Nebulosa recovers single-cell gene expression signals by kernel density estimation

Bioinformatics, Journal Year: 2021, Volume and Issue: 37(16), P. 2485 - 2487

Published: Jan. 7, 2021

Data sparsity in single-cell experiments prevents an accurate assessment of gene expression when visualized a low-dimensional space. Here, we introduce Nebulosa, R package that uses weighted kernel density estimation to recover signals lost through drop-out or low expression.Nebulosa can be easily installed from www.github.com/powellgenomicslab/Nebulosa.Supplementary data are available at Bioinformatics online.

Language: Английский

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Single-cell nascent RNA sequencing unveils coordinated global transcription

Nature, Journal Year: 2024, Volume and Issue: 631(8019), P. 216 - 223

Published: June 5, 2024

Abstract Transcription is the primary regulatory step in gene expression. Divergent transcription initiation from promoters and enhancers produces stable RNAs genes unstable 1,2 . Nascent RNA capture sequencing assays simultaneously measure enhancer activity cell populations 3 However, fundamental questions about temporal regulation of enhancer–gene coordination remain unanswered, primarily because absence a single-cell perspective on active transcription. In this study, we present scGRO–seq—a new nascent assay that uses click chemistry—and unveil coordinated throughout genome. We demonstrate episodic nature co-transcription functionally related genes. scGRO–seq can estimate burst size frequency by directly quantifying transcribing polymerases individual cells leverage replication-dependent non-polyadenylated histone to elucidate cycle dynamics. The single-nucleotide spatial resolution enables identification networks Our results suggest bursting at super-enhancers precedes associated By imparting insights into dynamic global origin propagation signals, ability investigate mechanisms role

Language: Английский

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PRC1 drives Polycomb-mediated gene repression by controlling transcription initiation and burst frequency

Nature Structural & Molecular Biology, Journal Year: 2021, Volume and Issue: 28(10), P. 811 - 824

Published: Oct. 1, 2021

Language: Английский

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The minimal intrinsic stochasticity of constitutively expressed eukaryotic genes is sub-Poissonian

Science Advances, Journal Year: 2023, Volume and Issue: 9(32)

Published: Aug. 9, 2023

Gene expression inherently gives rise to stochastic variation ("noise") in the production of gene products. Minimizing noise is crucial for ensuring reliable cellular functions. However, cannot be suppressed below a certain intrinsic limit. For constitutively expressed genes, this limit typically assumed Poissonian noise, wherein variance mRNA numbers equal their mean. Here, we demonstrate that several cell division genes fission yeast exhibit variances significantly The reduced can explained by model incorporating multiple transcription and degradation steps. Notably, sub-Poissonian regime, distinct from or super-Poissonian regimes, cytoplasmic effectively through higher export rate. Our findings redefine lower eukaryotic uncover molecular requirements achieving ultralow which expected important vital

Language: Английский

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The Polycomb system sustains promoters in a deep OFF state by limiting pre-initiation complex formation to counteract transcription

Nature Cell Biology, Journal Year: 2024, Volume and Issue: unknown

Published: Sept. 11, 2024

Language: Английский

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Real-time visualization of reconstituted transcription reveals RNA polymerase II activation mechanisms at single promoters

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2025, Volume and Issue: unknown

Published: Jan. 6, 2025

RNA polymerase II (RNAPII) is regulated by sequence-specific transcription factors (TFs) and the pre-initiation complex (PIC): TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, Mediator. TFs Mediator contain intrinsically-disordered regions (IDRs) form phase-separated condensates, but how IDRs control RNAPII function remains poorly understood. Using purified PIC factors, we developed a Real-time In-vitro Fluorescence Transcription assay (RIFT) for second-by-second visualization of at hundreds promoters simultaneously. We show rapid activation IDR-dependent, without condensate formation. For example, MED1-IDR can functionally replace native TF, activating with similar (not identical) kinetics; however, squelches as condensate, activates single-protein. cooperatively activate bursting re-initiation surprisingly, drive TF-promoter recruitment, TF-DNA binding. Collectively, RIFT addressed questions largely intractable cell-based methods, yielding mechanistic insights about IDRs, enhancer-promoter communication, that complement live-cell imaging data.

