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| Status |
Public on Jul 17, 2023 |
| Title |
H3K27me3 CUT&Tag analysis of EZH2 mutants in isogenic cell lines |
| Organism |
Homo sapiens |
| Experiment type |
Genome binding/occupancy profiling by high throughput sequencing
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| Summary |
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.
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| Overall design |
Genome-wide analysis of H3K27me3 via Cleavage Under Targets and Tagmentation (CUT&Tag) in a set of stable transfected HEK293T cell lines with EZH2 mutants, including EZH2(WT), EZH2(Y641F), EZH2(A677G), and EZH2(H689A/F667I) to investigate the effects of common single point mutations in the active site of EZH2.
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| Citation missing |
Has this study been published? Please login to update or notify GEO. |
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| Submission date |
Jun 15, 2023 |
| Last update date |
Jul 17, 2023 |
| Contact name |
Julian Aldana |
| E-mail(s) |
aldanaaroca.1@osu.edu
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| Organization name |
Ohio State University
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| Street address |
460 W 12th Ave
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| City |
Columbus |
| State/province |
Ohio |
| ZIP/Postal code |
43210 |
| Country |
USA |
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| Platforms (1) |
| GPL24676 |
Illumina NovaSeq 6000 (Homo sapiens) |
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| Samples (8)
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| GSM7493132 |
H3K27me3, CUT_Tag, HEK293T cells, EZH2 WT, replicate 1 |
| GSM7493133 |
H3K27me3, CUT_Tag, HEK293T cells, EZH2 WT, replicate 2 |
| GSM7493134 |
H3K27me3, CUT_Tag, HEK293T cells, EZH2 Y641F, replicate 1 |
| GSM7493135 |
H3K27me3, CUT_Tag, HEK293T cells, EZH2 Y641F, replicate 2 |
| GSM7493136 |
H3K27me3, CUT_Tag, HEK293T cells, EZH2 A677G, replicate 1 |
| GSM7493137 |
H3K27me3, CUT_Tag, HEK293T cells, EZH2 A677G, replicate 2 |
| GSM7493138 |
H3K27me3, CUT_Tag, HEK293T cells, EZH2 H689A/F667I, replicate 1 |
| GSM7493139 |
H3K27me3, CUT_Tag, HEK293T cells, EZH2 H689A/F667I, replicate 2 |
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| This SubSeries is part of SuperSeries: |
| GSE235014 |
EZH2 mutants in isogenic cell lines |
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| Relations |
| BioProject |
PRJNA984169 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE235011_RAW.tar |
2.1 Gb |
(http)(custom) |
TAR (of BROADPEAK, BW) |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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