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Abstract Transcription is the primary regulatory step in gene expression. Divergent transcription initiation from promoters and enhancers produces stable RNAs from genes and unstable RNAs from enhancers1,2. Nascent RNA capture and sequencing assays simultaneously measure gene and enhancer activity in cell populations3. However, fundamental questions about the temporal regulation of transcription and enhancer–gene coordination remain unanswered, primarily because of the absence of a single-cell perspective on active transcription. In this study, we present scGRO–seq—a new single-cell nascent RNA sequencing assay that uses click chemistry—and unveil coordinated transcription throughout the genome. We demonstrate the episodic nature of transcription and the co-transcription of functionally related genes. scGRO–seq can estimate burst size and frequency by directly quantifying transcribing RNA polymerases in individual cells and can leverage replication-dependent non-polyadenylated histone gene transcription to elucidate cell cycle dynamics. The single-nucleotide spatial and temporal resolution of scGRO–seq enables the identification of networks of enhancers and genes. Our results suggest that the bursting of transcription at super-enhancers precedes bursting from associated genes. By imparting insights into the dynamic nature of global transcription and the origin and propagation of transcription signals, we demonstrate the ability of scGRO–seq to investigate the mechanisms of transcription regulation and the role of enhancers in gene expression.
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Nascent transcription reveals regulatory changes in extremophile fishes inhabiting hydrogen sulfide-rich environments
Regulating transcription allows organisms to respond to their environment, both within a single generation (plasticity) and across generations (adaptation). We examined transcriptional differences in gill tissues of fishes in thePoecilia mexicanaspecies complex (family Poeciliidae), which have colonized toxic springs rich in hydrogen sulfide (H2S) in southern Mexico. There are gene expression differences between sulfidic and non-sulfidic populations, yet regulatory mechanisms mediating this gene expression variation remain poorly studied. We combined capped-small RNA sequencing (csRNA-seq), which captures actively transcribed (i.e. nascent) transcripts, and messenger RNA sequencing (mRNA-seq) to examine how variation in transcription, enhancer activity, and associated transcription factor binding sites may facilitate adaptation to extreme environments. csRNA-seq revealed thousands of differentially initiated transcripts between sulfidic and non-sulfidic populations, many of which are involved in H2S detoxification and response. Analyses of transcription factor binding sites in promoter and putative enhancer csRNA-seq peaks identified a suite of transcription factors likely involved in regulating H2S-specific shifts in gene expression, including several key transcription factors known to respond to hypoxia. Our findings uncover a complex interplay of regulatory processes that reflect the divergence of extremophile populations ofP. mexicanafrom their non-sulfidic ancestors and suggest shared responses among evolutionarily independent lineages.
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- PAR ID:
- 10539436
- Publisher / Repository:
- The Royal Society Publishing
- Date Published:
- Journal Name:
- Proceedings of the Royal Society B: Biological Sciences
- Volume:
- 291
- Issue:
- 2025
- ISSN:
- 0962-8452
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1098/rspb.2024.0412
