CoChat
Sign Up
Cited by 346Circular RNAs in diseasesRNA Research and SplicingPluripotent Stem Cells Research

The DOI matches its Crossref record; 43 of the 43 references it cites were found in a registry or an index.

The circular RNA circBIRC6 participates in the molecular circuitry controlling human pluripotency

Open access
Yu, Chun-Ying, Li, Tung-Cheng, Wu, Yi-Ying, Yeh, Chan-Hsien, Chiang, Wei, Chuang, Ching-Yu, Kuo, Hung-Chih·2017·Nature Communications
DOI 10.1038/s41467-017-01216-wPMC PMC5658440
Open this paper in CoChat — free account

Abstract

Accumulating evidence indicates that circular RNAs (circRNAs) are abundant in the human transcriptome. However, their involvement in biological processes, including pluripotency, remains mostly undescribed. We identified a subset of circRNAs that are enriched in undifferentiated human embryonic stem cells (hESCs) and demonstrated that two, circBIRC6 and circCORO1C, are functionally associated with the pluripotent state. Mechanistically, we found that circBIRC6 is enriched in the AGO2 complex and directly interacts with microRNAs, miR-34a, and miR-145, which are known to modulate target genes that maintain pluripotency. Correspondingly, circBIRC6 attenuates the downregulation of these target genes and suppresses hESC differentiation. We further identified hESC-enriched splicing factors (SFs) and demonstrated that circBIRC6 biogenesis in hESCs is promoted by the SF ESRP1, whose expression is controlled by the core pluripotency-associated factors, OCT4 and NANOG. Collectively, our data suggest that circRNA serves as a microRNA "sponge" to regulate the molecular circuitry, which modulates human pluripotency and differentiation.

