor binding site strengths. We then fit a statistical mechanics model to measured expression that accurately models gene expression and reveals properties of theoretically optimal inducible promoters. Furthermore, we characterize three alternative promoter architectures and show that repositioning binding sites within promoters influences the types of combinatorial effects observed between promoter elements. In total, this approach enables us to deconstruct relationships between inducible promoter elements and discover practical insights for engineering inducible promoters with desirable characteristics.

Remote enhancers are thought to interact with their target promoters via physical proximity, yet the importance of this proximity for enhancer function remains unclear. Although some enhancer-promoter loops are cell-type-specific, others are stable across tissues or even display reduced physical proximity upon activation. A major challenge is that relatively few enhancers are functionally characterized in vivo, making it difficult to draw generalized conclusions about enhancer-promoter looping during developmental gene activation. Here, we investigate the 3D conformation of enhancers during mammalian development by generating high-resolution tissue-resolved contact maps for nearly a thousand mammalian enhancers with known in vivo activities in ten murine embryonic tissues. We performed enhancer knockouts in mice, which validated newly identified enhancer-promoter chromatin interactions. The majority of enhancers display tissue-specific 3D conformations, and both enhancer-promoter and enhancer-enhancer interactions are significantly stronger upon enhancer activation in vivo. Less than 14% of enhancer-promoter interactions form stably across tissues; however, corresponding enhancers still display highly-tissue-specific activities indicating that their activity is uncoupled from a chromatin interaction with promoters. We find that these invariant interactions form in the absence of the enhancer and are mediated by adjacent CTCF binding. We also identified putative in vivo target genes for enhancers linked to congenital malformations, neurodevelopmental disorders, and autism, demonstrating the utility of our dataset for understanding human congenital disorders. Our results highlight the general significance of enhancer-promoter physical proximity for developmental gene activation in mammals.

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A crucial step towards engineering biological systems is the ability to precisely tune the genetic response to environmental stimuli. In the case of Escherichia coli inducible promoters, our incomplete understanding of the relationship between sequence composition and gene expression hinders our ability to predictably control transcriptional responses. Here, we profile the expression dynamics of 8,269 rationally designed IPTG-inducible promoters that collectively explore the individual and combinatorial effects of RNA polymerase and LacI repressor binding site strengths. Using these data, we fit a statistical mechanics model that accurately models gene expression and reveals properties of theoretically optimal inducible promoters. Furthermore, we characterize three novel promoter architectures and show that repositioning binding sites within promoters influences the types of combinatorial effects observed between promoter elements. In total, this approach enables us to deconstruct relationships between inducible promoter elements and discover practical insights for engineering inducible promoters with desirable characteristics.