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Transcriptional noise, gene activation, and roles of SAGA and Mediator Tail measured using nucleotide recoding single cell RNA-seq

ABSTRACT: 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.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE247795 | GEO | 2024/06/02

REPOSITORIES: GEO

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Project description:TFIID and SAGA complexes play a critical role in RNA Polymerase II dependent activated transcription. Although the two regulatory complexes are recruited to promoters by activation domain-interactions, the contribution of the different subunits or the different domains of the individual subunits is not completely understood. Taf9 is a shared subunit in TFIID and SAGA and has an N-terminal H3-like histone fold domain and a highly conserved C-terminal domain, Taf9-CTD. In this study, we have uncovered an essential role for the Taf9-CTD in transcriptional activation. The Taf9-CTD was not essential for the histone-fold mediated interaction with Taf6, SAGA and TFIID integrity or Gcn4 interaction with SAGA. Transcriptome profiling performed under Gcn4 activating conditions showed that the Taf9-CTD is required for expression of ~17% of the yeast genome and provides a coactivator function to recruit TFIID and SAGA complexes to the promoters in vivo during transcriptional activation. Integrated genome-wide data analysis showed that the Taf9-CTD is required for activation of promoters bound by several transcription factors indicating a broad role for Taf9-CTD in promoter occupancy of TFIID or SAGA complexes. Interestingly, only a subset of the promoters seemed to be dependent on the Taf9-CTD for assembly of the pre-initiation complex indicating redundancy in activator targets to assemble PIC in vivo. Together these results indicate that evolutionarily conserved domains in shared subunits of TFIID and SAGA have a pervasive role in genome-wide transcription. This GEO series consists of 14 microarray hybridizations using the Agilent two-color experiment with the Agilent Custom Saccharomyces cerevisiae 8x15k gene expression array. Four biological replicates each for the wild-type (TAF9), the mutant taf9-tCRD2 strain treated or untreated with SM, and the wild-type (TAF9) versus mutant taf9-tCRD2 treated with SM hybridized as dye-swapped replicates. Two biological replicates for wild-type (SPT20) vs spt20D strains treated with SM, and hybridized as dye-swapped replicates to identify the fraction of SAGA dependent genes under amino-acid starvation conditions. The overall aim was to identify genes dependent on the conserved C-terminal region domain of TAF9 and determine their dependence on the SAGA subunit Spt20 for expression.

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Transcriptional noise, gene activation, and roles of SAGA and Mediator Tail measured using nucleotide recoding single cell RNA-seq

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