Overview

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Integrity signals

Registry

Crossref · 10.1038/s41467-017-01216-w

Cites

43 of 43 references verified0 citation errors · 0 not checkable

Statistics

Figures

Fig. 1 Identification and validation of hESC-enriched circRNAs. a Flowchart illustrating the sequence of steps for identifying and validating circRNAs enriched in hESCs. b RT-PCR validation of circRNAs and their linear isoforms from RNaseR-treated RNA samples isolated from hESCs. Specific primers were used to distinguish the indicated circRNAs from their linear isoforms. c The expression level of indicated circRNAs in hESCs by RT-qPCR analysis. The expression of circRNAs was normalized to GAPDH. d Comparison of the expression levels of indicated circRNAs and linear isoforms between hESCs and their differentiated derivatives (H9-EB) by RT-qPCR analysis. e Comparison of the expression levels of the indicated circRNAs between OSKM-reprogrammed iPSCs and their parental fibroblasts (HFs) by RT-qPCR analysis. Quantitative data from three independent experiments is presented as mean ± SD (error bars). P-values were determined by two-tailed two-sample t-tests (*P < 0.05; **P < 0.01; ***P < 0.001)
Fig. 1 Identification and validation of hESC-enriched circRNAs. a Flowchart illustrating the sequence of steps for identifying and validating circRNAs enriched in hESCs. b RT-PCR validation of circRNAs and their linear isoforms from RNaseR-treated RNA samples isolated from hESCs. Specific primers were used to distinguish the indicated circRNAs from their linear isoforms. c The expression level of indicated circRNAs in hESCs by RT-qPCR analysis. The expression of circRNAs was normalized to GAPDH. d Comparison of the expression levels of indicated circRNAs and linear isoforms between hESCs and their differentiated derivatives (H9-EB) by RT-qPCR analysis. e Comparison of the expression levels of the indicated circRNAs between OSKM-reprogrammed iPSCs and their parental fibroblasts (HFs) by RT-qPCR analysis. Quantitative data from three independent experiments is presented as mean ± SD (error bars). P-values were determined by two-tailed two-sample t-tests (*P < 0.05; **P < 0.01; ***P < 0.001)CC BY · PubMed Central
Fig. 2 Disrupting circBIRC6 or circCORO1C impairs hESC pluripotency. a The schematic illustrates how circular junction sites were targeted for circRNA-specific shRNAs (shcircRNAs). The schematic also shows the targeted regions for RT-qPCR primers. Divergent primers were used for circRNAs and convergent primers for the linear isoforms. b–d Total RNA isolated from hESCs transfected with shcircB, shcircC, shcircMa, or paired control virus 3 days post transduction was analyzed by RT-qPCR to determine the expression levels of b circBIRC6 (circB), circCORO1C (circC), circMAN1A2 (circMa), and their linear counterparts (LinB, LinC, and LinMa), and the indicated c pluripotency-associated genes and d lineage-associated genes. e AP staining of shcircB, shcircC, and shcircMa-transduced H9 hESCs. Scale bar: 200 μm. f ICC analysis of shcircB, shcircC, or shcircMa-transduced H9 hESCs 3 days post virus infection using antibodies against the pluripotency and lineage markers as indicated. The corresponding bar graphs show quantitative analysis of the percentage of cells expressing pluripotency-associated markers or lineage-associated markers. Scale bar: 20 μm. g ICC and quantitative comparison of NANOG and OCT4 expression between shcircB-transduced and shcircC-transduced hESCs and their counterparts rescued by overexpression of circBIRC6 (H9-shcircB-res) or circCORO1C (H9-shcircC-res). Nuclei were stained with DAPI (blue). Scale bar: 20 μm. h RT-qPCR analysis of the expression of the indicated pluripotency-associated genes in shcircB-transduced or shcircC-transduced H9 hESCs and their rescued counterparts, H9-shcircB-res and H9-shcircC-res. Quantitative data from three independent experiments is presented as mean ± SD (error bars). P-values were determined by two-tailed two-sample t-tests (*P < 0.05; **P < 0.01; ***P < 0.001)
Fig. 2 Disrupting circBIRC6 or circCORO1C impairs hESC pluripotency. a The schematic illustrates how circular junction sites were targeted for circRNA-specific shRNAs (shcircRNAs). The schematic also shows the targeted regions for RT-qPCR primers. Divergent primers were used for circRNAs and convergent primers for the linear isoforms. b–d Total RNA isolated from hESCs transfected with shcircB, shcircC, shcircMa, or paired control virus 3 days post transduction was analyzed by RT-qPCR to determine the expression levels of b circBIRC6 (circB), circCORO1C (circC), circMAN1A2 (circMa), and their linear counterparts (LinB, LinC, and LinMa), and the indicated c pluripotency-associated genes and d lineage-associated genes. e AP staining of shcircB, shcircC, and shcircMa-transduced H9 hESCs. Scale bar: 200 μm. f ICC analysis of shcircB, shcircC, or shcircMa-transduced H9 hESCs 3 days post virus infection using antibodies against the pluripotency and lineage markers as indicated. The corresponding bar graphs show quantitative analysis of the percentage of cells expressing pluripotency-associated markers or lineage-associated markers. Scale bar: 20 μm. g ICC and quantitative comparison of NANOG and OCT4 expression between shcircB-transduced and shcircC-transduced hESCs and their counterparts rescued by overexpression of circBIRC6 (H9-shcircB-res) or circCORO1C (H9-shcircC-res). Nuclei were stained with DAPI (blue). Scale bar: 20 μm. h RT-qPCR analysis of the expression of the indicated pluripotency-associated genes in shcircB-transduced or shcircC-transduced H9 hESCs and their rescued counterparts, H9-shcircB-res and H9-shcircC-res. Quantitative data from three independent experiments is presented as mean ± SD (error bars). P-values were determined by two-tailed two-sample t-tests (*P < 0.05; **P < 0.01; ***P < 0.001)CC BY · PubMed Central
