Limits...
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 abolishes histone H3K9 methylation of the TESC promoter. a The location of mature ROR. ROR was mainly present in the nucleus. U2 RNA was used as a positive control for nuclear RNA. b Schematic diagram of the TESC promoter region and ROR lncRNA. T7 through T12 and S1 through S6: primer names, arrow: transcriptional direction, pTESC-1 and pTESC-2: two different biotinylated TESC promoter fragments; pTESC-3: biotinylated DNA fragments 10 kb upstream of TESC. Sites a, b, and c: different detecting locations of RNA IP. c The interaction of ROR and the TESC promoter. The TESC promoter (pTESC-1 and pTESC-2) specifically interacts with exon 3 of the ROR (site b). pTESC-3 was used as a negative control locus. Fibroblasts and lncRNA-KCNQ1OT1 were used as negative controls. Input: total RNA was reverse transcribed before incubation with labeled pTESC fragments and amplified with GAPDH primers. d TaqMan real time-PCR used to quantify the enrichment of ROR on the TESC promoter. TESC binds near exon 3 of ROR (site b). pTESC-3 was used as a negative control locus. KCNQ1OT1 was used a negative control. e-g ChIP assay detecting H3K9 trimethylation of the TESC promoter. Sites X, Y, and Z: different sites used in this assay. IgG: native control. All data are presented as the means ± SD. *P <0.05: compared with the control and mock
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4499915&req=5

Fig5: ROR abolishes histone H3K9 methylation of the TESC promoter. a The location of mature ROR. ROR was mainly present in the nucleus. U2 RNA was used as a positive control for nuclear RNA. b Schematic diagram of the TESC promoter region and ROR lncRNA. T7 through T12 and S1 through S6: primer names, arrow: transcriptional direction, pTESC-1 and pTESC-2: two different biotinylated TESC promoter fragments; pTESC-3: biotinylated DNA fragments 10 kb upstream of TESC. Sites a, b, and c: different detecting locations of RNA IP. c The interaction of ROR and the TESC promoter. The TESC promoter (pTESC-1 and pTESC-2) specifically interacts with exon 3 of the ROR (site b). pTESC-3 was used as a negative control locus. Fibroblasts and lncRNA-KCNQ1OT1 were used as negative controls. Input: total RNA was reverse transcribed before incubation with labeled pTESC fragments and amplified with GAPDH primers. d TaqMan real time-PCR used to quantify the enrichment of ROR on the TESC promoter. TESC binds near exon 3 of ROR (site b). pTESC-3 was used as a negative control locus. KCNQ1OT1 was used a negative control. e-g ChIP assay detecting H3K9 trimethylation of the TESC promoter. Sites X, Y, and Z: different sites used in this assay. IgG: native control. All data are presented as the means ± SD. *P <0.05: compared with the control and mock

Mentions: To determine the precise mechanism underlying ROR regulation of TESC expression in tumors, we examined the cellular location of the mature ROR transcript. It has been reported that the small nuclear RNA (snRNA) U2 is common in the nucleus and participates in RNA splicing in the assembly and function of canonical spliceosomes [22]. Thus, U2 snRNA was utilized as a positive reference for the examination of ROR location. By isolating both nuclear and cytoplasmic RNA, we showed that ROR was mainly present in the nucleus, at least in AGS (Fig. 5a, panel 1, lane 1) and HT29 cells (Fig. 5a, panel 4, lane 1). Thus, we investigated the possibility of ROR interacting with the TESC promoter. In a DNA pull-down assay, two biotin-labeled DNA fragments (pTESC-1 and pTESC-2) overlapping the TESC core promoter were used to incubate with total RNA. A control DNA fragment (pTESC-3) from 10 kb upstream of TESC was used as a negative control (Fig. 5b). After the pull-down and cDNA synthesis, we showed that the center of the ROR transcript, near exon 3 of ROR, could interact with the TESC promoter in ROR-expressing tumor cells (Fig. 5c, lanes 1-2 and 4-5). Fibroblasts (Fig. 5c, lanes 3 and 6) and non-biotin controls failed to show this DNA-RNA interaction (Fig. 5c, lanes 7-9). An interaction between ROR and a negative locus (pTESC-3) was not observed in AGS and HT29 cells (Fig. 5c, right, lanes 13-14). The non-biotin controls did not show an interaction too (Fig. 5c, lanes 16-18). To further validate the binding of ROR and TESC promoter, we performed a TaqMan qPCR analysis to quantitate the enrichment of ROR at the TESC promoter and showed that ROR was bound to the core promoter of TESC compared with that of the controls (Fig. 5d), suggesting that ROR may regulate TESC expression via chromatin-level machinery.Fig. 5


