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| Status |
Public on May 29, 2023 |
| Title |
A monoclonal antibody raised against human EZH2 cross-reacts with the RNA-binding protein SAFB [ChIP-seq] |
| Organism |
Mus musculus |
| Experiment type |
Genome binding/occupancy profiling by high throughput sequencing
|
| Summary |
The Polycomb Repressive Complex 2 (PRC2) is a conserved enzyme that tri-methylates Lysine 27 on Histone 3 (H3K27me3) to promote gene silencing. PRC2 is remarkably responsive to the expression of certain long noncoding RNAs (lncRNAs). In the most notable example, PRC2 is recruited to the X-chromosome shortly after expression of the lncRNA Xist begins during X-chromosome inactivation. However, the mechanisms by which lncRNAs recruit PRC2 to chromatin are not yet clear. We report that a broadly used rabbit monoclonal antibody raised against human EZH2, a catalytic subunit of PRC2, cross-reacts with an RNA-binding protein called Scaffold Attachment Factor B (SAFB) in mouse embryonic stem cells (ESCs) under buffer conditions that are commonly used for chromatin immunoprecipitation (ChIP). Knockout of EZH2 in ESCs demonstrated that the antibody is specific for EZH2 by western blot (no cross-reactivity). Likewise, comparison to previously published datasets confirmed that the antibody recovers PRC2-bound sites by ChIP-Seq. However, RNA-IP from formaldehyde-crosslinked ESCs using ChIP wash conditions recovers distinct peaks of RNA association that co-localize with peaks of SAFB and whose enrichment disappears upon knockout of SAFB but not EZH2. IP and mass spectrometry-based proteomics in wild-type and EZH2 knockout ESCs confirm that the EZH2 antibody recovers SAFB in an EZH2-independent manner. Our data highlight the importance of orthogonal assays when studying interactions between chromatin-modifying enzymes and RNA.
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| Overall design |
CS5246 EZH2 ChIP in WT mESCs
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| Contributor(s) |
Cherney RE, Mills CA, Herring LE, Braceros AK, Calabrese J |
| Citation(s) |
37066147, 37283223 |
| |
| Submission date |
Mar 21, 2023 |
| Last update date |
Sep 08, 2023 |
| Contact name |
Joseph Mauro Calabrese |
| Organization name |
University of North Carolina at Chapel Hill
|
| Department |
Pharmacology
|
| Lab |
Calabrese
|
| Street address |
120 Mason Farm Road
|
| City |
Chapel Hill |
| State/province |
North Carolina |
| ZIP/Postal code |
27599 |
| Country |
USA |
| |
|
| Platforms (1) |
| GPL19057 |
Illumina NextSeq 500 (Mus musculus) |
|
| Samples (1) |
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| This SubSeries is part of SuperSeries: |
| GSE227893 |
A monoclonal antibody raised against human EZH2 cross-reacts with the RNA-binding protein SAFB |
|
| Relations |
| BioProject |
PRJNA947239 |