Fig. 3 C ircBIRC6 or circCORO1C promote hESC pluripotency. a Schematic illustrating the major components of circRNA minigene constructs. Arrows indicate the placement and direction of RT-qPCR primers used to amplify circRNA or precursor transcripts. b–d RT-qPCR analysis of the expression of b circRNAs and their precursor transcripts, and the indicated c pluripotency-associated genes and d lineage-associated genes in hESCs transfected with a circRNA minigene (H9-circB, H9-circC, and H9-circMa) cultured under differentiation conditions. e ICC analysis and f quantitative analysis of the expression of the pluripotency and lineage markers as indicated in hESCs transfected with a circRNA minigene (H9-circB, H9-circC, and H9-circMa) and cultured under differentiation conditions. Nuclei were stained with DAPI (blue). Scale bar: 20 μm. Quantitative data from three independent experiments is presented as mean ± SD (error bars). P-values were determined by two-tailed two-sample t-tests (*P < 0.05; **P < 0.01; ***P < 0.001)
Fig. 3 C ircBIRC6 or circCORO1C promote hESC pluripotency. a Schematic illustrating the major components of circRNA minigene constructs. Arrows indicate the placement and direction of RT-qPCR primers used to amplify circRNA or precursor transcripts. b–d RT-qPCR analysis of the expression of b circRNAs and their precursor transcripts, and the indicated c pluripotency-associated genes and d lineage-associated genes in hESCs transfected with a circRNA minigene (H9-circB, H9-circC, and H9-circMa) cultured under differentiation conditions. e ICC analysis and f quantitative analysis of the expression of the pluripotency and lineage markers as indicated in hESCs transfected with a circRNA minigene (H9-circB, H9-circC, and H9-circMa) and cultured under differentiation conditions. Nuclei were stained with DAPI (blue). Scale bar: 20 μm. Quantitative data from three independent experiments is presented as mean ± SD (error bars). P-values were determined by two-tailed two-sample t-tests (*P < 0.05; **P < 0.01; ***P < 0.001)CC BY · PubMed Central
Fig. 4 CircBIRC6 or circCORO1C promotes reprogramming. a RT-qPCR analysis of the expression of the indicated pluripotency-associated genes in H9-OCT4pGFP, H9-OCT4pGFPdF, and H9-OCT4pGFPdF cells transduced with a circBIRC6 (H9-OCT4pGFPdF+circB) or circCORO1C (H9-OCT4pGFPdF+circC) minigene. b RT-qPCR analysis of the expression of circRNA and its precursor transcripts in H9-OCT4pGFPdF+circB and H9-OCT4pGFPdF+circC cells using primers specific for the indicated circRNAs and circRNA precursors (pre-circB and pre-circC). c AP staining and quantitative analysis of AP+ colonies among H9-OCT4pGFPdF cells transfected with OSKM or co-transfected with OSKM and circRNA minigenes (OSKMB and OSKMC). Scale bar: 400 μm. d Reactivation of OCT4pGFP in H9-OCT4pGFPdF cells and quantitative analysis of OCT4pGFP+ colonies of H9-OCT4pGFPdF cells transfected with OSKM or co-transfected with OSKM and circRNA minigenes (OSKMB and OSKMC) 21 days post transduction. Scale bar: 50 μm. e Quantitative FACS analysis of OCT4pGFP+ cells in OSKM, OSKMB, and OSKMC 28 days post reprogramming. Quantitative data from three independent experiments is presented as mean ± SD (error bars). P-values were determined by two-tailed two-sample t-tests (*P < 0.05; **P < 0.01; ***P < 0.001)
Fig. 4 CircBIRC6 or circCORO1C promotes reprogramming. a RT-qPCR analysis of the expression of the indicated pluripotency-associated genes in H9-OCT4pGFP, H9-OCT4pGFPdF, and H9-OCT4pGFPdF cells transduced with a circBIRC6 (H9-OCT4pGFPdF+circB) or circCORO1C (H9-OCT4pGFPdF+circC) minigene. b RT-qPCR analysis of the expression of circRNA and its precursor transcripts in H9-OCT4pGFPdF+circB and H9-OCT4pGFPdF+circC cells using primers specific for the indicated circRNAs and circRNA precursors (pre-circB and pre-circC). c AP staining and quantitative analysis of AP+ colonies among H9-OCT4pGFPdF cells transfected with OSKM or co-transfected with OSKM and circRNA minigenes (OSKMB and OSKMC). Scale bar: 400 μm. d Reactivation of OCT4pGFP in H9-OCT4pGFPdF cells and quantitative analysis of OCT4pGFP+ colonies of H9-OCT4pGFPdF cells transfected with OSKM or co-transfected with OSKM and circRNA minigenes (OSKMB and OSKMC) 21 days post transduction. Scale bar: 50 μm. e Quantitative FACS analysis of OCT4pGFP+ cells in OSKM, OSKMB, and OSKMC 28 days post reprogramming. Quantitative data from three independent experiments is presented as mean ± SD (error bars). P-values were determined by two-tailed two-sample t-tests (*P < 0.05; **P < 0.01; ***P < 0.001)CC BY · PubMed Central
