Tuning Methylation-Dependent Silencing Dynamics by Synthetic Modulation of CpG Density.

Methylation of cytosines in CG dinucleotides (CpGs) within promoters has been shown to lead to gene silencing in mammals in natural contexts. Recently, engineered recruitment of methyltransferases (DNMTs) at specific loci was shown to be sufficient to silence synthetic and endogenous gene expression through this mechanism. A critical parameter for DNA methylation-based silencing is the distribution of CpGs within the target promoter. However, how the number or density of CpGs in the target promoter affects the dynamics of silencing by DNMT recruitment has remained unclear. Here, we constructed a library of promoters with systematically varying CpG content, and analyzed the rate of silencing in response to recruitment of DNMT. We observed a tight correlation between silencing rate and CpG content. Further, methylation-specific analysis revealed a constant accumulation rate of methylation at the promoter after DNMT recruitment. We identified a single CpG site between TATA box and transcription start site (TSS) that accounted for a substantial part of the difference in silencing rates between promoters with differing CpG content, indicating that certain residues play disproportionate roles in controlling silencing. Together, these results provide a library of promoters for synthetic epigenetic and gene regulation applications, as well as insights into the regulatory link between CpG content and silencing rate.

Tuning methylation-dependent silencing dynamics by synthetic modulation of CpG density.

Methylation of cytosines in CG dinucleotides (CpGs) within promoters has been shown to lead to gene silencing in mammals in natural contexts. Recently, engineered recruitment of methyltransferases (DNMTs) at specific loci was shown to be sufficient to silence synthetic and endogenous gene expression through this mechanism. A critical parameter for DNA methylation-based silencing is the distribution of CpGs within the target promoter. However, how the number or density of CpGs in the target promoter affects the dynamics of silencing by DNMT recruitment has remained unclear. Here we constructed a library of promoters with systematically varying CpG content, and analyzed the rate of silencing in response to recruitment of DNMT. We observed a tight correlation between silencing rate and CpG content. Further, methylation-specific analysis revealed a constant accumulation rate of methylation at the promoter after DNMT recruitment. We identified a single CpG site between TATA box and transcription start site (TSS) that accounted for a substantial part of the difference in silencing rates between promoters with differing CpG content, indicating that certain residues play disproportionate roles in controlling silencing. Together, these results provide a library of promoters for synthetic epigenetic and gene regulation applications, as well as insights into the regulatory link between CpG content and silencing rate.

Synthetic mammalian signaling circuits for robust cell population control.

In multicellular organisms, cells actively sense and control their own population density. Synthetic mammalian quorum-sensing circuits could provide insight into principles of population control and extend cell therapies. However, a key challenge is reducing their inherent sensitivity to "cheater" mutations that evade control. Here, we repurposed the plant hormone auxin to enable orthogonal mammalian cell-cell communication and quorum sensing. We designed a paradoxical population control circuit, termed "Paradaux," in which auxin stimulates and inhibits net cell growth at different concentrations. This circuit limited population size over extended timescales of up to 42 days of continuous culture. By contrast, when operating in a non-paradoxical regime, population control became more susceptible to mutational escape. These results establish auxin as a versatile "private" communication system and demonstrate that paradoxical circuit architectures can provide robust population control.

Imaging cell lineage with a synthetic digital recording system.

During multicellular development, spatial position and lineage history play powerful roles in controlling cell fate decisions. Using a serine integrase-based recording system, we engineered cells to record lineage information in a format that can be read out in situ. The system, termed integrase-editable memory by engineered mutagenesis with optical in situ readout (intMEMOIR), allowed in situ reconstruction of lineage relationships in cultured mouse cells and flies. intMEMOIR uses an array of independent three-state genetic memory elements that can recombine stochastically and irreversibly, allowing up to 59,049 distinct digital states. It reconstructed lineage trees in stem cells and enabled simultaneous analysis of single-cell clonal history, spatial position, and gene expression in Drosophila brain sections. These results establish a foundation for microscopy-readable lineage recording and analysis in diverse systems.

