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Long non-coding RNA ROR decoys gene-specific histone methylation to promote tumorigenesis.

Fan J, Xing Y, Wen X, Jia R, Ni H, He J, Ding X, Pan H, Qian G, Ge S, Hoffman AR, Zhang H, Fan X - Genome Biol. (2015)

Bottom Line: Suppression of ROR in tumors results in silencing of TESC expression, and G9A-mediated histone H3K9 methylation in the TESC promoter is restored, which significantly reduces tumor growth and metastasis.Without ROR silencing, TESC knockdown presents consistent and significant reductions in tumor progression.Our results reveal a novel mechanism by which ROR may serve as a decoy oncoRNA that blocks binding surfaces, preventing the recruitment of histone modifying enzymes, thereby specifying a new pattern of histone modifications that promote tumorigenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Ninth People's Hospital, Shanghai JiaoTong University School of Medicine, Shanghai, 200025, P. R. China.

ABSTRACT

Background: Long non-coding RNAs (lncRNAs) are not translated into proteins and were initially considered to be part of the 'dark matter' of the genome. Recently, it has been shown that lncRNAs play a role in the recruitment of chromatin modifying complexes and can influence gene expression. However, it is unknown if lncRNAs function in a similar way in cancer.

Results: Here, we show that the lncRNA ROR occupies and activates the TESC promoter by repelling the histone G9A methyltransferase and promoting the release of histone H3K9 methylation. Suppression of ROR in tumors results in silencing of TESC expression, and G9A-mediated histone H3K9 methylation in the TESC promoter is restored, which significantly reduces tumor growth and metastasis. Without ROR silencing, TESC knockdown presents consistent and significant reductions in tumor progression.

Conclusions: Our results reveal a novel mechanism by which ROR may serve as a decoy oncoRNA that blocks binding surfaces, preventing the recruitment of histone modifying enzymes, thereby specifying a new pattern of histone modifications that promote tumorigenesis.

No MeSH data available.


