The Intronic Long Noncoding RNA <i>ANRASSF1</i> Recruits PRC2 to the <i>RASSF1A</i> Promoter, Reducing the Expression of <i>RASSF1A</i> and Increasing Cell Proliferation

<div><p>The down-regulation of the tumor-suppressor gene <i>RASSF1A</i> has been shown to increase cell proliferation in several tumors. <i>RASSF1A</i> 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 <i>RASSF1</i> locus remain largely unknown. Here, we identify and characterize <i>ANRASSF1</i>, an endogenous unspliced long noncoding RNA (lncRNA) that is transcribed from the opposite strand on the <i>RASSF1</i> gene locus in several cell lines and tissues and binds PRC2. <i>ANRASSF1</i> 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. <i>ANRASSF1</i> endogenous expression is higher in breast and prostate tumor cell lines compared with non-tumor, and an opposite pattern is observed for <i>RASSF1A</i>. <i>ANRASSF1</i> ectopic overexpression reduces <i>RASSF1A</i> abundance and increases the proliferation of HeLa cells, whereas <i>ANRASSF1</i> silencing causes the opposite effects. These changes in <i>ANRASSF1</i> levels do not affect the <i>RASSF1C</i> isoform abundance. <i>ANRASSF1</i> overexpression causes a marked increase in both PRC2 occupancy and histone H3K27me3 repressive marks, specifically at the <i>RASSF1A</i> promoter region. No effect of <i>ANRASSF1</i> overexpression was detected on PRC2 occupancy and histone H3K27me3 at the promoter regions of <i>RASSF1C</i> and the four other neighboring genes, including two well-characterized tumor suppressor genes. Additionally, we demonstrated that <i>ANRASSF1</i> forms an RNA/DNA hybrid and recruits PRC2 to the <i>RASSF1A</i> promoter. Together, these results demonstrate a novel mechanism of epigenetic repression of the <i>RASSF1A</i> tumor suppressor gene involving antisense unspliced lncRNA, in which <i>ANRASSF1</i> selectively represses the expression of the <i>RASSF1</i> 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.</p></div

Inverse correlation between <i>ANRASSF1</i> and <i>RASSF1A</i> expression in non-tumor and tumor cell lines.

<p>Expression of <i>ANRASSF1</i> and of <i>RASSF1A</i> in (A) breast and (B) prostate cell lines. Tumor cell lines (white bars) and non-tumor immortalized cell lines (hatched bars) were tested. These data show the means ± SD from two or three independent biological replicates for each cell line. The expression values in tumors are shown compared with the expression in the non-tumor cell line. These data are calculated relative to <i>HPRT1</i> expression.</p

<i>ANRASSF1</i> mediates recruitment of SUZ12 to the <i>RASSF1A</i> promoter.

<p>(A) RNase assay for detection of <i>ANRASSF1</i> using RT-qPCR in permeabilized HeLa cells treated with RNase inhibitor (black bar), RNase H (red bar) or RNase A (blue bar). RNA% for each of the two RNase treatments was calculated relative to the corresponding values for the RNase inhibitor. These data show the means ± SD from three independent experiments. (B) As a control, alpha-tubulin mRNA was measured using RT-qPCR in parallel under the same conditions as described in (A). These data show the means ± SD from three independent experiments. (C) RNase-ChIP assay with anti-SUZ12 antibody in permeabilized HeLa cells treated with either RNase inhibitor (black bar), RNase H (red bar) or RNase A (blue bar). The amount of DNA at the <i>RASSF1A</i> promoter region detected through qPCR in anti-SUZ12 samples was calculated in relation to the input. These data show the means ± SD from two independent experiments that were performed in triplicate. (D) The amount of DNA at the <i>RASSF1C</i> promoter region was measured under the same conditions as described in (C). (E–F) The amount of DNA at the <i>RASSF1A</i> and <i>RASSF1C</i> promoter regions was measured under the same conditions as in (C–D), except that an anti-DNMT3B antibody was used. (G–H) The amount of DNA at the <i>RASSF1A</i> and <i>RASSF1C</i> promoter regions was measured under the same conditions as in (C–D), except that an anti-RNA Pol II antibody was used.</p

Proposed model for <i>ANRASSF1</i> function at the <i>RASSF1</i> genomic <i>locus</i>.