Publisher Correction: In situ readout of DNA barcodes and single base edits facilitated by in vitro transcription.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

In situ readout of DNA barcodes and single base edits facilitated by in vitro transcription.

Molecular barcoding technologies that uniquely identify single cells are hampered by limitations in barcode measurement. Readout by sequencing does not preserve the spatial organization of cells in tissues, whereas imaging methods preserve spatial structure but are less sensitive to barcode sequence. Here we introduce a system for image-based readout of short (20-base-pair) DNA barcodes. In this system, called Zombie, phage RNA polymerases transcribe engineered barcodes in fixed cells. The resulting RNA is subsequently detected by fluorescent in situ hybridization. Using competing match and mismatch probes, Zombie can accurately discriminate single-nucleotide differences in the barcodes. This method allows in situ readout of dense combinatorial barcode libraries and single-base mutations produced by CRISPR base editors without requiring barcode expression in live cells. Zombie functions across diverse contexts, including cell culture, chick embryos and adult mouse brain tissue. The ability to sensitively read out compact and diverse DNA barcodes by imaging will facilitate a broad range of barcoding and genomic recording strategies.

Synthetic recording and in situ readout of lineage information in single cells.

Reconstructing the lineage relationships and dynamic event histories of individual cells within their native spatial context is a long-standing challenge in biology. Many biological processes of interest occur in optically opaque or physically inaccessible contexts, necessitating approaches other than direct imaging. Here we describe a synthetic system that enables cells to record lineage information and event histories in the genome in a format that can be subsequently read out of single cells in situ. This system, termed memory by engineered mutagenesis with optical in situ readout (MEMOIR), is based on a set of barcoded recording elements termed scratchpads. The state of a given scratchpad can be irreversibly altered by CRISPR/Cas9-based targeted mutagenesis, and later read out in single cells through multiplexed single-molecule RNA fluorescence hybridization (smFISH). Using MEMOIR as a proof of principle, we engineered mouse embryonic stem cells to contain multiple scratchpads and other recording components. In these cells, scratchpads were altered in a progressive and stochastic fashion as the cells proliferated. Analysis of the final states of scratchpads in single cells in situ enabled reconstruction of lineage information from cell colonies. Combining analysis of endogenous gene expression with lineage reconstruction in the same cells further allowed inference of the dynamic rates at which embryonic stem cells switch between two gene expression states. Finally, using simulations, we show how parallel MEMOIR systems operating in the same cell could enable recording and readout of dynamic cellular event histories. MEMOIR thus provides a versatile platform for information recording and in situ, single-cell readout across diverse biological systems.

Pydna: a simulation and documentation tool for DNA assembly strategies using python.

BACKGROUND: Recent advances in synthetic biology have provided tools to efficiently construct complex DNA molecules which are an important part of many molecular biology and biotechnology projects. The planning of such constructs has traditionally been done manually using a DNA sequence editor which becomes error-prone as scale and complexity of the construction increase. A human-readable formal description of cloning and assembly strategies, which also allows for automatic computer simulation and verification, would therefore be a valuable tool. RESULTS: We have developed pydna, an extensible, free and open source Python library for simulating basic molecular biology DNA unit operations such as restriction digestion, ligation, PCR, primer design, Gibson assembly and homologous recombination. A cloning strategy expressed as a pydna script provides a description that is complete, unambiguous and stable. Execution of the script automatically yields the sequence of the final molecule(s) and that of any intermediate constructs. Pydna has been designed to be understandable for biologists with limited programming skills by providing interfaces that are semantically similar to the description of molecular biology unit operations found in literature. CONCLUSIONS: Pydna simplifies both the planning and sharing of cloning strategies and is especially useful for complex or combinatorial DNA molecule construction. An important difference compared to existing tools with similar goals is the use of Python instead of a specifically constructed language, providing a simulation environment that is more flexible and extensible by the user.

HIF-1 and SKN-1 coordinate the transcriptional response to hydrogen sulfide in Caenorhabditis elegans.

