Integrative proteomics reveals increased non-degradative ubiquitylation during CD4+ T cell activation
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ABSTRACT:
PROVIDER: PRJNA526619 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR8713223_1.fastq.gz | Fastqsanger.gz | |||
| SRR8713223_2.fastq.gz | Fastqsanger.gz | |||
| SRR8713224_1.fastq.gz | Fastqsanger.gz | |||
| SRR8713224_2.fastq.gz | Fastqsanger.gz | |||
| SRR8713225_1.fastq.gz | Fastqsanger.gz |
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Project description:In spite of gathering evidence that ubiquitylation can direct non-degradative outcomes, most investigations of ubiquitylation in T cells remain focused on degradation. Here we integrated proteomic and transcriptomic datasets to establish a framework for predicting degradative or non-degradative outcomes of ubiquitylation in primary CD4+ T cells. Di-glycine remnant proteomics revealed ubiquitylated proteins, while whole cell proteomic and transcriptomic data were used to predict whether ubiquitylated proteins showed evidence of degradation. Applying this analysis to ubiquitylated proteins with functions in TCR signaling led us to the prediction that early T cell activation promotes increased non-degradative ubiquitylation. Supporting this, we observed an increase in non-proteasome targeted K29, K33 and K63 polyubiquitin chains during T cell activation, while K48 chains appeared unchanged. This study reveals over 1,200 proteins that are ubiquitylated in primary CD4+ T cells and supports the relevance of non-proteasomally targeted ubiquitin chains in T cell signaling.
2020-03-11 | GSE128154 | GEO
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2022-09-26 | GSE138071 | GEO
Project description:Despite gathering evidence that ubiquitylation can direct non-degradative outcomes, most investigations of ubiquitylation in T cells have focused on degradation. Here, we integrated proteomic and transcriptomic datasets from primary mouse CD4+ T cells to establish a framework for predicting degradative or non-degradative outcomes of ubiquitylation. Di-glycine remnant profiling was used to reveal ubiquitylated proteins, which in combination with whole-cell proteomic and transcriptomic data allowed prediction of protein degradation. Analysis of ubiquitylated proteins identified by di-glycine remnant profiling indicated that activation of CD4+ T cells led to an increase in non-degradative ubiquitylation. This correlated with an increase in non-proteasome-targeted K29, K33 and K63 polyubiquitin chains. This study revealed over 1,200 proteins that were ubiquitylated in primary mouse CD4+ T cells and highlighted the relevance of non-proteasomally targeted ubiquitin chains in T cell signaling.
| S-EPMC7007700 | biostudies-literature
Project description:Deciphering lineage specification during early embryogenesis using integrative multi-layered proteomics [RNA-Seq]
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Project description:Histone variant H2A.Z occupies the promoters of active and poised, bivalent genes in ESCs to regulate developmental programs, yet how it contributes to these contrasting states is poorly understood. Here, we investigate the function of H2A.Z.1 mono-ubiquitylation (H2A.Z.1ub) by mutation of the PRC1 target residues (H2A.Z.1K3R3). We show that H2A.Z.1K3R3 is properly incorporated at target promoters in murine ESCs (mESCs), however, loss of mono-ubiquitylation leads to de-repression of bivalent genes, loss of Polycomb binding, and to faulty lineage commitment. Using quantitative proteomics, we find that tandem bromodomain proteins, including the BET family member Brd2, are enriched in H2A.Z.1 chromatin. We further show that Brd2 is gained at de-repressed promoters in H2A.Z.1K3R3 mESCs whereas Brd2 inhibition restores gene silencing at these sites. Together, our study reveals an antagonistic relationship between H2A.Z.1ub and Brd2 to regulate the transcriptional balance at bivalent genes to enable proper execution of developmental programs. ChIP-Seq analysis on mouse embryonic stem cells harboring H2A.Z or H2A.Z.K3R3 (3 C-terminal lysines mutated to arginines) tagged with YFP, in the presence of a knockdown hairpin targeting the endogenous H2A.Z transcript.
