Transcriptome analysis in sumoylation deficient (ubc9-6) budding yeast and in a strain with a sumoylation-impairing mutation in the TFIIF subunit, Tfg1
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ABSTRACT:
PROVIDER: PRJNA704640 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR13777492_1.fastq.gz | Fastqsanger.gz | |||
| SRR13777492_2.fastq.gz | Fastqsanger.gz | |||
| SRR13777493_1.fastq.gz | Fastqsanger.gz | |||
| SRR13777493_2.fastq.gz | Fastqsanger.gz | |||
| SRR13777494_1.fastq.gz | Fastqsanger.gz |
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Project description:Transcription related proteins are among the most frequently identified targets of SUMO post-translational modification, but the gene-specific and genome-wide effects of transcription machinery sumoylation are not well-understood. To explore this, we examined whether dramatically reducing cellular sumoylation levels affects the levels of mRNAs in budding yeast. RNA-seq was performed using polyadenylate (polyA)-enriched RNAs from the sumoylation-deficient ubc9-6 strain and its wild-type parent. Indeed, the abundance of hundreds of mRNAs were upregulated or downregulated in the mutant strain, implying that sumoylation has a widespread influence on RNA polymerase II (RNAPII) transcription or mRNA stability. Interestingly, about one third of mRNAs that were significantly upregulated in the ubc9-6 strain encode proteins that are involved in small molecule metabolic processes, suggesting that sumoylation is normally involved in restricting these pathways. As part of our analysis of transcription-related sumoylation, we determined that the large subunit of TFIIF, Tfg1, is a major target of sumoylation among the general transcription factors (GTFs) of RNAPII. To determine whether sumoylation of Tfg1 specifically is involved in regulating mRNA levels, we performed RNA-seq on polyA-enriched RNAs derived from strains expressing wild-type (Tfg1-HA) or sumoylation-deficient (Tfg1-K60,61R-HA) forms of Tfg1. However, with few exceptions, impairing Tfg1 sumoylation had no effect on the mRNA transcriptome. Together, our data demonstrate that gene expression is extensively regulated by sumoylation, but this likely involves the coordinated modification of multiple proteins at multiple levels.
2021-09-21 | GSE167427 | GEO
Project description:Many transcription-related proteins, including components of the RNA polymerase II (RNAPII) general transcription factors (GTFs), have been shown through proteomics studies to be modifiable by SUMO conjugation. However, we determined that the large subunit of TFIIF, Tfg1, is likely the major target of sumoylation among GTFs in budding yeast. The primary site of sumoylation on Tfg1 is Lys residues 60 or 61, which lies in a region of the protein that is proximal to the Rpb2 subunit of RNAPII when the GTF-RNAPII preinitiation complex (PIC) assembles at promoters. As TFIIF and RNAPII are highly functionally and physically linked, we examined whether blocking Tfg1 sumoylation through Lys-to-Arg mutations could affect the recruitment or association of RNAPII with chromatin. RNAPII ChIP-seq was performed in strains expressing wild-type (Tfg1-HA) or sumoylation-deficient (Tfg1-K60,61R-HA) forms of Tfg1. Indeed, nearly half of all protein-coding genes showed significantly reduced RNAPII levels in the mutant strain. These data suggest that sumoylation of TFIIF plays an important role in regulating the dynamics of RNAPII association with genes.
