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Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Influence of SUMOylation on chromatin structure in hIPSCs (ChiPS4) assessed by time-resolved ATAC-seq in response to ML792 treatment

ABSTRACT: SUMOylation is thought to regulate chromatin structure and accessibility thus affecting gene expression. In order to assess these potential roles of SUMOylation in human pluripotent stem cells, ChiPS4 cells were treated with ML792 (selective SUMO E1 inhibitor) or DMSO (vehicle control) for the following times: 4, 8, 24 and 48h or left untreated for 48h. At each time point samples were collected for western blotting (control for the efficacy of treatment), RNA-seq (total RNA extracted using RNeasy Mini Kit) and ATAC-seq (using Omni-ATAC protocol, 3 replicates for each time point). Samples were further used for library preparation and sequenced using the Illumina NovaSeq 6000 S4.

ORGANISM(S): Homo sapiens

SUBMITTER: Marek Gierlinski

PROVIDER: E-MTAB-9961 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Influence of SUMOylation on transcriptional signature of hIPSCs (ChiPS4) assessed by time-resolved RNA-seq in response to ML792 treatment

Project description:SUMOylation is thought to regulate chromatin structure and accessibility thus affecting gene expression. In order to assess these potential roles of SUMOylation in human pluripotent stem cells, ChiPS4 cells were treated with ML792 (selective SUMO E1 inhibitor) or DMSO (vehicle control) for the following times: 4, 8, 24 and 48h. At each time point samples were collected for western blotting (control for the efficacy of treatment), RNA-seq (total RNA extracted using RNeasy Mini Kit, samples in 4 replicates per time point) and ATAC-seq (using Omni-ATAC protocol). Samples were further used for library preparation and sequenced using the Illumina NovaSeq 6000 S4.

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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.

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Project description:In addition to the glucocorticoids, the glucocorticoid receptor (GR) is regulated by post-translational modifications, including SUMOylation. We have analyzed how SUMOylation influences the activity of endogenous GR target genes and the receptor chromatin binding by using isogenic HEK293 cells expressing wild-type GR (wtGR) or SUMOylation-defective GR (GR3KR). Gene expression profiling revealed that both dexamethasone up- and down-regulated genes are affected by the GR sumoylation and that the affected genes are significantly associated with pathways of cellular proliferation and survival. The GR3KR-expressing cells proliferated more rapidly and their anti-proliferative response to dexamethasone was less pronounced than in the wtGR-expressing cells. ChIP-seq analyses indicated that the SUMOylation modulates the chromatin occupancy of GR on several loci associated with cellular growth in a fashion which parallels with their differential dexamethasone-regulated expression between the two cell lines. Moreover, genome-wide SUMO-2/3 marks, which were generally associated with active chromatin, showed markedly higher overlap with the wtGR cistrome than with the GR3KR cistrome. In sum, our results indicate that the SUMOylation does not simply repress the GR activity, but regulates the activity of the receptor in a target locus selective fashion, playing an important role in controlling the GR activity on genes influencing cell growth. Total RNA isolated from isogenic HEK293 cell lines stably expressing either wild-type GR (wtGR) or SUMOylation-defective GR (GR3KR) treated with either vehicle (EtOH) or 100 nM of dexamethasone (dex) for 6h. All conditions are performed in triplicate

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The HSV-1 ICP22 protein selectively impairs histone repositioning upon Pol II transcription downstream of genes (Omni-ATAC-seq I)

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