Introduction
When genes are transcribed within a cell, their activity is not continuous but instead alternates between ON (active) and OFF (inactive) states. For instance, a gene may randomly switch to an ON state for several minutes, producing a burst of RNA, and then abruptly turn OFF. This irregular, dynamic transcription behavior is known as transcriptional bursting and serves as a critical mechanism for fine-tuning gene activity in individual cells (see Figure 1).
Figure 1. Schematic illustration of transcriptional bursting.
Transcriptional bursting leads to variability in gene expression levels even among cells in the same tissue or culture environment. This phenomenon is thought to contribute to diverse cellular behaviors observed during early embryogenesis and cancer evolution, making it extremely important in the context of biological processes and diseases. However, the precise mechanisms that control transcriptional bursting remain insufficiently understood (see Figure 2). Why do some cells exhibit frequent bursts of a particular gene while others remain quiet? What factors determine the timing and duration of these bursts? Answering these questions will open up new avenues in cell biology and gene regulation.
Figure 2. Mechanisms controlling transcriptional bursting.
Objective of the Research
This research project aims to elucidate the regulatory mechanisms governing transcriptional bursting. By uncovering the molecular basis behind transcriptional bursting, we can better understand how cells modulate gene expression levels and the inherent fluctuations (noise) in transcription. This knowledge not only deepens our grasp of the fundamental principles of gene expression but also provides clues for explaining the variability observed among cells with identical genetic backgrounds.
Variations in gene expression among cells significantly influence cell fate decisions and stress responses. For example, within a population of undifferentiated stem cells, differences in transcriptional bursting may cause only some cells to express key genes that trigger differentiation, while others remain undifferentiated. Understanding the regulatory mechanisms of transcriptional bursting is therefore crucial for predicting and controlling cell state transitions and fate decisions—an insight that has implications from developmental biology to regenerative medicine. Moreover, the inherent fluctuations resulting from transcriptional bursting may enhance the adaptability of multicellular organisms to environmental changes, thereby maintaining overall organismal health. Conversely, excessive noise might lead to tissue dysfunction or disease; hence, deciphering how cells balance these fluctuations is a central goal of this research.
Research Approach and Methods
To address the complex phenomenon of transcriptional bursting, our laboratory employs a multiscale and multimodal approach by integrating several advanced techniques:
Live Imaging:
We observe living cells in real time to directly visualize the switching between transcription ON and OFF states (i.e., the occurrence of bursts). By using fluorescent protein reporters and in vivo labeling techniques, we can track transcription dynamics on a scale of seconds to minutes and quantitatively assess burst frequency and duration.
Using our STREAMING-tag system, we capture real-time videos of transcription at the single-gene level Ohishi et al., Nat Commun, 2022). For example, when applied to the Nanog gene in mouse ES cells, this technology records transcriptional activity at 15-second intervals, providing insight into the spatiotemporal dynamics of transcriptional bursting.
Using our STREAMING-tag system, we capture real-time videos of transcription at the single-gene level Ohishi et al., Nat Commun, 2022). For example, when applied to the Nanog gene in mouse ES cells, this technology records transcriptional activity at 2-min intervals, providing insight into the spatiotemporal dynamics of transcriptional bursting.
Single-Molecule Fluorescence In Situ Hybridization (smFISH):
This method detects individual mRNA molecules in fixed cells. By counting each transcript produced from a gene, smFISH provides a snapshot of the distribution of mRNA numbers in individual cells, allowing us to visualize the variability resulting from transcriptional bursts.
An image of mouse ES cells stained for Nanog mRNA with a green fluorescent dye using smFISH. The blue color indicates the nucleus, and the arrow points to the gene region (transcriptional spot) where transcriptional bursting is occurring.
Multimodal seqFISH (DNA/RNA/IF-FISH):
This advanced sequential FISH technique simultaneously visualizes DNA, RNA, and proteins within the same cell. Specifically, it involves sequentially labeling specific genomic regions (e.g., enhancers and promoters), corresponding RNA transcripts, and transcription factors (via immunofluorescence). This approach enables us to observe changes in DNA structure (such as alterations in inter-domain distances) and local accumulation of regulatory factors near genes undergoing transcriptional bursts, thereby revealing the spatial context of burst regulation.
A schematic diagram of DNA/RNA/IF-seqFISH.
A representative image of seq-DNA/RNA/IF-FISH. This video was created from maximum intensity projections of images acquired during each round of seq-DNA/RNA/IF-FISH. In each round, three sets of secondary probes and readout probes were used, enabling image acquisition through three channels. Each channel was used to observe the localization of a single RNA, a genomic region, a protein, or post-translational modifications. (Ohishi et al., Sci Adv, 2024)
Data from these complementary techniques are integrated to develop mathematical models of transcriptional bursting. For instance, we model burst occurrence using a two-state (ON/OFF) or probabilistic framework and fit these models to our experimental data to estimate key regulatory parameters. This combined experimental and computational approach allows us to comprehensively dissect the phenomenon of transcriptional bursting.
Related Research and Achievements
Our laboratory has been at the forefront of transcriptional bursting research. Recent key publications include:
- Ochiai, H. et al. (2020). Genome-wide kinetic properties of transcriptional bursting in mouse embryonic stem cells. Sci Adv 6, eaaz6699.
– This study revealed, via genome-wide analysis in mouse ES cells, that the magnitude and frequency of transcriptional bursts are regulated by specific factors such as polycomb complexes and elongation factors. A CRISPR screen further identified that the Akt/MAPK signaling pathway modulates burst dynamics through effects on transcription elongation. - Ohishi, H. et al. (2024). Transcription-coupled changes in genomic region proximities during transcriptional bursting. Sci Adv 10, eadn0020.
– Using an advanced seq-DNA/RNA/IF-FISH approach, this study demonstrated that changes in the distance between enhancers and promoters contribute to the maintenance and stabilization of transcriptional bursts, highlighting the role of three-dimensional genome architecture in dynamic gene regulation. - Ohishi, H. et al. (2022). STREAMING-tag system reveals spatiotemporal relationships between transcriptional regulatory factors and transcriptional activity. Nat Commun 13, 7672.
– Our STREAMING-tag system enabled high-resolution (100 nm scale) simultaneous visualization of the transcriptional activity and nuclear positioning of endogenous genes, such as Nanog, along with the nanoscale distribution of co-regulatory factors like BRD4. This breakthrough has paved the way for elucidating the direct causal links between regulatory factor clustering and transcriptional bursting.
Future Perspectives
Moving forward, we will expand our approach to examine whether the regulatory factors and mechanisms identified in mouse ES cells are universally applicable across different cell types and developmental stages. For example, we plan to compare the control of transcriptional bursting in various tissues and during distinct phases of development to understand both universal principles and cell type–specific differences.
Moreover, we aim to investigate whether aberrations in transcriptional bursting contribute to disease—such as the increased gene expression variability observed in cancer—and explore interventions that restore normal burst dynamics. Ultimately, insights gained from deciphering the mechanisms controlling transcriptional bursts may lead to innovative strategies for stabilizing or modulating gene expression, with implications for drug discovery and regenerative medicine.
Our goal is to translate these foundational insights into applications that improve early diagnosis, preventive strategies, and treatments for age-related and other diseases.