2021-09-21 | GSE167426 | GEO
Project description:Ageing and mutations of transthyretin (TTR), the thyroid hormones and retinol transporting protein lead to amyloidosis by destabilizing the structure of TTR. Because protein structure is regulated through posttranslational modifications, we investigated the Small Ubiquitin-like Modifier (SUMO)ylation of TTR. We chose the widely used Ubc9 fusion-directed SUMOylation system, which is based on a fusion of the SUMOylation substrate of interest with Ubc9, a sole SUMO conjugating enzyme. Surprisingly, despite our presumptions, we found that Ubc9 fused to TTR was SUMOylated at a unique set of lysine residues. Three unknown SUMOylation sites of Ubc9—K154, K18 and K65—were revealed by mass spectrometry (MS). The previously reported SUMOylation at K49 of Ubc9 was also observed. SUMOylation of the lysine residues of TTR fused to Ubc9 was hardly detectable. However, non-fused TTR was SUMOylated via trans-SUMOylation by Ubc9 fused to TTR. Interestingly, mutating the catalytic residue of Ubc9 fused to TTR did not result in complete loss of the SUMOylation signal, suggesting that Ubc9 linked to TTR is directly cross-SUMOylated by the SUMO-activating enzyme E1. Ubc9, TTR or fusion proteins composed of TTR and Ubc9 specifically affected the global SUMOylation of cellular proteins. TTR or Ubc9 alone increased global SUMOylation, whereas TTR and Ubc9 together decreased the amount of high-molecular weight (HMW) SUMO conjugates. Our data suggest that TTR may influence the SUMOylation of Ubc9, thereby altering signalling pathways in the cell.
2016-08-15 | PXD004515 | Pride
Project description:Ageing and mutations of transthyretin (TTR), the thyroid hormones and retinol transporting protein lead to amyloidosis by destabilizing the structure of TTR. Because protein structure is regulated through posttranslational modifications, we investigated the Small Ubiquitin-like Modifier (SUMO)ylation of TTR. We chose the widely used Ubc9 fusion-directed SUMOylation system, which is based on a fusion of the SUMOylation substrate of interest with Ubc9, a sole SUMO conjugating enzyme. Surprisingly, despite our presumptions, we found that Ubc9 fused to TTR was SUMOylated at a unique set of lysine residues. Three unknown SUMOylation sites of Ubc9—K154, K18 and K65—were revealed by mass spectrometry (MS). The previously reported SUMOylation at K49 of Ubc9 was also observed. SUMOylation of the lysine residues of TTR fused to Ubc9 was hardly detectable. However, non-fused TTR was SUMOylated via trans-SUMOylation by Ubc9 fused to TTR. Interestingly, mutating the catalytic residue of Ubc9 fused to TTR did not result in complete loss of the SUMOylation signal, suggesting that Ubc9 linked to TTR is directly cross-SUMOylated by the SUMO-activating enzyme E1. Ubc9, TTR or fusion proteins composed of TTR and Ubc9 specifically affected the global SUMOylation of cellular proteins. TTR or Ubc9 alone increased global SUMOylation, whereas TTR and Ubc9 together decreased the amount of high-molecular weight (HMW) SUMO conjugates. Our data suggest that TTR may influence the SUMOylation of Ubc9, thereby altering signalling pathways in the cell.
2017-03-29 | MSV000080754 | MassIVE
Project description:Effects of sumoylation-impairing mutation of TFIIF subunit Tfg1 on genome-wide RNA polymerase II (RNAPII) levels in budding yeast
| PRJNA704639 | ENA
Project description:UBC9 is the sole conjugating enzyme E2 in the sumoylation and plays a pivotal role in maintaining homeostasis and restraining stress reaction. Targeting UBC9 is emerging as a novel strategy for cancer therapy. However, the role of UBC9 in bladder cancer is not clear. Here, we sought to determine the alterations of trancriptome after shRNA-mediated knockdown UBC9 in T24 cell lines．
2018-12-25 | GSE117143 | GEO
Project description:Calorie restriction (CR) provides anti-aging benefits through diverse processes, such as reduced metabolism and growth and increased mitochondrial activity. Although controversy still exists regarding CR-mediated lifespan effects, many researchers are seeking interventions that mimic the effects of CR. Yeast has proven to be a useful model system for aging studies, including CR effects. We report here that yeast adapted through in vitro evolution to the severe cellular stress caused by loss of the Ulp2 SUMO-specific protease exhibit both enhanced growth rates and replicative lifespan, and they have altered gene expression profiles similar to those observed in CR. Notably, in certain evolved ulp2Δ lines, a dramatic increase in the auto-sumoylation of Ubc9 E2 SUMO-conjugating enzyme results in altered regulation of multiple targets involved in energy metabolism and translation at both transcriptional and post-translational levels. This increase is essential for the survival of aged cells and CR-mediated lifespan extension. Thus, we suggest that high Ubc9 auto-sumoylation exerts potent anti-aging effects by promoting efficient energy metabolism-driven improvements in cell replication abilities. This potential could be therapeutically explored for the development of novel CR-mimetic strategies.
