A monoclonal antibody raised against human EZH2 cross-reacts with the RNA-binding protein SAFB

• Published in: Biology open Vol. 12; no. 6
• Main Authors: Cherney, Rachel E, Mills, Christine A, Herring, Laura E, Braceros, Aki K, Calabrese, J Mauro
• Format: Journal Article
• Language: English
• Published: England The Company of Biologists Ltd 15.06.2023; The Company of Biologists
• Subjects: Animals; Antibodies, Monoclonal - genetics; Antibodies, Monoclonal - metabolism; Chromatin; Enhancer of Zeste Homolog 2 Protein - genetics; Enhancer of Zeste Homolog 2 Protein - metabolism; ezh2; Humans; lncrna; Matrix Attachment Region Binding Proteins - genetics; Matrix Attachment Region Binding Proteins - metabolism; Methods & Techniques; Mice; Mice, Knockout; Nuclear Matrix-Associated Proteins - genetics; Nuclear Matrix-Associated Proteins - metabolism; polycomb; Polycomb Repressive Complex 2 - genetics; Polycomb Repressive Complex 2 - metabolism; prc2; Receptors, Estrogen - genetics; Receptors, Estrogen - metabolism; RNA, Long Noncoding - genetics; RNA-Binding Proteins - genetics; safb; xist; LncRNA; Polycomb; Xist; PRC2; SAFB; EZH2
• ISSN: 2046-6390; 2046-6390
• DOI: 10.1242/bio.059955

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.