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| Status |
Public on Jun 02, 2024 |
| Title |
Transcriptional noise, gene activation, and roles of SAGA and Mediator Tail measured using nucleotide recoding single cell RNA-seq |
| Organism |
Saccharomyces cerevisiae |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
The populational variance of eukaryotic transcription differs by gene, and can be range from constitutive to bursty in nature. Exemplary bursty yeast genes tend to rely on SAGA and Mediator Tail (coactivator-redundant, CR) for transcription, and often contain TATA boxes in their promoters. To dissect gene-specific transcriptional bursting and the roles of coactivator complexes in regulating bursting in yeast, we performed genome-wide nascent single-cell RNA-seq. Examining the transcriptional variance-mean relationship (Fano factor) for thousands of yeast genes across cell populations, we found that CR-class genes generally display burstier transcription than TFIID-class genes, regardless of expression level. We also found that the cell cycle-dependent activation of yeast histone genes correlates with an increase in Fano. Finally, we assessed the consequences of rapid depletion of SAGA or Mediator Tail coactivator complex subunits. On a per-gene basis, we observed a strong relationship between bulk transcriptional fold change and the change in fraction of yeast cells actively transcribing (Fon), and a weak relationship between bulk transcriptional fold change and the change in mean expression per cell (MPC). We also observe gene-specific variation in Fon, MPC, and Fano following coactivator depletion. These results provide insight into gene-specific transcriptional kinetics and the roles of coactivator complexes in gene activation and transcriptional bursting.
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| Overall design |
Time courses of transcriptional metabolic labeling with 4-thiouracil were performed in S. cerevisiae, followed by in-cell nucleotide recoding (NR) chemistry treatment to induce U-to-C mutations in nascent RNAs. NR-recoded yeast were then subjected to scRNA-seq, and nascent RNAs were identified by their T-to-C mutation content in cDNAs. The functions of SAGA and Mediator Tail were also assessed by performing rapid coactivator depletion (Spt3/7 or Med15) using the auxin-inducible degron approach immediately prior to metabolic labeling, NR chemistry treament and scRNA-seq.
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| Contributor(s) |
Schofield JA, Hahn S |
| Citation(s) |
39102335 |
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| Submission date |
Nov 15, 2023 |
| Last update date |
Oct 09, 2024 |
| Contact name |
Jeremy Aspin Schofield |
| E-mail(s) |
jschofie@fredhutch.org
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| Organization name |
Fred Hutchinson Cancer Center
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| Department |
Basic Sciences Division
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| Street address |
1100 Fairview Ave N
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| City |
Seattle |
| State/province |
Washington |
| ZIP/Postal code |
98109 |
| Country |
USA |
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| Platforms (1) |
| GPL31112 |
NextSeq 2000 (Saccharomyces cerevisiae) |
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| Samples (8)
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| GSM7900890 |
WT yeast, 15 minute 4-TU treatment |
| GSM7900891 |
Med15-degron yeast, plus auxin treatment |
| GSM7900892 |
Med15-degron yeast, minus auxin treatment (negative control) |
| GSM7900893 |
Spt3/7-degron yeast, plus auxin treatment |
| GSM7900894 |
Spt3/7-degron yeast, minus auxin treatment (negative control) |
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| Relations |
| BioProject |
PRJNA1040497 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE247795_RAW.tar |
158.9 Mb |
(http)(custom) |
TAR (of MTX, TSV) |
SRA Run Selector |
| Raw data are available in SRA |
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