Abstract 3724: RAI2 controls polycomb-mediated repression of CDKN1A by its interaction with CtBP1

Abstract RAI2 has initially been identified as a metastasis-associated gene in breast cancer. Recently, we found that increased RAI2 protein in primary tumors predicts early biochemical relapse of prostate cancer patients. On the molecular level, RAI2 interacts with CtBP1 as transcriptional co-repressors via a non-consensus tandem ALDLS-motif in hormone-dependent cancer cells. In this study, we aim to investigate the cell-biological relevance of this molecular interaction in prostate cancer cells using the CDKN1A gene as an example of a transcriptional target of CtBP-mediated gene regulation. To analyze a molecular relation between the RAI2/CtBP1 interaction and repressor of the CDKN1A gene, we first applied a transactivation assay in 293T cells, transiently transfected with CtBP1 and different RAI2 protein variants. Next, we depleted the RAI2 protein in VCaP prostate cancer cells. We used this cell line model to analyze gene expression and protein concertation of RAI2, CtBPs and p21 under different conditions. We studied the subcellular localization and chromatin binding of the proteins of interest by confocal laser-scanning microscopy and ChIP analysis. The gene reporter assay revealed that combined overexpression of RAI2 and CtBP1 reliefs the repression of the proximal CDKN1A-promotor and that RAI2 with an intact ALDLS tandem motif is required for this process. RAI2 depletion in VCaP cells resulted in a significant reduction of both CtBP1 and CtBP2 and almost complete abolishment of p21 protein levels, which is accompanied by reduced interaction of RAI2 with CtBP1 in nuclei. In parental VCaP cells, genotoxic stress significantly induced CDKNA1 gene expression and p21 protein concentration. In contrast, in RAI2-depleted VCaP cells we did not observe any of these effects, demonstrating the requirement of RAI2 to cause the relief of CDKNA1 repression. RAI2 together with CtBP1 appeared as definite foci in the nuclei of VCaP cells that are co-localized with key factors of polycomb 1 and 2. The ChIP analysis revealed that RAI2-depletion significantly induced binding of CtBPs to the CDKN1A promoter. In contrast, the binding of the polycomb protein LCoR to chromatin is significantly reduced in RAI2-depleted cells. Irrespective of the RAI2 status, the EZH2 binding to the chromatin remained unaltered, whereas a significant increase in H3K27me3 in RAI2-depleted cells was observed. In summary, we found that interruption of the interaction of RAI2 with CtBP1 is leading to increased chromatin binding of CtBP1 and trimethylation of H3K27 of the CDKN1A promotor, which is associated with repression of CDKN1A gene expression. We conclude that the molecular interaction of RAI2 with CtBP1 is a new molecular mechanism of corepression by modulating the histone-modifications of the target gene. Citation Format: Sarah Greimeier, Bettina Steinbach, Simon Sander, Lina Merkens, Nishit Goradia, Matthias Wilmanns, Eric Metzger, Klaus Pantel, Stefan Werner. RAI2 controls polycomb-mediated repression of CDKN1A by its interaction with CtBP1. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3724.