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Series GSE235010 Query DataSets for GSE235010
Status Public on Jul 17, 2023
Title ATAC-Seq analysis of EZH2 mutants in isogenic cell lines
Organism Homo sapiens
Experiment type Genome binding/occupancy profiling by high throughput sequencing
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.
 
Overall design Genome-wide analysis of chromatin accesibility was performed via via Assay for Transposase-Accessible Chromatin with sequencing (ATAC-Seq) 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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Submission date Jun 15, 2023
Last update date Jul 17, 2023
Contact name Julian Aldana
E-mail(s) aldanaaroca.1@osu.edu
Organization name Ohio State University
Street address 460 W 12th Ave
City Columbus
State/province Ohio
ZIP/Postal code 43210
Country USA
 
Platforms (1)
GPL24676 Illumina NovaSeq 6000 (Homo sapiens)
Samples (8)
GSM7493124 ATACSeq, HEK293T cells, EZH2 WT, replicate 1
GSM7493125 ATACSeq, HEK293T cells, EZH2 WT, replicate 2
GSM7493126 ATACSeq, HEK293T cells, EZH2 Y641F, replicate 1
This SubSeries is part of SuperSeries:
GSE235014 EZH2 mutants in isogenic cell lines
Relations
BioProject PRJNA984170

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Supplementary file Size Download File type/resource
GSE235010_RAW.tar 1.4 Gb (http)(custom) TAR (of BROADPEAK, BW)
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file
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