ATAC-Seq analysis of EZH2 mutants in isogenic cell lines
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA984170 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR24927137.fastq.gz | Fastqsanger.gz | |||
| SRR24927138.fastq.gz | Fastqsanger.gz | |||
| SRR24927139_1.fastq.gz | Fastqsanger.gz | |||
| SRR24927139_2.fastq.gz | Fastqsanger.gz | |||
| SRR24927140.fastq.gz | Fastqsanger.gz |
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Project description:EZH2 mutants in isogenic cell lines
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Project description:The Enhancer of Zeste 2 Polycomb Repressive Complex 2 Subunit (EZH2) is an essential epigenetic modifier able to methylate lysine 27 on histone H3 (H3K27) to induce chromatin compaction, protein complex recruitment and ultimately transcriptional repression. Hematologic malignancies, including Diffuse Large B cell lymphoma (DLBCL) and Acute myeloid leukemia (AML) have shown a high EZH2-mutation frequency (>20%) associated with poor clinical outcomes. Particularly, two distinct oncogenic mutations, so-called gain-of-function (Y641F and A677G) and loss-of-function (H689A and F667I) are found in the catalytic domain of EZH2. In this study, a comprehensive multi-omics approach was employed to characterize downstream effects of H3K27me3 deposition driven by EZH2 mutations. Human embryonic kidney cells (HEK293T) were transfected to generate three stable EZH2 mutants: EZH2(Y641F), EZH2(A677G), and EZH2(H689A/F667I), which were validated via immunoblotting and DIA-MS-based histone profiling assay. Subsequently, constructs were analyzed under a comprehensive multi-omics approach including: 1) Cleavage Under Targets and Tagmentation (CUT&Tag); 2) chromatin accessibility characterization using the assay for transposase-accessible chromatin with sequencing (ATAC-Seq); 3) transcriptomics (RNA-Seq); 4) label-free whole-cell proteomics, acquired with a Bruker timsTOF Pro HPLC-MS/MS with Ion Mobility, and 5) MS-based untargeted metabolomics, in positive and negative ionization MS/MS mode, acquired with an Agilent 6545 QTOF with a 1290 UHPLC system and HILIC column. Total coverage comprised over 21,000 chromatin regions, 18,000 transcripts, 6,000 proteins and 400 metabolic features. Pair-wise comparison using univariate and supervised multivariate statistical methods revealed significant differences between constructs in each omic level. Furthermore, effector pathway analysis combining omics data revealed distinctive reprogramming effects for each EZH2 mutant.
2023-07-17 | GSE235010 | GEO
Project description:The Enhancer of Zeste 2 Polycomb Repressive Complex 2 Subunit (EZH2) is an essential epigenetic modifier able to methylate lysine 27 on histone H3 (H3K27) to induce chromatin compaction, protein complex recruitment and ultimately transcriptional repression. Hematologic malignancies, including Diffuse Large B cell lymphoma (DLBCL) and Acute myeloid leukemia (AML) have shown a high EZH2-mutation frequency (>20%) associated with poor clinical outcomes. Particularly, two distinct oncogenic mutations, so-called gain-of-function (Y641F and A677G) and loss-of-function (H689A and F667I) are found in the catalytic domain of EZH2. In this study, a comprehensive multi-omics approach was employed to characterize downstream effects of H3K27me3 deposition driven by EZH2 mutations. Human embryonic kidney cells (HEK293T) were transfected to generate three stable EZH2 mutants: EZH2(Y641F), EZH2(A677G), and EZH2(H689A/F667I), which were validated via immunoblotting and DIA-MS-based histone profiling assay. Subsequently, constructs were analyzed under a comprehensive multi-omics approach including: 1) Cleavage Under Targets and Tagmentation (CUT&Tag); 2) chromatin accessibility characterization using the assay for transposase-accessible chromatin with sequencing (ATAC-Seq); 3) transcriptomics (RNA-Seq); 4) label-free whole-cell proteomics, acquired with a Bruker timsTOF Pro HPLC-MS/MS with Ion Mobility, and 5) MS-based untargeted metabolomics, in positive and negative ionization MS/MS mode, acquired with an Agilent 6545 QTOF with a 1290 UHPLC system and HILIC column. Total coverage comprised over 21,000 chromatin regions, 18,000 transcripts, 6,000 proteins and 400 metabolic features. Pair-wise comparison using univariate and supervised multivariate statistical methods revealed significant differences between constructs in each omic level. Furthermore, effector pathway analysis combining omics data revealed distinctive reprogramming effects for each EZH2 mutant.