2016-01-21 | E-GEOD-53206 | biostudies-arrayexpress
Project description:Histone variant H2A.Z occupies the promoters of active and poised, bivalent genes in ESCs to regulate developmental programs, yet how it contributes to these contrasting states is poorly understood. Here, we investigate the function of H2A.Z.1 mono-ubiquitylation (H2A.Z.1ub) by mutation of the PRC1 target residues (H2A.Z.1K3R3). We show that H2A.Z.1K3R3 is properly incorporated at target promoters in murine ESCs (mESCs), however, loss of mono-ubiquitylation leads to de-repression of bivalent genes, loss of Polycomb binding, and to faulty lineage commitment. Using quantitative proteomics, we find that tandem bromodomain proteins, including the BET family member Brd2, are enriched in H2A.Z.1 chromatin. We further show that Brd2 is gained at de-repressed promoters in H2A.Z.1K3R3 mESCs whereas Brd2 inhibition restores gene silencing at these sites. Together, our study reveals an antagonistic relationship between H2A.Z.1ub and Brd2 to regulate the transcriptional balance at bivalent genes to enable proper execution of developmental programs. RNA-Seq analysis on mouse embryonic stem cells harboring H2A.Z or H2A.Z.K3R3 (3 C-terminal lysines mutated to arginines) tagged with YFP, in the presence of a knockdown hairpin targeting the endogenous H2A.Z transcript.
2016-01-21 | E-GEOD-53207 | biostudies-arrayexpress
Project description:Deciphering lineage specification during early embryogenesis in mouse gastruloids using integrative multi-layered proteomics
| PRJNA952600 | ENA
Project description:Histone variant H2A.Z occupies the promoters of active and poised, bivalent genes in ESCs to regulate developmental programs, yet how it contributes to these contrasting states is poorly understood. Here, we investigate the function of H2A.Z.1 mono-ubiquitylation (H2A.Z.1ub) by mutation of the PRC1 target residues (H2A.Z.1K3R3). We show that H2A.Z.1K3R3 is properly incorporated at target promoters in murine ESCs (mESCs), however, loss of mono-ubiquitylation leads to de-repression of bivalent genes, loss of Polycomb binding, and to faulty lineage commitment. Using quantitative proteomics, we find that tandem bromodomain proteins, including the BET family member Brd2, are enriched in H2A.Z.1 chromatin. We further show that Brd2 is gained at de-repressed promoters in H2A.Z.1K3R3 mESCs whereas Brd2 inhibition restores gene silencing at these sites. Together, our study reveals an antagonistic relationship between H2A.Z.1ub and Brd2 to regulate the transcriptional balance at bivalent genes to enable proper execution of developmental programs.
2016-01-21 | GSE67944 | GEO
Project description:The large family of SCF ubiquitin ligases catalyze ubiquitylation by bridging protein substrates and ubiquitin-modifying enzymes. S. cerevisiae SCFs employ a sole, essential enzyme, Cdc34, to build poly-ubiquitin chains required for degradation. However, humans have no less than six chain building enzymes associated with SCFs, including the long assumed to be essential Cdc34 orthologs, UBE2R1 and UBE2R2. Furthermore, uncertainty regarding the physiological concentrations of ubiquitin-modifying enzymes has hindered in vitro mechanistic work. Proteomics was used to estimate enzyme levels in cells, and ubiquitylation assays were employed to quantitatively compare enzyme activities. The results show that UBE2R2 alone has negligible ubiquitylation activity at physiological concentrations, and the ablation of UBE2R1/2 had no effect on the stability of SCF substrates in cells. A genome-wide CRISPR screen revealed that UBE2G1 buffers against the deletion of UBE2R1/2. UBE2G1 had robust in vitro activity with SCF, and UBE2G1 knockdown in cells lacking UBE2R1/2 resulted in stabilization of the SCF substrate p27 as well as the Cul3-RING ligase substrate NRF2. The results reveal the human SCF enzyme system is heavily buffered and suggest that SCF specificity is diversified by association with multiple enzyme partners.
2020-01-01 | GSE136175 | GEO