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, 12 (12), 1818-28

Metastasis-associated Protein Ribosomal RNA Processing 1 Homolog B (RRP1B) Modulates Metastasis Through Regulation of Histone Methylation

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Metastasis-associated Protein Ribosomal RNA Processing 1 Homolog B (RRP1B) Modulates Metastasis Through Regulation of Histone Methylation

Minnkyong Lee et al. Mol Cancer Res.

Abstract

Overexpression of ribosomal RNA processing 1 homolog B (RRP1B) induces a transcriptional profile that accurately predicts patient outcome in breast cancer. However, the mechanism by which RRP1B modulates transcription is unclear. Here, the chromatin-binding properties of RRP1B were examined to define how it regulates metastasis-associated transcription. To identify genome-wide RRP1B-binding sites, high-throughput ChIP-seq was performed in the human breast cancer cell line MDA-MB-231 and HeLa cells using antibodies against endogenous RRP1B. Global changes in repressive marks such as histone H3 lysine 9 trimethylation (H3K9me3) were also examined by ChIP-seq. Analysis of these samples identified 339 binding regions in MDA-MB-231 cells and 689 RRP1B-binding regions in HeLa cells. Among these, 136 regions were common to both cell lines. Gene expression analyses of these RRP1B-binding regions revealed that transcriptional repression is the primary result of RRP1B binding to chromatin. ChIP-reChIP assays demonstrated that RRP1B co-occupies loci with decreased gene expression with the heterochromatin-associated proteins, tripartite motif-containing protein 28 (TRIM28/KAP1), and heterochromatin protein 1-α (CBX5/HP1α). RRP1B occupancy at these loci was also associated with higher H3K9me3 levels, indicative of heterochromatinization mediated by the TRIM28/HP1α complex. In addition, RRP1B upregulation, which is associated with metastasis suppression, induced global changes in histone methylation.

Implications: RRP1B, a breast cancer metastasis suppressor, regulates gene expression through heterochromatinization and transcriptional repression, which helps our understanding of mechanisms that drive prognostic gene expression in human breast cancer.

Conflict of interest statement

The authors disclose no potential conflicts of interest.

Figures

Figure 1
Figure 1. RRP1B suppresses metastasis in vivo and decreases cell invasiveness in vitro
A cell proliferation assay. B, soft agar assay. C, trans-well invasion assays. D, pulmonary surface metastases from intravenous injection of RRP1B over-expressing cells and control cells in NU/J mice. All data represent the average of three biological replicates.
Figure 2
Figure 2. Over-expression of RRP1B in MDA-MB-231 cells causes a significant change in gene expression
Global gene expression was quantified in five clonal isolates of MDA-MB-231 cells ectopically expressing RRP1B and control. Heatmap represents relative gene expression levels in the five clonal isolates of MDA-MB-231 cells ectopically expressing RRP1B and control detected by microarray analysis (WT; RRP1B, ctrl; control).
Figure 3
Figure 3. RRP1B interacts with multiple chromatin regions
A, Venn diagram of unique and common binding regions detected by ChIP-seq in MDA-MB-231 and HeLa cells. B, division of chromatin regions based on relative genic locations. C, verification of common RRP1B binding regions via ChIP-qPCR in HeLa cells, and D, MDA-MB-231 cells. *; P ≤ 0.05 compared to IgG controls.
Figure 4
Figure 4. RRP1B-binding regions are frequently associated with decreased gene expression
The consequences of RRP1B binding were examined in RRP1B over-expressing cells and compared to that of RRP1B knockdown cells. Fold change was calculated using the control for each treatment. RRP1B siRNA represents the average fold change of cells transfected with either one of the two different RRP1B siRNA sequences. *; P ≤ 0.05, **; P ≤ 0.005 compared to RRP1B over-expressed.
Figure 5
Figure 5. RRP1B interacts with TRIM28 and HP1α at regions associated with H3K9me3 and decreased gene expression
A, ChIP-reChIP with TRIM28 and RRP1B antibodies in MDA-MB-231 cells. *; P ≤ 0.05 compared to both IgG/TRIM28 and IgG/RRP1B controls. B, ChIP-reChIP with HP1α and RRP1B antibodies in MDA-MB-231 cells. *; P ≤ 0.05 compared to both IgG/HP1α and IgG/RRP1B controls. C, H3K9me3 ChIP-qPCR in MDA-MB-231 clonal isolates over-expressing RRP1B and control cell lines. D, H3K9me3 ChIP-qPCR in MDA-MB-231 cells transfected with either RRP1B siRNA or negative control siRNA. E, Venn diagram of unique and common H3K9me3-enriched regions identified by ChIP-seq in MDA-MB-231 clonal isolates over-expressing RRP1B and control cell lines.

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