Language: Английский

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Live imaging of transcription sites using an elongating RNA polymerase II–specific probe

The Journal of Cell Biology, Journal Year: 2021, Volume and Issue: 221(2)

Published: Dec. 2, 2021

In eukaryotic nuclei, most genes are transcribed by RNA polymerase II (RNAP2), whose regulation is a key to understanding the genome and cell function. RNAP2 has long heptapeptide repeat (Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7), Ser2 phosphorylated on an elongation form. To detect phosphorylation (RNAP2 Ser2ph) in living cells, we developed genetically encoded modification-specific intracellular antibody (mintbody) probe. The Ser2ph-mintbody exhibited numerous foci, possibly representing transcription “factories,” foci were diminished during mitosis kinase inhibitor. An vitro binding assay using phosphopeptides confirmed mintbody’s specificity. colocalized with proteins associated elongating compared factors involved initiation. These results support view that mintbody localization represents sites of Ser2ph cells. showed constrained diffusional motion like chromatin, but they more mobile than DNA replication domains p300-enriched suggesting complexes separated from confined chromatin domains.

Language: Английский

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Genome-wide inference reveals that feedback regulations constrain promoter-dependent transcriptional burst kinetics

Nucleic Acids Research, Journal Year: 2022, Volume and Issue: 51(1), P. 68 - 83

Published: Dec. 30, 2022

Gene expression in mammalian cells is highly variable and episodic, resulting a series of discontinuous bursts mRNAs. A challenge to understand how static promoter architecture dynamic feedback regulations dictate bursting on genome-wide scale. Although single-cell RNA sequencing (scRNA-seq) provides an opportunity address this challenge, effective analytical methods are scarce. We developed interpretable scalable inference framework, which combined experimental data with mechanistic model infer transcriptional burst kinetics (sizes frequencies) regulations. Applying framework scRNA-seq generated from embryonic mouse fibroblast cells, we found Simpson's paradoxes, i.e. exhibit different characteristics two cases without distinguishing also showed that feedbacks differently modulate frequencies sizes conceal the effects transcription start site distributions kinetics. Notably, only presence positive feedback, TATA genes expressed high enhancer-promoter interactions mainly frequencies. The method provided flexible efficient way investigate obtained results would be helpful for understanding cell development fate decision.

Language: Английский

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Probing transient memory of cellular states using single-cell lineages

Frontiers in Microbiology, Journal Year: 2023, Volume and Issue: 13

Published: Feb. 7, 2023

The inherent stochasticity in the gene product levels can drive single cells within an isoclonal population to different phenotypic states. dynamic nature of this intercellular variation, where individual transition between states over time, makes it a particularly hard phenomenon characterize. We reviewed recent progress leveraging classical Luria-Delbrück experiment infer transient heritability cellular Similar original experiment, were first grown into cell colonies, and then, fraction residing was assayed for each colony. discuss modeling approaches capturing state transitions growing highlight formulas that identify kinetics switching from extent colony-to-colony fluctuations. utility method identifying multi-generational memory both expression is illustrated across diverse biological systems cancer drug resistance, reactivation human viruses, immune responses. In summary, fluctuation-based methodology provides powerful approach elucidating cell-state

Language: Английский

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Inferring transcriptional bursting kinetics from single-cell snapshot data using a generalized telegraph model

Royal Society Open Science, Journal Year: 2023, Volume and Issue: 10(4)

Published: April 1, 2023

Gene expression has inherent stochasticity resulting from transcription's burst manners. Single-cell snapshot data can be exploited to rigorously infer transcriptional kinetics, using mathematical models as blueprints. The classical telegraph model (CTM) been widely used explain bursting with Markovian assumptions. However, growing evidence suggests that the gene-state dwell times are generally non-exponential, switching is a multi-step process in organisms. Therefore, interpretable non-Markovian and efficient statistical inference methods urgently required investigating kinetics. We develop an tractable model, generalized (GTM), characterize allows arbitrary dwell-time distributions, rather than exponential incorporated into ON OFF process. Based on GTM, we propose method for kinetics approximate Bayesian computation framework. This demonstrates scalable estimation of frequency size synthetic data. Further, application genome-wide mouse embryonic fibroblasts reveals GTM would estimate lower higher those estimated by CTM. In conclusion, corresponding effective tools dynamic static single-cell

Language: Английский

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