Chromatin endogenous cleavage provides a global view of yeast RNA polymerase II transcription kinetics DOI Creative Commons
Jake VanBelzen, Bennet Sakelaris,

Donna Garvey Brickner

et al.

eLife, Journal Year: 2024, Volume and Issue: 13

Published: Nov. 28, 2024

Chromatin immunoprecipitation (ChIP-seq) is the most common approach to observe global binding of proteins DNA in vivo. The occupancy transcription factors (TFs) from ChIP-seq agrees well with an alternative method, chromatin endogenous cleavage (ChEC-seq2). However, and ChEC-seq2 reveal strikingly different patterns enrichment yeast RNA polymerase II (RNAPII). We hypothesized that this reflects distinct populations RNAPII, some which are captured by ChEC-seq2. RNAPII association enhancers promoters - predicted biochemical studies detected but not ChIP-seq. Enhancer/promoter-bound correlates levels matches based on published rates enhancer recruitment, preinitiation assembly, initiation, elongation, termination. allowed us develop a stochastic model for kinetics both data changes upon chemical-genetic perturbations transcription. Finally, kinetic modeling suggests mutation Gcn4 factor blocks interaction NPC destabilizes promoter-associated without altering its recruitment enhancer.

Language: Английский

Time will tell: comparing timescales to gain insight into transcriptional bursting DOI Creative Commons
Joseph V.W. Meeussen, Tineke L. Lenstra

Trends in Genetics, Journal Year: 2024, Volume and Issue: 40(2), P. 160 - 174

Published: Jan. 12, 2024

Recent imaging studies have captured the dynamics of regulatory events transcription inside living cells. These include factor (TF) DNA binding, chromatin remodeling and modification, enhancer-promoter (E-P) proximity, cluster formation, preinitiation complex (PIC) assembly. Together, these molecular culminate in stochastic bursts RNA synthesis, but their kinetic relationship remains largely unclear. In this review, we compare timescales upstream steps (input) with kinetics transcriptional bursting (output) to generate mechanistic models single We highlight open questions potential technical advances guide future endeavors toward a quantitative understanding regulation.

Language: Английский

Citations

24
Emerging insights into transcriptional condensates DOI Creative Commons
Kwangmin Ryu, Gunhee Park,

Won‐Ki Cho

et al.

Experimental & Molecular Medicine, Journal Year: 2024, Volume and Issue: 56(4), P. 820 - 826

Published: April 25, 2024

Abstract Eukaryotic transcription, a fundamental process that governs cell-specific gene expression, has long been the subject of extensive investigations in fields molecular biology, biochemistry, and structural biology. Recent advances microscopy techniques have led to fascinating concept known as “transcriptional condensates.” These dynamic assemblies are result phenomenon called liquid‒liquid phase separation, which is driven by multivalent interactions between constituent proteins cells. The essential associated with transcription concentrated transcriptional condensates. studies shed light on temporal dynamics condensates their potential role enhancing efficiency transcription. In this article, we explore properties condensates, investigate how they evolve over time, evaluate significant impact Furthermore, highlight innovative allow us manipulate these thus demonstrating responsiveness cellular signals connection bursting. As our understanding continues grow, poised revolutionize eukaryotic regulation.

Language: Английский

Citations

12
Correlative single molecule lattice light sheet imaging reveals the dynamic relationship between nucleosomes and the local chromatin environment DOI Creative Commons
Timothy A. Daugird, Yu Shi, Katie L. Holland

et al.

Nature Communications, Journal Year: 2024, Volume and Issue: 15(1)

Published: May 16, 2024

Abstract In the nucleus, biological processes are driven by proteins that diffuse through and bind to a meshwork of nucleic acid polymers. To better understand this interplay, we present an imaging platform simultaneously visualize single protein dynamics together with local chromatin environment in live cells. Together super-resolution imaging, new fluorescent probes, biophysical modeling, demonstrate nucleosomes display differential diffusion packing arrangements as density increases whereas viscoelastic properties accessibility interchromatin space remain constant. Perturbing nuclear functions impacts nucleosome diffusive manner is dependent both on relative location within nucleus. Our results support model wherein transcription locally stabilizes while allowing for free exchange proteins. Additionally, they reveal heterogeneity arises from active passive highlight need account different organizational principles when modeling environments.

Language: Английский

Citations

12
Transcription-coupled changes in genomic region proximities during transcriptional bursting DOI Creative Commons
Hiroaki Ohishi, Soya Shinkai, Hitoshi Owada

et al.

Science Advances, Journal Year: 2024, Volume and Issue: 10(49)

Published: Dec. 6, 2024

The orchestration of our genes heavily relies on coordinated communication between enhancers and promoters, yet the mechanisms behind this dynamic interplay during active transcription remain unclear. Here, we investigated enhancer-promoter (E-P) interactions in relation to transcriptional bursting mouse embryonic stem cells using sequential DNA/RNA/immunofluorescence–fluorescence situ hybridization analyses. Our data reveal that state specific is characterized by proximities different genomic regions accumulation regulatory factors. Mathematical simulations suggest an increase local viscosity could potentially contribute stabilizing duration these E-P proximities. study provides insights into association among proximity, protein accumulation, dynamics, paving way for a more nuanced understanding gene-specific mechanisms.

Language: Английский

Citations

8
Live-cell imaging of chromatin contacts opens a new window into chromatin dynamics DOI Creative Commons
Jente van Staalduinen,

Thomas van Staveren,

Frank Grosveld

et al.

