Abstract
The down-regulation of the tumor-suppressor gene RASSF1A has been shown to increase cell proliferation in several tumors. RASSF1A expression is regulated through epigenetic events involving the polycomb repressive complex 2 (PRC2); however, the molecular mechanisms modulating the recruitment of this epigenetic modifier to the RASSF1 locus remain largely unknown. Here, we identify and characterize ANRASSF1, an endogenous unspliced long noncoding RNA (lncRNA) that is transcribed from the opposite strand on the RASSF1 gene locus in several cell lines and tissues and binds PRC2. ANRASSF1 is transcribed through RNA polymerase II and is 5′-capped and polyadenylated; it exhibits nuclear localization and has a shorter half-life compared with other lncRNAs that bind PRC2. ANRASSF1 endogenous expression is higher in breast and prostate tumor cell lines compared with non-tumor, and an opposite pattern is observed for RASSF1A. ANRASSF1 ectopic overexpression reduces RASSF1A abundance and increases the proliferation of HeLa cells, whereas ANRASSF1 silencing causes the opposite effects. These changes in ANRASSF1 levels do not affect the RASSF1C isoform abundance. ANRASSF1 overexpression causes a marked increase in both PRC2 occupancy and histone H3K27me3 repressive marks, specifically at the RASSF1A promoter region. No effect of ANRASSF1 overexpression was detected on PRC2 occupancy and histone H3K27me3 at the promoter regions of RASSF1C and the four other neighboring genes, including two well-characterized tumor suppressor genes. Additionally, we demonstrated that ANRASSF1 forms an RNA/DNA hybrid and recruits PRC2 to the RASSF1A promoter. Together, these results demonstrate a novel mechanism of epigenetic repression of the RASSF1A tumor suppressor gene involving antisense unspliced lncRNA, in which ANRASSF1 selectively represses the expression of the RASSF1 isoform overlapping the antisense transcript in a location-specific manner. In a broader perspective, our findings suggest that other non-characterized unspliced intronic lncRNAs transcribed in the human genome might contribute to a location-specific epigenetic modulation of genes.
Highlights
RASSF1A (RAS-association domain family member 1A) is a tumor suppressor gene that modulates a broad range of cellular functions essential for normal growth, such as the maintenance of genomic stability, cell cycle control, the modulation of apoptosis, and cell motility and invasion [1]
Repression is effected by DNA hypermethylation of the RASSF1A promoter, which in turn is triggered through histone H3K9/H3K27 trimethylation repressive marks
We identified a long noncoding RNA, termed ANRASSF1 for antisense noncoding RASSF1, that is transcribed from the opposite strand of the RASSF1A gene and is responsible for recruiting polycomb repressive complex 2 (PRC2) to the RASSF1A promoter region in a highly location-specific manner
Summary
RASSF1A (RAS-association domain family member 1A) is a tumor suppressor gene that modulates a broad range of cellular functions essential for normal growth, such as the maintenance of genomic stability, cell cycle control, the modulation of apoptosis, and cell motility and invasion [1]. The biological relevance of only two isoforms, RASSF1A and RASSF1C, has been demonstrated. Both isoforms are ubiquitously expressed in non-tumor tissues, whereas in tumors and tumor cell lines, the expression of RASSF1A is frequently low, leading to increased cell proliferation [3]. The epigenetic silencing of RASSF1A requires the HOXB3-mediated induction of DNMT3B DNA methyltransferase expression and the recruitment of the DNMT3B protein to the RASSF1A promoter [10]. MYC is required for PRC2 recruitment to the RASSF1A promoter [10], MYC is generally not sufficient to recruit PRC2 [10,11], and other regulatory factors and mechanisms underlying the recruitment of this epigenetic silencing machinery have not yet been identified
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