Dynamics and Spatial Genomics of the Nascent Transcriptome by Intron seqFISH

Area of Science:

• Molecular Biology
• Genomics
• Cell Biology

Background:

• Visualizing the transcriptome and nuclear organization in single cells remains a significant challenge.
• Existing methods struggle to capture the dynamics of active transcription sites in situ.

Purpose of the Study:

• To develop a novel method for multiplexed, single-molecule visualization of the nascent transcriptome in situ.
• To investigate the spatial organization and dynamics of active transcription sites within single cells.
• To identify cell types and states using nascent transcriptome profiling.

Main Methods:

• Developed intron sequencing by fluorescence in situ hybridization (intron seqFISH), a multiplexed single-molecule in situ method.
• Enabled imaging of 10,421 genes at nascent transcription active sites.
• Combined with mRNA and lncRNA seqFISH and immunofluorescence for comprehensive analysis.

Main Results:

• Successfully identified different cell types and states in mouse embryonic stem cells and fibroblasts.
• Demonstrated that nascent RNA synthesis sites are typically located on chromosome territory surfaces with high cell-to-cell variability.
• Discovered asynchronous oscillations in global nascent transcription with a 2-hour period in both cell types.

Conclusions:

• Intron seqFISH provides unprecedented spatial genomics of the nascent transcriptome in single cells.
• Reveals fundamental principles of nuclear organization and rapid transcriptional dynamics previously obscured.
• Offers a powerful tool for dissecting cellular heterogeneity and gene regulation.