<p>We postulate that lncRNA <i>ANRASSF1</i> (blue line) interacts with genomic DNA at the transcription site, forming an RNA/DNA hybrid, and recruits the chromatin-modifying PRC2 complex to the protein-coding <i>RASSF1A</i> gene promoter region. The recruitment of the PCR2 complex results in the selective modification of the histone H3K27 pattern of methylation (red circles) at the <i>RASSF1A</i> promoter, leading to a specific reduction in <i>RASSF1A</i> transcriptional activity with no effect on the <i>RASSF1C</i> transcription.</p

<i>ANRASSF1</i> interacts with PRC2 and affects its occupancy at the <i>RASSF1A</i> promoter.

<p>(A) Endogenous <i>ANRASSF1</i> levels bound to PRC2 were measured in HeLa cells through RNA IP with anti-SUZ12 relative to the input. A control IP with non-specific IgG was performed in parallel. As a negative control, <i>GAPDH</i> mRNA, which was not expected to bind to PRC2, was used. The percent input in the IP fractions was shown as the <i>ANRASSF1</i>/<i>GAPDH</i> ratio. These data show the means ± SD from three independent experiments. (B) lincRNA S<i>FPQ</i> is a positive control that binds to PRC2; RNA IP was assayed as in (A). These data show the means ± SD from three independent experiments. (C) ChIP assay using an anti-SUZ12 antibody in HeLa cells overexpressing <i>ANRASSF1</i> (pCEP4 <i>ANRASSF1</i>, black bars) or control cells (empty pCEP4, white bars). The promoter regions of the <i>RASSF1A</i> and <i>RASSF1C</i> isoforms and two other genes on either side of the <i>RASSF1</i> locus on chr 3 were investigated (the promoters are indicated with vertical lines in the scheme shown at the bottom of the figure). Control <i>GAPDH</i> was included as a gene not expected to be regulated through SUZ12. Control <i>HOXA9</i> is a gene regulated through SUZ12 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003705#pgen.1003705-Cao1" target="_blank">[34]</a> and encoded on chr 7. The amount of DNA in anti-SUZ12 samples at each promoter region detected through qPCR analysis was calculated in relation to the input. These data show the means ± SD from three independent experiments. *<i>t</i>-test <i>p</i><0.02 relative to control at the <i>RASSF1A</i> locus. No significant changes were detected at other loci. (D) ChIP analysis using an anti-H3K27me3 antibody in an assay similar to that described in (C), except that the enrichment was calculated relative to anti-H3 ChIP. These data show the means ± SD from three independent experiments. *<i>t</i>-test <i>p</i><0.02 relative to control at the <i>RASSF1A</i> locus. No significant changes were detected at the other loci. (E) ChIP analysis using an anti-DNMT3B antibody in an assay similar to that described in (C). These data show the means ± SD from three independent experiments. No significant change was observed. (F) DNA methylation at the <i>RASSF1A</i> promoter region was detected through qPCR with a methylation-dependent McrBC endonuclease assay in the <i>ANRASSF1</i>-overexpressing or control cells. The percentage of DNA remaining was calculated after comparing the amount of DNA amplified through qPCR following treatment with McrBC endonuclease with that following no-endonuclease treatment. These data show the means ± SD from three independent experiments. No significant change was observed.</p

Antisense noncoding RNA <i>ANRASSF1</i> is expressed within the <i>RASSF1</i> genomic locus and inversely correlated with <i>RASSF1</i> expression.