Hydrogen sulfide (H2S) has dramatic physiological effects on animals that are associated with improved survival. C. elegans grown in H2S are long-lived and thermotolerant. To identify mechanisms by which adaptation to H2S effects physiological functions, we have measured transcriptional responses to H2S exposure. Using microarray analysis we observe rapid changes in the abundance of specific mRNAs. The number and magnitude of transcriptional changes increased with the duration of H2S exposure. Functional annotation suggests that genes associated with protein homeostasis are upregulated upon prolonged exposure to H2S. Previous work has shown that the hypoxia-inducible transcription factor, HIF-1, is required for survival in H2S. In fact, we show that hif-1 is required for most, if not all, early transcriptional changes in H2S. Moreover, our data demonstrate that SKN-1, the C. elegans homologue of NRF2, also contributes to H2S-dependent changes in transcription. We show that these results are functionally important, as skn-1 is essential to survive exposure to H2S. Our results suggest a model in which HIF-1 and SKN-1 coordinate a broad transcriptional response to H2S that culminates in a global reorganization of protein homeostasis networks.

The response of Caenorhabditis elegans to hydrogen sulfide and hydrogen cyanide.

Hydrogen sulfide (H2S), an endogenously produced small molecule, protects animals from various stresses. Recent studies demonstrate that animals exposed to H2S are long lived, resistant to hypoxia, and resistant to ischemia-reperfusion injury. We performed a forward genetic screen to gain insights into the molecular mechanisms Caenorhabditis elegans uses to appropriately respond to H2S. At least two distinct pathways appear to be important for this response, including the H2S-oxidation pathway and the hydrogen cyanide (HCN)-assimilation pathway. The H2S-oxidation pathway requires two distinct enzymes important for the oxidation of H2S: the sulfide:quinone reductase sqrd-1 and the dioxygenase ethe-1. The HCN-assimilation pathway requires the cysteine synthase homologs cysl-1 and cysl-2. A low dose of either H2S or HCN can activate hypoxia-inducible factor 1 (HIF-1), which is required for C. elegans to respond to either gas. sqrd-1 and cysl-2 represent the entry points in the H2S-oxidation and HCN-assimilation pathways, respectively, and expression of both of these enzymes is highly induced by HIF-1 in response to both H2S and HCN. In addition to their role in appropriately responding to H2S and HCN, we found that cysl-1 and cysl-2 are both essential mediators of innate immunity against fast paralytic killing by Pseudomonas. Furthermore, in agreement with these data, we showed that growing worms in the presence of H2S is sufficient to confer resistance to Pseudomonas fast paralytic killing. Our results suggest the hypoxia-independent hif-1 response in C. elegans evolved to respond to the naturally occurring small molecules H2S and HCN.

Hydrogen sulfide increases hypoxia-inducible factor-1 activity independently of von Hippel-Lindau tumor suppressor-1 in C. elegans.

Rapid alteration of gene expression in response to environmental changes is essential for normal development and behavior. The transcription factor hypoxia-inducible factor (HIF)-1 is well known to respond to alterations in oxygen availability. In nature, low oxygen environments are often found to contain high levels of hydrogen sulfide (H(2)S). Here, we show that Caenorhabditis elegans can have mutually exclusive responses to H(2)S and hypoxia, both involving HIF-1. Specifically, H(2)S results in HIF-1 activity throughout the hypodermis, whereas hypoxia causes HIF-1 activity in the gut as judged by a reporter for HIF-1 activity. C. elegans require hif-1 to survive in room air containing trace amounts of H(2)S. Exposure to H(2)S results in HIF-1 nuclear localization and transcription of HIF-1 targets. The effects of H(2)S on HIF-1 reporter activity are independent of von Hippel-Lindau tumor suppressor (VHL)-1, whereas VHL-1 is required for hypoxic regulation of HIF-1 reporter activity. Because H(2)S is naturally produced by animal cells, our results suggest that endogenous H(2)S may influence HIF-1 activity.