Related in: MedlinePlus

ROR competes with G9A at the TESC promoter in vitro. a Schematic diagram showing the first experimental design of purified ROR competing with purified G9A at the TESC promoter. We used 15 μg purified G9A protein and incubated it with 1 μg biotinylated TESC-1 DNA probe. After incubation, 0.1-0.5 μg purified ROR lncRNA was then added to the reaction mixture to compete with the G9A:TESC-1 hybrid. PCR and western blot were used to detect the residual amount of ROR lncRNA and G9A protein after biotin purification and precipitation, respectively. Red helix: TESC DNA; green cycle: G9A protein; small blue cycle: biotin labeled; red arrow: transcriptional direction of TESC. b Expression of purified G9A and purified ROR lncRNA. M: maker. c Different amount of purified ROR competing with the G9A:TESC-1 hybrid. In the presence of 0.4 μg purified ROR, G9A significantly reduced binding with the TESC DNA fragment. CTCF and KCNQ1OT1 were used as a negative control. Input: collected biotinylated TESC-1 fragments before pull-down and PCR with primers aligned with the TESC promoter. d, e The quantitive assay of RoR and G9A interaction. The image J software was used to quantitate the RNA:protein interaction, *P <0.05. f Schematic diagram showing the modified experimental design of total RNA containing ROR competing with purified G9A at the TESC promoter. We used 5 μg purified G9A incubated with 1 μg biotinylated TESC-1 probe. After incubation, different amounts of total RNA extracted from tumor cells were then added to the reaction mixture to compete with the G9A:TESC-1 hybrid. PCR and western blot were used to detect the amount of ROR lncRNA and G9A protein after biotin purification and precipitation, respectively. g, h Different amounts of total RNA containing ROR competed with the G9A:TESC-1 hybrid. In the presence of 30 μg total RNA extracted from tumor cells, the G9A protein significantly reduced binding with the TESC DNA fragment (g). The total RNA of ROR silencing AGS and HT29 tumor cells failed to compete with the G9A:TESC hybrid (h). CTCF and KCNQ1OT1 were used as a negative control. Input: collected biotinylated TESC-1 fragments before pull-down and PCR with primers aligned with the TESC promoter. i, j Competition of total RNA and G9A is shown in i and j. The RNA:protein interaction was quantitated by the image J software,*P <0.05
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Fig6: ROR competes with G9A at the TESC promoter in vitro. a Schematic diagram showing the first experimental design of purified ROR competing with purified G9A at the TESC promoter. We used 15 μg purified G9A protein and incubated it with 1 μg biotinylated TESC-1 DNA probe. After incubation, 0.1-0.5 μg purified ROR lncRNA was then added to the reaction mixture to compete with the G9A:TESC-1 hybrid. PCR and western blot were used to detect the residual amount of ROR lncRNA and G9A protein after biotin purification and precipitation, respectively. Red helix: TESC DNA; green cycle: G9A protein; small blue cycle: biotin labeled; red arrow: transcriptional direction of TESC. b Expression of purified G9A and purified ROR lncRNA. M: maker. c Different amount of purified ROR competing with the G9A:TESC-1 hybrid. In the presence of 0.4 μg purified ROR, G9A significantly reduced binding with the TESC DNA fragment. CTCF and KCNQ1OT1 were used as a negative control. Input: collected biotinylated TESC-1 fragments before pull-down and PCR with primers aligned with the TESC promoter. d, e The quantitive assay of RoR and G9A interaction. The image J software was used to quantitate the RNA:protein interaction, *P <0.05. f Schematic diagram showing the modified experimental design of total RNA containing ROR competing with purified G9A at the TESC promoter. We used 5 μg purified G9A incubated with 1 μg biotinylated TESC-1 probe. After incubation, different amounts of total RNA extracted from tumor cells were then added to the reaction mixture to compete with the G9A:TESC-1 hybrid. PCR and western blot were used to detect the amount of ROR lncRNA and G9A protein after biotin purification and precipitation, respectively. g, h Different amounts of total RNA containing ROR competed with the G9A:TESC-1 hybrid. In the presence of 30 μg total RNA extracted from tumor cells, the G9A protein significantly reduced binding with the TESC DNA fragment (g). The total RNA of ROR silencing AGS and HT29 tumor cells failed to compete with the G9A:TESC hybrid (h). CTCF and KCNQ1OT1 were used as a negative control. Input: collected biotinylated TESC-1 fragments before pull-down and PCR with primers aligned with the TESC promoter. i, j Competition of total RNA and G9A is shown in i and j. The RNA:protein interaction was quantitated by the image J software,*P <0.05

Mentions: We next determined whether ROR lncRNA was capable of competing with G9A at its target DNA, the TESC promoter by conducting a competition assay with G9A and ROR. First, 355 bp biotin-labeled double stranded DNA fragment from the TESC promoter (pTESC-1) was synthesized, and then 1 μg pTESC-1 DNA probe was incubated with purified G9A protein to form a protein:DNA hybrid. To examine the ability of ROR lncRNA to compete with the G9A protein, we then added purified ROR lncRNA produced by in vitro RNA synthesis to the reaction mixture. If the ROR was a real competitor of G9A, ROR would occupy the binding site of G9A, and free G9A protein would be released from the TESC DNA. Following this route, after the biotin-streptavidin pull down, PCR or western blot was used to determine the residual amount of ROR and G9A, respectively (Fig. 6a). Before the assay, we evaluated the amount of purified ROR and purified G9A used in this examination. As shown in Fig. 6b, we found that 5-20 μg purified G9A protein was sufficient for detection by western blot (Fig. 6b, lanes 2, 3, 4 and 5). We also showed that 0.3-0.5 μg purified ROR could be successfully detected via PCR assay (Fig. 6b, lanes 8, 9, and 10).Fig. 6


Long non-coding RNA ROR decoys gene-specific histone methylation to promote tumorigenesis.