<p>(A) Schematic representation of the entire <i>RASSF1 locus</i> presented at the top. Protein-coding <i>RASSF1</i> gene isoforms (RefSeq-annotated) are shown in black; <i>ANRASSF1</i> evidence from an assembly of public ESTs is shown in light gray; the portions extended through 5′-end RACE and primer-walking or 3′-end RACE are shown in red, with a poly(A) segment detected through sequencing. The arrowheads define the orientation of the sequences. (B) <i>ANRASSF1</i> was detected in several human cell lines using end-point PCR in the antisense (AS), and not the sense (S), direction relative to the protein-coding gene. Strand-specific primers were used for the reverse-transcription (RT) (short half-arrow numbers 2 and 3 on <i>ANRASSF1</i> in panel A). RT reaction in the absence of primer was used as a negative control (Ctl). The short half-arrows numbered 1 and 4 indicate the primers used for the end-point PCR. (C) Strand-specific RNA-seq from poly(A)+ RNA of LNCaP cells. The abundance of the reads mapping within the <i>RASSF1</i> genomic locus is displayed in the diagram, and the color corresponds to the DNA strand of the transcripts. The red horizontal bar represents the consensus sequence from the assembly of RNA-seq reads mapping to the positive strand in this locus, generated using the Cufflinks tool. The gray bar represents the full-length <i>ANRASSF1</i>, obtained by RACE-PCR and Sanger sequencing. The arrowheads define the orientation of the sequences. (D) Expression of <i>RASSF1</i> and <i>ANRASSF1</i> measured with Affymetrix microarrays in HeLa, MDA-MB-231 and MCF-7 cells from GSE5823 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003705#pgen.1003705-Cappellen1" target="_blank">[54]</a>. (E) The expression of <i>RASSF1</i> and <i>ANRASSF1</i> measured with Affymetrix microarrays in Jurkat cells under mitotic stress induced through treatment with phorbol ester and ionomycin for 30 and 60 min from GSE11118 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003705#pgen.1003705-Byun1" target="_blank">[55]</a>.</p

<i>ANRASSF1</i> knockdown increases the <i>RASSF1A</i> isoform expression and decreases cell proliferation.

<p>(A) Expression levels of <i>ANRASSF1</i> in cells treated with siRNA <i>ANRASSF1</i> relative to the expression of <i>ANRASSF1</i> in control cells treated with scrambled siRNA (siRNA control). The relative expression was detected using RT-qPCR normalized to α-tubulin. These data show the means ± SD from three independent experiments. *<i>p</i><0.01 relative to control. (B) The relative expression levels of <i>RASSF1A</i> in siRNA <i>ANRASSF1</i> cells and siRNA control cells, as described in (A). These data show the means ± SD from three independent experiments. *<i>p</i><0.01 relative to the control. (C) The relative expression levels of <i>RASSF1C</i> in siRNA <i>ANRASSF1</i> cells and siRNA control cells, as described in (A). (D) Cell proliferation coefficients of siRNA <i>ANRASSF1</i> cells relative to the control cells (siRNA control). These data show the means ± SD from three independent experiments. *<i>p</i>< 0.02 relative to control.</p

Overexpression of <i>ANRASSF1</i> increases cell growth and decreases UVC- and staurosporine-mediated cell death.

<p>(A) Cell proliferation coefficients of <i>ANRASSF1</i>-transfected cells (pCEP4 <i>ANRASSF1</i>) relative to control cells (empty pCEP4), as measured using MTS assay. These data show the means ± SD from three independent experiments. *<i>t</i>-test <i>p</i><0.03 relative to control. (B) Effect of <i>ANRASSF1</i> overexpression on the cell population growth, as measured by counting the number of cells over time in culture and calculating the population doubling time. These data show the means ± SD from two independent experiments in triplicate. * <i>t</i>-test <i>p</i><0.05. (C) DNA content histograms showing the effect of <i>ANRASSF1</i> overexpression on the cell cycle at 48 h after exposure to UVC irradiation (40 J/m²). The cells were labeled with propidium iodide. Light gray indicates the sub-G1 population. (D) Percent of sub-G₁ population from experiments identical to (C). These data show the means ± SD from three independent replicate experiments. *<i>t</i>-test <i>p</i><0.05 relative to control. (E) Effect of <i>ANRASSF1</i> overexpression on cell survival in the presence of the cytotoxic drug staurosporine (100 nM). The changes in impedance were measured using the xCELLigence system to continuously monitor cell attachment to the culture plates. Three independent biological replicates for either condition (pCEP4 <i>ANRASSF1</i> or empty pCEP4) are shown, each representing the means ± SD of two or three technical replicates; the red lines show cells overexpressing <i>ANRASSF1</i>, and the blue lines show control cells carrying empty pCEP4) vector. *<i>t</i>-test <i>p</i><0.001, for the cell indices at 170 h.</p

