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

Bioinformatics, Год журнала: 2021, Номер 37(16), С. 2485 - 2487

Опубликована: Янв. 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.

Язык: Английский

---

Single-cell nascent RNA sequencing unveils coordinated global transcription

Nature, Год журнала: 2024, Номер 631(8019), С. 216 - 223

Опубликована: Июнь 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

Язык: Английский

---

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

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2025, Номер unknown

Опубликована: Янв. 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.

Язык: Английский

---

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

Nature Structural & Molecular Biology, Год журнала: 2021, Номер 28(10), С. 811 - 824

Опубликована: Окт. 1, 2021

Язык: Английский

---

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

Science Advances, Год журнала: 2023, Номер 9(32)

Опубликована: Авг. 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

Язык: Английский

---

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

Nature Cell Biology, Год журнала: 2024, Номер unknown

Опубликована: Сен. 11, 2024

Язык: Английский

---

Priestia megaterium cells are primed for surviving lethal doses of antibiotics and chemical stress

Communications Biology, Год журнала: 2025, Номер 8(1)

Опубликована: Фев. 8, 2025

Antibiotic resistant infections kill millions worldwide yearly. However, a key factor in recurrent is antibiotic persisters. Persisters are not inherently antibiotic-resistant but can withstand exposure by entering non-dividing state. This tolerance often results prolonged usage, increasing the likelihood of developing strains. Here, we show existence "primed cells" Gram-positive bacterium Priestia megaterium, formerly known as Bacillus megaterium. These cells pre-adapted to become persisters prior lethal stress. Remarkably, this prepared state passed down through multiple generations via epigenetic memory, enhancing survival against antibiotics and other chemical Previously, two distinct types were proposed: Type I II, formed during stationary log phases, respectively. our findings reveal that primed contribute an increase transition with no evidence supporting phenotypes between II

Язык: Английский

---

Identification of molecular determinants of gene-specific bursting patterns by high-throughput imaging screens

Molecular Cell, Год журнала: 2025, Номер unknown

Опубликована: Фев. 1, 2025

Язык: Английский

---

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

The Journal of Cell Biology, Год журнала: 2021, Номер 221(2)

Опубликована: Дек. 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.

Язык: Английский

---

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

Nucleic Acids Research, Год журнала: 2022, Номер 51(1), С. 68 - 83

Опубликована: Дек. 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.

Язык: Английский

---