Fan J, Xing Y, Wen X, Jia R, Ni H, He J, Ding X, Pan H, Qian G, Ge S, Hoffman AR, Zhang H, Fan X - Genome Biol. (2015)

ROR competes with G9A at the TESC promoter in vitro. a Schematic diagram showing the first experimental design of purified ROR competing with purified G9A at the TESC promoter. We used 15 μg purified G9A protein and incubated it with 1 μg biotinylated TESC-1 DNA probe. After incubation, 0.1-0.5 μg purified ROR lncRNA was then added to the reaction mixture to compete with the G9A:TESC-1 hybrid. PCR and western blot were used to detect the residual amount of ROR lncRNA and G9A protein after biotin purification and precipitation, respectively. Red helix: TESC DNA; green cycle: G9A protein; small blue cycle: biotin labeled; red arrow: transcriptional direction of TESC. b Expression of purified G9A and purified ROR lncRNA. M: maker. c Different amount of purified ROR competing with the G9A:TESC-1 hybrid. In the presence of 0.4 μg purified ROR, G9A significantly reduced binding with the TESC DNA fragment. CTCF and KCNQ1OT1 were used as a negative control. Input: collected biotinylated TESC-1 fragments before pull-down and PCR with primers aligned with the TESC promoter. d, e The quantitive assay of RoR and G9A interaction. The image J software was used to quantitate the RNA:protein interaction, *P <0.05. f Schematic diagram showing the modified experimental design of total RNA containing ROR competing with purified G9A at the TESC promoter. We used 5 μg purified G9A incubated with 1 μg biotinylated TESC-1 probe. After incubation, different amounts of total RNA extracted from tumor cells were then added to the reaction mixture to compete with the G9A:TESC-1 hybrid. PCR and western blot were used to detect the amount of ROR lncRNA and G9A protein after biotin purification and precipitation, respectively. g, h Different amounts of total RNA containing ROR competed with the G9A:TESC-1 hybrid. In the presence of 30 μg total RNA extracted from tumor cells, the G9A protein significantly reduced binding with the TESC DNA fragment (g). The total RNA of ROR silencing AGS and HT29 tumor cells failed to compete with the G9A:TESC hybrid (h). CTCF and KCNQ1OT1 were used as a negative control. Input: collected biotinylated TESC-1 fragments before pull-down and PCR with primers aligned with the TESC promoter. i, j Competition of total RNA and G9A is shown in i and j. The RNA:protein interaction was quantitated by the image J software,*P <0.05
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Fig6: ROR competes with G9A at the TESC promoter in vitro. a Schematic diagram showing the first experimental design of purified ROR competing with purified G9A at the TESC promoter. We used 15 μg purified G9A protein and incubated it with 1 μg biotinylated TESC-1 DNA probe. After incubation, 0.1-0.5 μg purified ROR lncRNA was then added to the reaction mixture to compete with the G9A:TESC-1 hybrid. PCR and western blot were used to detect the residual amount of ROR lncRNA and G9A protein after biotin purification and precipitation, respectively. Red helix: TESC DNA; green cycle: G9A protein; small blue cycle: biotin labeled; red arrow: transcriptional direction of TESC. b Expression of purified G9A and purified ROR lncRNA. M: maker. c Different amount of purified ROR competing with the G9A:TESC-1 hybrid. In the presence of 0.4 μg purified ROR, G9A significantly reduced binding with the TESC DNA fragment. CTCF and KCNQ1OT1 were used as a negative control. Input: collected biotinylated TESC-1 fragments before pull-down and PCR with primers aligned with the TESC promoter. d, e The quantitive assay of RoR and G9A interaction. The image J software was used to quantitate the RNA:protein interaction, *P <0.05. f Schematic diagram showing the modified experimental design of total RNA containing ROR competing with purified G9A at the TESC promoter. We used 5 μg purified G9A incubated with 1 μg biotinylated TESC-1 probe. After incubation, different amounts of total RNA extracted from tumor cells were then added to the reaction mixture to compete with the G9A:TESC-1 hybrid. PCR and western blot were used to detect the amount of ROR lncRNA and G9A protein after biotin purification and precipitation, respectively. g, h Different amounts of total RNA containing ROR competed with the G9A:TESC-1 hybrid. In the presence of 30 μg total RNA extracted from tumor cells, the G9A protein significantly reduced binding with the TESC DNA fragment (g). The total RNA of ROR silencing AGS and HT29 tumor cells failed to compete with the G9A:TESC hybrid (h). CTCF and KCNQ1OT1 were used as a negative control. Input: collected biotinylated TESC-1 fragments before pull-down and PCR with primers aligned with the TESC promoter. i, j Competition of total RNA and G9A is shown in i and j. The RNA:protein interaction was quantitated by the image J software,*P <0.05
Mentions: We next determined whether ROR lncRNA was capable of competing with G9A at its target DNA, the TESC promoter by conducting a competition assay with G9A and ROR. First, 355 bp biotin-labeled double stranded DNA fragment from the TESC promoter (pTESC-1) was synthesized, and then 1 μg pTESC-1 DNA probe was incubated with purified G9A protein to form a protein:DNA hybrid. To examine the ability of ROR lncRNA to compete with the G9A protein, we then added purified ROR lncRNA produced by in vitro RNA synthesis to the reaction mixture. If the ROR was a real competitor of G9A, ROR would occupy the binding site of G9A, and free G9A protein would be released from the TESC DNA. Following this route, after the biotin-streptavidin pull down, PCR or western blot was used to determine the residual amount of ROR and G9A, respectively (Fig. 6a). Before the assay, we evaluated the amount of purified ROR and purified G9A used in this examination. As shown in Fig. 6b, we found that 5-20 μg purified G9A protein was sufficient for detection by western blot (Fig. 6b, lanes 2, 3, 4 and 5). We also showed that 0.3-0.5 μg purified ROR could be successfully detected via PCR assay (Fig. 6b, lanes 8, 9, and 10).Fig. 6

