Abstract 123: The breast cancer metastasis suppressor RRP1B modulates metastasis through regulation of histone methylation
Authors: Lee Minnkyong , Dworkin Amy M. , Lichtenberg Jens , Patel Shashank J. , Gildea Derek , Bodine David M. , Crawford Nigel PS 
Published: May 2015

Ribosomal RNA Processing 1 Homolog B (Rrp1b) is a germline breast cancer metastasis suppressor that regulates transcription to induce a highly prognostic gene expression signature. Although RRP1B interacts with chromatin- and mRNA splicing-associated factors, the mechanisms by which RRP1B modulates metastasis-associated gene expression are unclear. In this study, we examined the chromatin-binding properties of RRP1B to determine the mechanism by which it regulates metastasis-associated gene transcription.

Genome-wide chromatin binding sites for endogenous RRP1B were defined using chromatin immunoprecipitation-sequencing (ChIP-seq) in Hela and MDA-MB-231 cells. ChIP-seq analysis of these samples identified 689 RRP1B binding regions in Hela and 339 binding regions in MDA-MB-231 cells. Among these, 136 regions were common to both cell lines. The expression of 40 genes located at or near these binding regions was examined by qPCR in MDA-MB-231 cells stably over-expressing RRP1B. These experiments demonstrated that RRP1B binding is primarily associated with transcriptional repression. Co-occupancy of RRP1B with the heterochromatin-associated proteins TRIM28 and HP1α at these loci were observed through ChIP-reChIP assays. Interaction between RRP1B and TRIM28 and HP1α was confirmed through co-immunoprecipitation.

TRIM28 and HP1α have been shown to induce trimethylation of histone H3 at the lysine-9 position (H3K9me3) via the histone methyltransferase, SETDB1. Increases in H3K9me3 are typically associated with heterochromatinization and transcriptional repression. Given that RRP1B interacts with both TRIM28 and HP1α, and binds at common chromatin regions with these factors, we hypothesized that RRP1B is also implicated in increasing H3K9me3 levels at these loci. To examine the effect of RRP1B on H3K9me3, ChIP assays were performed in MDA-MB-231 cells stably over-expressing RRP1B and control cells with an antibody against H3K9me3. ChIP-qPCR demonstrated that chromatin regions that displayed co-occupancy of RRP1B with TRIM28 and HP1α have increased levels of H3K9me3 compared to that of the control. Additionally, ChIP-seq for H3K9me3 in cells stably over-expressing RRP1B demonstrated that RRP1B causes a global increase in H3K9me3 levels compared to control.

These data collectively demonstrate that RRP1B regulates gene expression at the transcriptional level through an association with the transcriptional repressors TRIM28 and HP1α. Furthermore, the prominent mechanism that the metastasis suppressor RRP1B uses to regulate transcription is through epigenetic modifications, such as histone methylation.

Citation Format: Minnkyong Lee, Amy M. Dworkin, Jens Lichtenberg, Shashank J. Patel, Derek Gildea, David M. Bodine, Nigel PS Crawford. The breast cancer metastasis suppressor RRP1B modulates metastasis through regulation of histone methylation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 123. doi:10.1158/1538-7445.AM2014-123