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. 2018 Mar 29;11(1):13.
doi: 10.1186/s13072-018-0184-2.

The SAGA/TREX-2 subunit Sus1 binds widely to transcribed genes and affects mRNA turnover globally

Varinia García-Molinero  1   2 , José García-Martínez  3 , Rohit Reja  4   5 , Pedro Furió-Tarí  6 , Oreto Antúnez  7 , Vinesh Vinayachandran  4 , Ana Conesa  6   8   9 , B Franklin Pugh  4 , José E Pérez-Ortín  7 , Susana Rodríguez-Navarro  10   11
Affiliations

The SAGA/TREX-2 subunit Sus1 binds widely to transcribed genes and affects mRNA turnover globally

Varinia García-Molinero et al. Epigenetics Chromatin. .

Abstract

Background: Eukaryotic transcription is regulated through two complexes, the general transcription factor IID (TFIID) and the coactivator Spt-Ada-Gcn5 acetyltransferase (SAGA). Recent findings confirm that both TFIID and SAGA contribute to the synthesis of nearly all transcripts and are recruited genome-wide in yeast. However, how this broad recruitment confers selectivity under specific conditions remains an open question.

Results: Here we find that the SAGA/TREX-2 subunit Sus1 associates with upstream regulatory regions of many yeast genes and that heat shock drastically changes Sus1 binding. While Sus1 binding to TFIID-dominated genes is not affected by temperature, its recruitment to SAGA-dominated genes and RP genes is significantly disturbed under heat shock, with Sus1 relocated to environmental stress-responsive genes in these conditions. Moreover, in contrast to recent results showing that SAGA deubiquitinating enzyme Ubp8 is dispensable for RNA synthesis, genomic run-on experiments demonstrate that Sus1 contributes to synthesis and stability of a wide range of transcripts.

Conclusions: Our study provides support for a model in which SAGA/TREX-2 factor Sus1 acts as a global transcriptional regulator in yeast but has differential activity at yeast genes as a function of their transcription rate or during stress conditions.

Keywords: ChIP-exo; GRO; SAGA; Sus1; Transcription.

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Figures

Fig. 1
Fig. 1
Positional organization of Sus1 before and after heat stress. a Heat map showing shifted 5′-end sequencing reads (tags) for Sus1-WT at 25 °C (blue) and after 15 min at 37 °C (red), aligned by the midpoint in between transcription start site (TSS) and transcription end site (TES). Data presented are divided into three subgroups: ribosomal protein genes (RP, n = 137), SAGA-dominated genes (SAGA, n = 471) and TFIID-dominated genes (TFIID, n = 4351) genes and sorted by gene length in each subgroup. The results of two replicates are shown. As a control, the signal of an isogenic strain bearing no-tagged Sus1 was also monitored (No-tag). b Gene-averaged 5′-ends of shifted relative read counts (representing points of cross-linking) of Sus1-WT at 25 °C (blue line) and at 37 °C (red line) around the transcription start site (TSS) in three gene classes: ribosomal protein (RP) genes, SAGA-dominated genes and TFIID-dominated genes, with TSS oriented to the right. Nucleosomes (based on MNase ChIP-seq) are plotted and based on values from [39]. Abrupt heat shock at 37 °C (yellow line) and 25 °C (grey fill) is shown. The resulting normalized ratios were plotted. Note that ordinate scales vary for the three gene classes due to differences in the number of genes in each class. c) Signal tracks, showing unshifted ChIP-exo tag 5′-end reads for Sus1-wt at 25 °C and 37 °C at RP-dependent gene RPL11 (upper panel), at SAGA-dependent gene CDC19 (middle panel) and at a TFIID-dominated gene SPB1 (lower panel) are shown
Fig. 2
Fig. 2
Genomic effects of Sus1 depletion on mRNA turnover. a Plot of the transcription rates (TR) of 3757 yeast genes in the sus1Δ deletion mutant versus the wild-type strain. b Plot of the mRNA levels (RA) of 5216 yeast genes in the sus1Δ deletion mutant versus the wild-type strain. c Plot of the fold change of TR of 3757 yeast genes in the sus1Δ mutant against the TR level of the WT. Note that all graphs are in log2 scales of arbitrary units. Pearson R of the cloud to a linear fitting is shown
Fig. 3
Fig. 3
Genomic effects of Sus1 depletion on changes in mRNA half-lives. a Box-and-whisker plot of the changes in mRNA stability half-lives (HL) in sus1Δ mutant (dark grey) and the wild-type strain (light grey). All comparisons show a statistically significant HL increase of sus1Δ in relation to wild-type strains (Wilcoxon signed-rank test p value < 10−5). Note that SAGA- and TFIID-dominated genes have similar average increase in HLs (3.3- vs. 4.73-fold) but that ribosomal protein-coding genes (RP) are much more increased in HLs (7.7-fold) than environmental stress response activated (ESR-up) genes (2.75-fold). b Box-and-whisker plot of the changes in TR in the sus1Δ mutant (dark grey) and the wild-type strain (light grey). All comparisons show a statistically significant TR decrease of sus1Δ in relation to wild-type strains (Wilcoxon signed-rank test p value < 10−5). Note that the decrease in RP genes is much higher (19.4-fold) than in SAGA (5.5-fold), TFIID (7.1-fold) or ESR-up (4.4-fold) genes. c Plot of the fold change in HL in the sus1Δ mutant against the TR level of each gene in the WT strain. Note that both axes are in log2 scales of arbitrary units. Pearson R of the cloud to a linear fitting is shown
Fig. 4
Fig. 4
The binding of Sus1 to UAS correlates with the gene TR. a A sliding window plot of the transcription rate (TR) in arbitrary units versus the total reads counted in each gene in the Sus1 (black dots) or the no-tag (grey dots) of the ChIP-exo experiments. A window of 100 genes was used in both cases. b Box-and-whisker plot of the Sus1 binding to yeast genes. Note that the binding to ESR-up genes is lower than the global genome average in a wild-type strain and increases with temperature, whereas the opposite trend is observed for RP genes. Differences between ESR-up or RP genes and all genes are statistically significant for both temperatures (Wilcoxon signed-rank test p value < 10−5)
Fig. 5
Fig. 5
Positional organization of Sus1 in SLIK-constitutive mutants. a Heat map showing shifted 5′-end sequencing reads (tags) for Sus1 in spt71180 (left panel) and spt8Δ (right panel) at 25 °C (blue) and after 15 min at 37 °C (red), aligned by the midpoint in between the transcription start site (TSS) and transcription end site (TES), split into three subgroups: RP, SAGA and TFIID genes and sorted by gene length in each subgroup. The results of two replicates are shown. b Gene-averaged 5′-ends of shifted relative read counts (representing points of cross-linking) of Sus1-WT (left panel), Sus1 in spt71180 (middle panel) and spt8Δ (right panel) at 25 °C (blue) and after 15 min at 37 °C (red) around the transcription start site (TSS) in three gene classes: ribosomal protein (RP) genes, SAGA-dominated genes and TFIID-dominated genes, with TSS oriented to the right. Nucleosomes (based on MNase ChIP-seq) are plotted and based on values from [39]. Abrupt heat shock at 37 °C (yellow line) and 25 °C (grey fill) is shown. The resulting normalized ratios were plotted. Note that ordinate scales vary for the three gene classes due to differences in the number of genes in each class
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