2025-01-15 | GSE278286 | GEO
Project description:Many transcription-related proteins are known to be modified by SUMO post-translational modification in yeast, plants, humans and other eukaryotes. We are interested in understanding how cells use sumoylation to regulate transcription of specific genes and globally. To explore this, we carried out ChIP-seq analysis to determine how genomic levels of RNA polymerase II (RNAPII) change when cellular levels of sumoylation are greatly reduced in budding yeast. Compared to wild-type yeast, RNAP occupancy in the sumoylation-deficient ubc9-6 strain was significantly altered for hundreds of genes. Among the genes with the most elevated levels of RNAPII are those involved in translation and ribosome biogenesis, including most ribosomal protein genes. On the other hand, many genes involved in small molecule and amino acid metabolism and biosynthesis showed reduced RNAPII levels in ubc9-6. Exposure of yeast to heat shock is known to cause a widespread change to RNAPII occupancy across the genome. To examine whether cellular sumoylation plays a role in this process, RNAPII ChIP-seq was performed in wild-type and ubc9-6 strains after a brief heat shock. Strikingly, the sumoylation deficient strain showed a far more dramatic redistribution of RNAPII across the genome, with significantly more genes showing a heat-shock induced increase or decrease in RNAPII occupancy. These results imply that cellular sumoylation levels have a significant impact on the regulation of transcription genome-wide, and that normal sumoylation levels are needed to restrain the vast redistribution of RNAPII that occurs in response to heat shock.
2021-09-21 | GSE167424 | GEO
Project description:Foxp3-expressing regulatory T (Treg) cells are essential regulators in the immune system; molecular mechanisms underlying Treg cell expansion and function are still not well understood. SUMOylation is an important post-translational modification characterized by covalent attachment of SUMO moieties to lysine within proteins. UBC9 is the only E2 conjugation enzyme involved in this process and loss of UBC9 completely impairs the SUMOylation pathway. Here we report that selective deletion of Ubc9 within the Treg cell lineage resulted in fatal early-onset autoimmunity as the Foxp3 mutant mice. Ubc9-deficient Treg cells exhibited severe defects in TCR-driven homeostatic proliferation, accompanied by impaired activation and compromised suppressor function. Importantly, TCR-enhanced SUMOylation of IRF4, a critical regulator of Treg cell function downstream of TCR signals, regulates its stability in Treg cells. Our data thus have demonstrated an essential role of SUMOylation in the expansion and function of Treg cells.
2016-05-31 | GSE82031 | GEO
Project description:Yeast has proven to be a useful model system for aging studies, including CR effects. We report here that yeast adapted through in vitro evolution to the severe cellular stress caused by loss of the Ulp2 SUMO-specific protease exhibit both enhanced growth rates and replicative lifespan, and they have altered gene expression profiles similar to those observed in CR. Notably, in certain evolved ulp2Δ lines, a dramatic increase in the auto-sumoylation of Ubc9 E2 SUMO-conjugating enzyme results in altered regulation of multiple targets involved in energy metabolism and translation at both transcriptional and post-translational levels. This increase is essential for the survival of aged cells and CR-mediated lifespan extension
2024-04-17 | GSE253055 | GEO