2023-07-17 | GSE235013 | GEO
Project description:The Enhancer of Zeste 2 Polycomb Repressive Complex 2 Subunit (EZH2) is an essential epigenetic modifier able to methylate lysine 27 on histone H3 (H3K27) to induce chromatin compaction, protein complex recruitment and ultimately transcriptional repression. Hematologic malignancies, including Diffuse Large B cell lymphoma (DLBCL) and Acute myeloid leukemia (AML) have shown a high EZH2-mutation frequency (>20%) associated with poor clinical outcomes. Particularly, two distinct oncogenic mutations, so-called gain-of-function (Y641F and A677G) and loss-of-function (H689A and F667I) are found in the catalytic domain of EZH2. In this study, a comprehensive multi-omics approach was employed to characterize downstream effects of H3K27me3 deposition driven by EZH2 mutations. Human embryonic kidney cells (HEK293T) were transfected to generate three stable EZH2 mutants: EZH2(Y641F), EZH2(A677G), and EZH2(H689A/F667I), which were validated via immunoblotting and DIA-MS-based histone profiling assay. Subsequently, constructs were analyzed under a comprehensive multi-omics approach including: 1) Cleavage Under Targets and Tagmentation (CUT&Tag); 2) chromatin accessibility characterization using the assay for transposase-accessible chromatin with sequencing (ATAC-Seq); 3) transcriptomics (RNA-Seq); 4) label-free whole-cell proteomics, acquired with a Bruker timsTOF Pro HPLC-MS/MS with Ion Mobility, and 5) MS-based untargeted metabolomics, in positive and negative ionization MS/MS mode, acquired with an Agilent 6545 QTOF with a 1290 UHPLC system and HILIC column. Total coverage comprised over 21,000 chromatin regions, 18,000 transcripts, 6,000 proteins and 400 metabolic features. Pair-wise comparison using univariate and supervised multivariate statistical methods revealed significant differences between constructs in each omic level. Furthermore, effector pathway analysis combining omics data revealed distinctive reprogramming effects for each EZH2 mutant.
2023-07-17 | GSE235011 | GEO
Project description:The Enhancer of Zeste 2 Polycomb Repressive Complex 2 Subunit (EZH2) is an essential epigenetic modifier able to methylate lysine 27 on histone H3 (H3K27) to induce chromatin compaction, protein complex recruitment and ultimately transcriptional repression. Hematologic malignancies, including Diffuse Large B cell lymphoma (DLBCL) and Acute myeloid leukemia (AML) have shown a high EZH2-mutation frequency (>20%) associated with poor clinical outcomes. Particularly, two distinct oncogenic mutations, so-called gain-of-function (Y641F and A677G) and loss-of-function (H689A and F667I) are found in the catalytic domain of EZH2. In this study, a comprehensive multi-omics approach was employed to characterize downstream effects of H3K27me3 deposition driven by EZH2 mutations. Human embryonic kidney cells (HEK293T) were transfected to generate three stable EZH2 mutants: EZH2(Y641F), EZH2(A677G), and EZH2(H689A/F667I), which were validated via immunoblotting and DIA-MS-based histone profiling assay. Subsequently, constructs were analyzed under a comprehensive multi-omics approach including label-free whole-cell proteomics, acquired with a Bruker timsTOF Pro HPLC-MS/MS with Ion Mobility. Important protein interactors at nuclear level were dysregulated, such as SMYD3 (SET and MYND domain containing 3), NSD2 (nuclear receptor binding SET domain protein 2) and CHD7 (chromodomain helicase DNA binding protein 7), suggesting a cooperative network of chromatin remodelers for gene expression reprogramming.
2023-10-24 | PXD042875 | Pride
Project description:The Enhancer of Zeste 2 Polycomb Repressive Complex 2 Subunit (EZH2) is an essential epigenetic modifier able to methylate lysine 27 on histone H3 (H3K27) to induce chromatin compaction, protein complex recruitment and ultimately transcriptional repression. Hematologic malignancies, including Diffuse Large B cell lymphoma (DLBCL) and Acute myeloid leukemia (AML) have shown a high EZH2-mutation frequency (>20%) associated with poor clinical outcomes. Particularly, two distinct oncogenic mutations, so-called gain-of-function (Y641F and A677G) and loss-of-function (H689A and F667I) are found in the catalytic domain of EZH2. In this study, a comprehensive multi-omics approach was employed to characterize downstream effects of H3K27me3 deposition driven by EZH2 mutations. Human embryonic kidney cells (HEK293T) were transfected to generate three stable EZH2 mutants: EZH2(Y641F), EZH2(A677G), and EZH2(H689A/F667I), which were validated via immunoblotting and DIA-MS-based histone profiling assay. The histone profiling assay demonstrated a significant increase of approximately two-fold in H3.1/H3.3K27me3 for Y641F EZH2 mutant. There was a modest increase in the combinatorial PTMs H3.1/H3.3K27me3K36me1 and a significant depletion in H3.1 and H3.3 K27me2. The most depleted peptide was H3.3K27me2K36me2. For the A677G EZH2 mutant, the assay demonstrated an enrichment on H3.1/H3.3K27me3, combinatorial K27me3K36me1 and a slight increase in K27me1 and K18ac but only on H3.1. The most depleted peptide was H3.3K27me2K36me2. The H689A/F667I cell line has the most alterations in global histone PTMs. The most highly enriched were H3.1/H3.3K36me2, H3.1K9me3 and H3.3K36me1. The most depleted modifications were H3.1K23ac and H3.1K27me3.
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