Bottom Line: Suppression of ROR in tumors results in silencing of TESC expression, and G9A-mediated histone H3K9 methylation in the TESC promoter is restored, which significantly reduces tumor growth and metastasis.Without ROR silencing, TESC knockdown presents consistent and significant reductions in tumor progression.Our results reveal a novel mechanism by which ROR may serve as a decoy oncoRNA that blocks binding surfaces, preventing the recruitment of histone modifying enzymes, thereby specifying a new pattern of histone modifications that promote tumorigenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Ninth People's Hospital, Shanghai JiaoTong University School of Medicine, Shanghai, 200025, P. R. China.

ABSTRACT

Background: Long non-coding RNAs (lncRNAs) are not translated into proteins and were initially considered to be part of the 'dark matter' of the genome. Recently, it has been shown that lncRNAs play a role in the recruitment of chromatin modifying complexes and can influence gene expression. However, it is unknown if lncRNAs function in a similar way in cancer.

Results: Here, we show that the lncRNA ROR occupies and activates the TESC promoter by repelling the histone G9A methyltransferase and promoting the release of histone H3K9 methylation. Suppression of ROR in tumors results in silencing of TESC expression, and G9A-mediated histone H3K9 methylation in the TESC promoter is restored, which significantly reduces tumor growth and metastasis. Without ROR silencing, TESC knockdown presents consistent and significant reductions in tumor progression.

Conclusions: Our results reveal a novel mechanism by which ROR may serve as a decoy oncoRNA that blocks binding surfaces, preventing the recruitment of histone modifying enzymes, thereby specifying a new pattern of histone modifications that promote tumorigenesis.

No MeSH data available.


Related in: MedlinePlus