Biology subjects

Johnstone, C. P.

Publications and source records attributed to Johnstone, C. P..

5 recordsLinked to original sources

bioRxiv2025

Gene syntax defines supercoiling-mediated transcriptional feedback

Gene syntax--the order and arrangement of genes and their regulatory elements--shapes the dynamic coordination of both natural and synthetic gene circuits. Transcription at one locus profoundly impacts the transcription of nearby adjacent genes, but the molecular basis of this effect remains poorly understood. Here, using integrated reporter circuits in human cells, we show that the reciprocal effects of transcription and DNA supercoiling, which we term supercoiling-mediated feedback, regulates expression of adjacent genes in a syntax-specific manner. Using a suite of chromatin state assays, we measure syntax-and induction-dependent formation of chromatin structures in human induced pluripotent stem cells. Applying syntax as a design parameter and without altering sequence or copy number, we built compact gene circuits, tuning the expression mean, noise, and stoichiometry across diverse delivery methods and cell types. Integrating supercoiling-mediated feedback into models of gene regulation will expand our understanding of native systems and enhance the design of synthetic gene circuits.

synthetic biology↗

bioRxiv2024

High-resolution profiling reveals coupled transcriptional and translational regulation of transgenes

Concentrations of RNAs and proteins provide important determinants of cell fate. Robust gene circuit design requires an understanding of how the combined actions of individual genetic components influence both mRNA and protein levels. Here, we simultaneously measure mRNA and protein levels in single cells using HCR Flow-FISH for a set of commonly used synthetic promoters. We find that promoters generate differences in both the mRNA abundance and the effective translation rate of these transcripts. Stronger promoters not only transcribe more RNA but also show higher effective translation rates. While the strength of the promoter is largely preserved upon genome integration with identical elements, the choice of polyadenylation signal and coding sequence can generate large differences in the profiles of the mRNAs and proteins. We used long-read direct RNA sequencing to characterize full-length mRNA isoforms and observe remarkable uniformity of mRNA isoforms from the transgenic units. Together, our high-resolution profiling of transgenic mRNAs and proteins offers insight into the impact of common synthetic genetic components on transcriptional and translational mechanisms. By developing a novel framework for quantifying expression profiles of transgenes, we have established a system for comparing native and synthetic gene regulation and for building more robust transgenic systems.

synthetic biology↗

bioRxiv2024

Cells transit through a quiescent-like state to convert to neurons at high rates

While transcription factors (TFs) provide essential cues for directing and redirecting cell fate, TFs alone are insufficient to drive cells to adopt alternative fates. Rather, transcription factors rely on receptive cell states to induce novel identities. Cell state emerges from and is shaped by cellular history and the activity of diverse processes. Here, we define the cellular and molecular properties of a highly receptive state amenable to transcription factor-mediated direct conversion from fibroblasts to induced motor neurons. Using a well-defined model of direct conversion to a post-mitotic fate, we identify the highly proliferative, receptive state that transiently emerges during conversion. Through examining chromatin accessibility, histone marks, and nuclear features, we find that cells reprogram from a state characterized by global reductions in nuclear size and transcriptional activity. Supported by globally increased levels of H3K27me3, cells enter a quiescent-like state of reduced RNA metabolism and elevated expression of REST and p27, markers of quiescent neural stem cells. From this transient state, cells convert to neurons at high rates. Inhibition of Ezh2, the catalytic subunit of PRC2 that deposits H3K27me3, abolishes conversion. Our work offers a roadmap to identify global changes in cellular processes that define cells with different conversion potentials that may generalize to other cell-fate transitions. HighlightsO_LIProliferation drives cells to a compact nuclear state that is receptive to TF-mediated conversion. C_LIO_LIIncreased receptivity to TFs corresponds to reduced nuclear volumes. C_LIO_LIReprogrammable cells display global, genome-wide increases in H3K27me3. C_LIO_LIHigh levels of H3K27me3 support cells transits through a state of altered RNA metabolism. C_LIO_LIInhibition of Ezh2 increases nuclear size, reduces the expression of the quiescence marker p27. C_LIO_LIAcute inhibition of Ezh2 abolishes motor neuron conversion. C_LI One Sentence SummaryCells transit through a quiescent-like state characterized by global reductions in nuclear size and transcriptional activity to convert to neurons at high rates. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=143 SRC="FIGDIR/small/624928v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@ee2127org.highwire.dtl.DTLVardef@1c2a39dorg.highwire.dtl.DTLVardef@183bf46org.highwire.dtl.DTLVardef@82b98d_HPS_FORMAT_FIGEXP M_FIG C_FIG

systems biology↗

bioRxiv2024

Model-guided design of microRNA-based gene circuits supports precise dosage of transgenic cargoes into diverse primary cells

To realize the potential of engineered cells in therapeutic applications, transgenes must be expressed within the window of therapeutic efficacy. Differences in copy number and other sources of extrinsic noise generate variance in transgene expression and limit the performance of synthetic gene circuits. In a therapeutic context, supraphysiological expression of transgenes can compromise engineered phenotypes and lead to toxicity. To ensure a narrow range of transgene expression, we design and characterize Compact microRNA-Mediated Attenuator of Noise and Dosage (ComMAND), a single-transcript, microRNA-based incoherent feedforward loop. We experimentally tune the ComMAND output profile, and we model the system to explore additional tuning strategies. By comparing ComMAND to two-gene implementations, we highlight the precise control afforded by the single-transcript architecture, particularly at relatively low copy numbers. We show that ComMAND tightly regulates transgene expression from lentiviruses and precisely controls expression in primary human T cells, primary rat neurons, primary mouse embryonic fibroblasts, and human induced pluripotent stem cells. Finally, ComMAND effectively sets levels of the clinically relevant transgenes FMRP1 and FXN within a narrow window. Together, ComMAND is a compact tool well-suited to precisely specify expression of therapeutic cargoes.

synthetic biology↗

bioRxiv2022

Supercoiling-mediated feedback rapidly couples and tunes transcription

Transcription induces a wave of DNA supercoiling, altering the binding affinity of RNA polymerases and reshaping the biochemical landscape of gene regulation. As supercoiling rapidly diffuses, transcription dynamically reshapes the regulation of proximal genes, forming a complex feedback loop. The resulting intergene coupling may provide a mechanism to control transcriptional variance in engineered gene networks and explain the behavior of co-localized native circuits. However, a theoretical framework is needed for integrating both biophysical and biochemical transcriptional regulation to investigate the role of supercoiling-mediated feedback within multi-gene systems. Here, we model transcriptional regulation under the influence of supercoiling-mediated polymerase dynamics, allowing us to identify patterns of expression that result from physical intergene coupling and explore integration of this biophysical model with a set of canonical biochemical gene regulatory systems. We find that gene syntax--the relative ordering and orientation of genes--defines the expression profiles, variance, burst dynamics, and intergene correlation of two-gene systems. By applying our model to both a synthetic toggle switch and the endogenous zebrafish segmentation network, we find that supercoiling can enhance or weaken conventional biochemical regulatory strategies such as mRNA- and protein-mediated feedback loops. Together, our results suggest that supercoiling couples behavior between neighboring genes, representing a novel regulatory mechanism. Integrating biophysical regulation into the analysis and design of gene regulation provides a framework for enhanced understanding of native networks and engineering of synthetic gene circuits.

synthetic biology↗