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 abolishes histone H3K9 methylation of the TESC promoter. a The location of mature ROR. ROR was mainly present in the nucleus. U2 RNA was used as a positive control for nuclear RNA. b Schematic diagram of the TESC promoter region and ROR lncRNA. T7 through T12 and S1 through S6: primer names, arrow: transcriptional direction, pTESC-1 and pTESC-2: two different biotinylated TESC promoter fragments; pTESC-3: biotinylated DNA fragments 10 kb upstream of TESC. Sites a, b, and c: different detecting locations of RNA IP. c The interaction of ROR and the TESC promoter. The TESC promoter (pTESC-1 and pTESC-2) specifically interacts with exon 3 of the ROR (site b). pTESC-3 was used as a negative control locus. Fibroblasts and lncRNA-KCNQ1OT1 were used as negative controls. Input: total RNA was reverse transcribed before incubation with labeled pTESC fragments and amplified with GAPDH primers. d TaqMan real time-PCR used to quantify the enrichment of ROR on the TESC promoter. TESC binds near exon 3 of ROR (site b). pTESC-3 was used as a negative control locus. KCNQ1OT1 was used a negative control. e-g ChIP assay detecting H3K9 trimethylation of the TESC promoter. Sites X, Y, and Z: different sites used in this assay. IgG: native control. All data are presented as the means ± SD. *P <0.05: compared with the control and mock
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4499915&req=5

Fig5: ROR abolishes histone H3K9 methylation of the TESC promoter. a The location of mature ROR. ROR was mainly present in the nucleus. U2 RNA was used as a positive control for nuclear RNA. b Schematic diagram of the TESC promoter region and ROR lncRNA. T7 through T12 and S1 through S6: primer names, arrow: transcriptional direction, pTESC-1 and pTESC-2: two different biotinylated TESC promoter fragments; pTESC-3: biotinylated DNA fragments 10 kb upstream of TESC. Sites a, b, and c: different detecting locations of RNA IP. c The interaction of ROR and the TESC promoter. The TESC promoter (pTESC-1 and pTESC-2) specifically interacts with exon 3 of the ROR (site b). pTESC-3 was used as a negative control locus. Fibroblasts and lncRNA-KCNQ1OT1 were used as negative controls. Input: total RNA was reverse transcribed before incubation with labeled pTESC fragments and amplified with GAPDH primers. d TaqMan real time-PCR used to quantify the enrichment of ROR on the TESC promoter. TESC binds near exon 3 of ROR (site b). pTESC-3 was used as a negative control locus. KCNQ1OT1 was used a negative control. e-g ChIP assay detecting H3K9 trimethylation of the TESC promoter. Sites X, Y, and Z: different sites used in this assay. IgG: native control. All data are presented as the means ± SD. *P <0.05: compared with the control and mock
Mentions: To determine the precise mechanism underlying ROR regulation of TESC expression in tumors, we examined the cellular location of the mature ROR transcript. It has been reported that the small nuclear RNA (snRNA) U2 is common in the nucleus and participates in RNA splicing in the assembly and function of canonical spliceosomes [22]. Thus, U2 snRNA was utilized as a positive reference for the examination of ROR location. By isolating both nuclear and cytoplasmic RNA, we showed that ROR was mainly present in the nucleus, at least in AGS (Fig. 5a, panel 1, lane 1) and HT29 cells (Fig. 5a, panel 4, lane 1). Thus, we investigated the possibility of ROR interacting with the TESC promoter. In a DNA pull-down assay, two biotin-labeled DNA fragments (pTESC-1 and pTESC-2) overlapping the TESC core promoter were used to incubate with total RNA. A control DNA fragment (pTESC-3) from 10 kb upstream of TESC was used as a negative control (Fig. 5b). After the pull-down and cDNA synthesis, we showed that the center of the ROR transcript, near exon 3 of ROR, could interact with the TESC promoter in ROR-expressing tumor cells (Fig. 5c, lanes 1-2 and 4-5). Fibroblasts (Fig. 5c, lanes 3 and 6) and non-biotin controls failed to show this DNA-RNA interaction (Fig. 5c, lanes 7-9). An interaction between ROR and a negative locus (pTESC-3) was not observed in AGS and HT29 cells (Fig. 5c, right, lanes 13-14). The non-biotin controls did not show an interaction too (Fig. 5c, lanes 16-18). To further validate the binding of ROR and TESC promoter, we performed a TaqMan qPCR analysis to quantitate the enrichment of ROR at the TESC promoter and showed that ROR was bound to the core promoter of TESC compared with that of the controls (Fig. 5d), suggesting that ROR may regulate TESC expression via chromatin-level machinery.Fig. 5

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