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Genome-wide localization of SUMO-modified proteins in untreated or heat-shocked budding yeast

ABSTRACT: Most SUMO-modified proteins in budding yeast and human cells are associated with chromatin. To determine specifically where sumoylated proteins are found across the genome, we performed ChIP-seq with a SUMO antibody in budding yeast. We identified 603 loci that each contain a stable SUMO peak. In agreement with a previous study, most of these sites correspond to tRNA genes or genes that encode ribosomal proteins (RPGs). However, 147 of the SUMO peaks in our analysis are found at promoters of protein-coding genes that are not RPGs (non-RPGs), many of which are among the most highly expressed genes. By comparing our data with other ChIP-seq studies, we determined that non-RPG SUMO peaks are situated precisely where general transcription factors (GTFs) assemble, but not where RNA polymerase II (RNAPII) or other promoter-proximal proteins bind. In support of this, we determined that the large subunit of TFIIF is the major SUMO target among GTFs and propose that its sumoylation controls the dynamics of RNAPII gene association. Additionally, we found that exposing yeast to a brief heat shock significantly reduces the intensity of SUMO peaks at most genes of all classes, which likely reflects the vast transcriptional reprogramming that occurs in response to temperature stress.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE167425 | GEO | 2021/09/21

REPOSITORIES: GEO

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Effects of sumoylation-impairing mutation of TFIIF subunit Tfg1 on genome-wide RNA polymerase II (RNAPII) levels in budding yeast

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

Effect of ribosomal protein intron deletion on gene expression

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Two distinct promoter architectures centered on dynamic nucleosomes control ribosomal protein gene transcription

Project description:In yeast, ribosome production is controlled transcriptionally by tight coregulation of the 138 ribosomal protein genes (RPGs). RPG promoters display limited sequence homology, and the molecular basis for their coregulation remains largely unknown. Here we identify two prevalent RPG promoter types, both characterized by upstream binding of the general transcription factor (TF) Rap1 followed by the RPG-specific Fhl1/Ifh1 pair, with one type also binding the HMG-B protein Hmo1. We show that the regulatory properties of the two promoter types are remarkably similar, suggesting that they are determined to a large extent by Rap1 and the Fhl1/Ifh1 pair. Rapid depletion experiments allowed us to define a hierarchy of TF binding in which Rap1 acts as a pioneer factor required for binding of all other TFs. We also uncovered unexpected features underlying recruitment of Fhl1, whose forkhead DNA-binding domain is not required for binding at most promoters, and Hmo1, whose binding is supported by repeated motifs. Finally, we describe unusually micrococcal nuclease (MNase)-sensitive nucleosomes at all RPG promoters, located between the canonical +1 and M-bM-^@M-^S1 nucleosomes, which coincide with sites of Fhl1/Ifh1 and Hmo1 binding. We speculate that these M-bM-^@M-^XM-bM-^@M-^XfragileM-bM-^@M-^YM-bM-^@M-^Y nucleosomes play an important role in regulating RPG transcriptional output. Genome-wide examination of binding sites for transcription factors controlling ribosomal protein genes expression and nucleosome positions in budding yeast cells

2014-10-01 | E-GEOD-61596 | biostudies-arrayexpress

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Transcriptome analysis of budding yeast strains manipulated in RPL22A and RPL22B genes

Project description:Most Saccharomyces cerevisiae ribosomal protein genes (RPGs) are present as two paralogous copies that emerged during a whole genome duplication. The preservation of two copies of the same gene during the past ~100 million years enabled diversification of the regulation of their expression, and also partially of their functional properties. One example of such paralogous genes are RPL22A and RPL22B that are asymmetrically expressed and their protein sequences represent one of the most divergent pairs among budding yeast RPG paralogs. It was shown that introns in the RPL22A and RPL22B genes are important for regulation of their expression levels. To investigate functional differences between RPL22A and RPL22B we performed RNA-seq analysis of strains with complete deletions of either RPL22A or RPL22B, and also of strains with intron deletions in the RPL22 genes, in comparison with a wild-type strain. Cells were grown to mid-exponential phase in the rich YPAD medium. Total RNA was isolated by combining phenol-chlorophorm extraction with MasterPure Yeast RNA Purification Kit (Epicentre). Ribodepletion, library preparation and sequencing were performed by BGI Genomics.

2018-03-07 | E-MTAB-5275 | biostudies-arrayexpress

The DYRK1A protein kinase is recruited to a subset of ribosomal protein gene promoters in human and mouse cells [RNA-seq]

Project description:Ribosomal protein genes (RPGs) coding sequences are highly conserved along evolution; however, promoter features and the machinery involved in their transcriptional regulation are not. In eukaryotes, the main genomic elements and players involved in RPG transcriptional regulation have been mostly characterized in Saccharomyces cerevisiae. However, given the lack of evolutionary conservation of the yeast factors, studies in higher eukaryotes have focused on searching for differential enrichment of transcription factor-binding motifs within the RPG promoters. Among them, the palindromic motif TCTCGCGAGA, which is currently acknowledged as a ZBTB33/KAISO motif, also matches the genomic sequence bound by the protein kinase DYRK1A. DYRK1A, a member of the human family of DYRK kinases, fulfills many diverse functions by phosphorylating a broad set of proteins involved in different cellular processes. One of such functions is to be a chromatin-associated kinase capable of regulating gene expression. Here, we analyze in-depth the presence of DYRK1A at the promoters of human and mouse RPGs and explore its functional consequences. Our results indicate that DYRK1A is a positive regulator of RPGs’ expression at the transcriptional level, and further expand the functional spectrum of the kinase as a contributor to the regulation of cell growth in mammalian cells.

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The DYRK1A protein kinase is recruited to a subset of ribosomal protein gene promoters in human and mouse cells [HeLa RNA-seq]

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The DYRK1A protein kinase is recruited to a subset of ribosomal protein gene promoters in human and mouse cells [ChIP-seq]

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Genome-wide levels of RNA polymerase II (RNAPII) in normally growing or heat-shocked wild-type and sumolyation-deficient ubc9-6 yeast strains

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

Transcriptome analysis in sumoylation deficient (ubc9-6) budding yeast and in a strain with a sumoylation-impairing mutation in the TFIIF subunit, Tfg1

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

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