PRC2 is specifically directed by <i>ANRASSF1</i> lncRNA to the <i>RASSF1A</i> promoter.

<p>(A) ChIP assay using an anti-SUZ12 antibody in HeLa cells transfected with an RNAi oligonucleotide antisense to <i>ANRASSF1</i> or with a control scrambled oligonucleotide. The amount of DNA at the <i>RASSF1A</i> promoter region detected using qPCR in the anti-SUZ12 samples was calculated in relation to the input. These data show the means ± SD from two independent experiments performed in triplicate. *<i>p</i><0.03 relative to control. (B) ChIP assay using an anti-DNMT3B antibody in an assay similar to (A). (C) DNA methylation at the <i>RASSF1A</i> promoter region was detected through qPCR in a methylation-dependent McrBC endonuclease assay in cells transfected with an RNAi oligonucleotide antisense to <i>ANRASSF1</i> or with a control scrambled oligonucleotide. The percent remaining DNA was calculated by comparing the amount of DNA at the promoter of <i>RASSF1A</i> amplified through qPCR following treatment with McrBC endonuclease against that amplified in the no-endonuclease treatment. These data show the means ± SD from three independent experiments. No significant change was observed.</p

Synergy of Omeprazole and Praziquantel <i>In Vitro</i> Treatment against <i>Schistosoma mansoni</i> Adult Worms

<div><p>Background</p><p>Treatment and morbidity control of schistosomiasis relies on a single drug, praziquantel (PZQ), and the selection of resistant worms under repeated treatment is a concern. Therefore, there is a pressing need to understand the molecular effects of PZQ on schistosomes and to investigate alternative or synergistic drugs against schistosomiasis.</p><p>Methodology</p><p>We used a custom-designed <i>Schistosoma mansoni</i> expression microarray to explore the effects of sublethal doses of PZQ on large-scale gene expression of adult paired males and females and unpaired mature females. We also assessed the efficacy of PZQ, omeprazole (OMP) or their combination against <i>S</i>. <i>mansoni</i> adult worms with a survival <i>in vitro</i> assay.</p><p>Principal Findings</p><p>We identified sets of genes that were affected by PZQ in paired and unpaired mature females, however with opposite gene expression patterns (up-regulated in paired and down-regulated in unpaired mature females), indicating that PZQ effects are heavily influenced by the mating status. We also identified genes that were similarly affected by PZQ in males and females. Functional analyses of gene interaction networks were performed with parasite genes that were differentially expressed upon PZQ treatment, searching for proteins encoded by these genes whose human homologs are targets of different drugs used for other diseases. Based on these results, OMP, a widely prescribed proton pump inhibitor known to target the ATP1A2 gene product, was chosen and tested. Sublethal doses of PZQ combined with OMP significantly increased worm mortality <i>in vitro</i> when compared with PZQ or OMP alone, thus evidencing a synergistic effect.</p><p>Conclusions</p><p>Functional analysis of gene interaction networks is an important approach that can point to possible novel synergistic drug candidates. We demonstrated the potential of this strategy by showing that PZQ in combination with OMP displayed increased efficiency against <i>S</i>. <i>mansoni</i> adult worms <i>in vitro</i> when compared with either drug alone.</p></div