Epigenetics & Chromatin, Journal Year: 2023, Volume and Issue: 16(1)

Published: June 23, 2023

Abstract Our understanding of the organization chromatin fiber within cell nucleus has made great progress in last few years. High-resolution techniques based on next-generation sequencing as well optical imaging that can investigate conformations down to single level have revealed structure is highly heterogeneous at individual allele. While TAD boundaries and enhancer–promoter pairs emerge hotspots 3D proximity, spatiotemporal dynamics these different types contacts remain largely unexplored. Investigation live cells necessary close this knowledge gap further enhance current models genome communication. In review, we first discuss potential locus labeling study architectural provide an overview available such FROS, TALE, CRISPR–dCas9 ANCHOR, latest developments applications systems.

Language: Английский

Citations

12
AI-assisted protein design to rapidly convert antibody sequences to intrabodies targeting diverse peptides and histone modifications DOI Creative Commons
Gabriel Galindo,

Daiki Maejima,

Jacob DeRoo

et al.

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

Published: Feb. 8, 2025

ABSTRACT Intrabodies are engineered antibodies that function inside living cells, enabling therapeutic, diagnostic, and imaging applications. While powerful, their development has been hindered by challenges associated with folding, solubility, stability in the reduced intracellular environment. Here, we present an AI-driven pipeline integrating AlphaFold2, ProteinMPNN, live-cell screening to optimize antibody framework regions while preserving epitope-binding complementarity-determining regions. Using this approach, successfully converted 19 out of 26 sequences into functional single-chain variable fragment (scFv) intrabodies, including a panel targeting diverse histone modifications for real-time chromatin dynamics gene regulation. Notably, 18 these had failed convert using standard demonstrating unique effectiveness our method. As sequence databases expand, method will accelerate intrabody design, making easier, more cost-effective, broadly accessible biological research.

Language: Английский

Citations

0
Anti-Phase Clustering of Regulatory Factors Shapes Gene Transcription Burst DOI
Bitong Li, Yew Yan Wong, Neftali Flores‐Rodriguez

et al.

Published: Jan. 1, 2025

Language: Английский

Citations

0
Repetitive CREB-DNA interactions at gene loci predetermined by CBP induce activity-dependent gene expression in human cortical neurons DOI Creative Commons

Yuri Atsumi,

Ryohei Iwata, Hiroshi Kimurâ

et al.

Cell Reports, Journal Year: 2023, Volume and Issue: 43(1), P. 113576 - 113576

Published: Dec. 20, 2023

Neuronal activity-dependent transcription plays a key role in plasticity and pathology the brain. An intriguing question is how neuronal activity controls gene expression via interactions of factors with DNA chromatin modifiers nucleus. By utilizing single-molecule imaging human embryonic stem cell (ESC)-derived cortical neurons, we demonstrate that increases repetitive emergence cAMP response element-binding protein (CREB) at histone acetylation sites nucleus, where RNA polymerase II (RNAPII) accumulation FOS occur rapidly. also enhances co-localization CREB CREB-binding (CBP). Increased binding constitutively active to CBP efficiently induces emergence. On other hand, formation dependent on modification acetyltransferase (HAT) but not affected by activity. Taken together, our results suggest promotes CREB-CRE CREB-CBP predetermined sites, leading rapid expression.

Language: Английский

Citations

7
Transcription-coupled changes in higher-order genomic structure and transcription hub viscosity prolong enhancer-promoter connectivity DOI Creative Commons
Hiroaki Ohishi, Soya Shinkai, Hitoshi Owada

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2023, Volume and Issue: unknown

Published: Nov. 27, 2023

Abstract The orchestration of our genes heavily relies on a coordinated communication between enhancers and promoters, yet how this dynamic interplay remains elusive while transcription is active. Here, we investigated enhancer-promoter (E-P) interactions relative to transcriptional bursting in mouse embryonic stem cells using sequential DNA/RNA/immunofluorescence (IF)-FISH analyses computational simulations. Our data reveal that the active state specific characterized by higher-order genomic structures local condensates regulatory factors, leading an elevation viscosity highly stabilizes duration E-P interactions. study underscores pivotal role dynamics paves way for more nuanced understanding gene-specific mechanisms.

Language: Английский

Citations

5
Amphiphiles Formed from Synthetic DNA-Nanomotifs Mimic the Stepwise Dispersal of Transcriptional Clusters in the Cell Nucleus DOI Creative Commons
Xenia Tschurikow, Aaron Gadzekpo, Mai P. Tran

et al.

Nano Letters, Journal Year: 2023, Volume and Issue: 23(17), P. 7815 - 7824

Published: Aug. 16, 2023

Stem cells exhibit prominent clusters controlling the transcription of genes into RNA. These form by a phase-separation mechanism, and their size shape are controlled via an amphiphilic effect transcribed genes. Here, we construct amphiphile-nanomotifs purely from DNA, achieve similar control for phase-separated droplets formed fully synthetic, self-interacting DNA-nanomotifs. Increasing amphiphile concentrations induce rounding droplets, prevent droplet fusion, and, at high concentrations, cause full dispersal droplets. Super-resolution microscopy data obtained zebrafish embryo stem reveal comparable transition transcriptional with increasing levels. Brownian dynamics lattice simulations further confirm that addition particles is sufficient to explain observed changes in size. Our work reproduces key aspects cluster formation biological using relatively simple DNA sequence-programmable nanostructures, opening novel ways mesoscopic organization synthetic nanomaterials.

Language: Английский

Citations

4