Fig. 5 C ircBIRC6 interacts with and sequester miR-34a and miR-145. a RT-qPCR analysis of cytoplasmic-to-nuclear expression ratios of circBIRC6, circCORO1C, CDR1as, GAPDH, and U6 in hESCs. b RIP analysis of circBIRC6, linear BIRC6 (linB), circCORO1C, linear CORO1C (LinC), and CDR1as in hESCs using antibodies against AGO2. Immunoblotting (IB) of immunoprecipitated (IP) AGO2 protein is shown. The RIP enrichment of the AGO2-associated circular RNAs (as indicated) was measured by RT-qPCR, and each value was normalized to the level of input RNA used in RIP analysis. c RT-qPCR analysis of circBIRC6 or circCORO1C pulled-down by biotinylated miR-34a or miR-145 mimics in hESCs. d Schematic illustration showing the 3′ UTR of luciferase reporters containing the complete circBIRC6 sequence (luc-circB) or circBIRC6 sequence with deletions of miR-34a (luc-circBm1 and luc-circBm2) or miR-145 (luc-circBm3 and luc-circBm4) binding sites. e, f Reporter assays showing the luciferase activity of luc-circB and luc-circBm1-m4 in 293T cells co-transfected with e miR-34a or f miR-145 mimics, or a scrambled oligonucleotide (control). g RT-qPCR analysis of circBIRC6, miR-34a, and miR-145 expression during hESC differentiation. IVD: in vitro differentiation day. h RT-qPCR and i immunoblot analyses of the indicated gene and protein expression in hESCs transfected with miR-34a or miR-145 mimics, or scrambled oligonucleotide 3 days post transfection. Protein level quantification was normalized to actin and shown below each panel. Quantitative data from three independent experiments is presented as mean ± SD (error bars). P-values were determined by two-tailed two-sample t-tests (*P < 0.05; **P < 0.01; ***P < 0.001)
Fig. 5 C ircBIRC6 interacts with and sequester miR-34a and miR-145. a RT-qPCR analysis of cytoplasmic-to-nuclear expression ratios of circBIRC6, circCORO1C, CDR1as, GAPDH, and U6 in hESCs. b RIP analysis of circBIRC6, linear BIRC6 (linB), circCORO1C, linear CORO1C (LinC), and CDR1as in hESCs using antibodies against AGO2. Immunoblotting (IB) of immunoprecipitated (IP) AGO2 protein is shown. The RIP enrichment of the AGO2-associated circular RNAs (as indicated) was measured by RT-qPCR, and each value was normalized to the level of input RNA used in RIP analysis. c RT-qPCR analysis of circBIRC6 or circCORO1C pulled-down by biotinylated miR-34a or miR-145 mimics in hESCs. d Schematic illustration showing the 3′ UTR of luciferase reporters containing the complete circBIRC6 sequence (luc-circB) or circBIRC6 sequence with deletions of miR-34a (luc-circBm1 and luc-circBm2) or miR-145 (luc-circBm3 and luc-circBm4) binding sites. e, f Reporter assays showing the luciferase activity of luc-circB and luc-circBm1-m4 in 293T cells co-transfected with e miR-34a or f miR-145 mimics, or a scrambled oligonucleotide (control). g RT-qPCR analysis of circBIRC6, miR-34a, and miR-145 expression during hESC differentiation. IVD: in vitro differentiation day. h RT-qPCR and i immunoblot analyses of the indicated gene and protein expression in hESCs transfected with miR-34a or miR-145 mimics, or scrambled oligonucleotide 3 days post transfection. Protein level quantification was normalized to actin and shown below each panel. Quantitative data from three independent experiments is presented as mean ± SD (error bars). P-values were determined by two-tailed two-sample t-tests (*P < 0.05; **P < 0.01; ***P < 0.001)CC BY · PubMed Central
Fig. 6 C ircBIRC6 serves as a sponge for miR-34a and miR-145 in hESC. a, b RT-qPCR analysis of the expression of the indicated a pluripotency-associated genes and b lineage-associated genes in hESCs transfected with miR-34a mimics, or co-transfected with circBIRC6 minigene construct (miR-34a+circB (1 μg or 3 μg)) or circBIRC6 minigenes harboring deleted miR-34a and miR-145 binding sites (circB4m). c, d RT-qPCR analysis of the expression of the indicated c pluripotency-associated genes and d early lineage-associated genes in hESCs transfected with miR-145 mimics, or co-transfected with circBIRC6 minigene construct (miR-145+circB (1 μg or 3 μg)) or co-transfected with circB4m. e ICC analysis of the indicated pluripotency-associated markers and lineage-associated markers in hESCs transfected with miR-34a or miR-145 mimics, or co-transfected with a circBIRC6 minigene construct (miR-34a+circB or miR-145+circB). Nuclei were stained with DAPI. Scale bar: 20 μm. A quantitative analysis of cells expressing the indicated pluripotency-associated or early lineage-associated markers is shown in the right-hand panel. Quantitative data from three independent experiments is presented as mean ± SD (error bars). P-values were determined by two-tailed two-sample t-tests (*P < 0.05; **P < 0.01; ***P < 0.001)
Fig. 6 C ircBIRC6 serves as a sponge for miR-34a and miR-145 in hESC. a, b RT-qPCR analysis of the expression of the indicated a pluripotency-associated genes and b lineage-associated genes in hESCs transfected with miR-34a mimics, or co-transfected with circBIRC6 minigene construct (miR-34a+circB (1 μg or 3 μg)) or circBIRC6 minigenes harboring deleted miR-34a and miR-145 binding sites (circB4m). c, d RT-qPCR analysis of the expression of the indicated c pluripotency-associated genes and d early lineage-associated genes in hESCs transfected with miR-145 mimics, or co-transfected with circBIRC6 minigene construct (miR-145+circB (1 μg or 3 μg)) or co-transfected with circB4m. e ICC analysis of the indicated pluripotency-associated markers and lineage-associated markers in hESCs transfected with miR-34a or miR-145 mimics, or co-transfected with a circBIRC6 minigene construct (miR-34a+circB or miR-145+circB). Nuclei were stained with DAPI. Scale bar: 20 μm. A quantitative analysis of cells expressing the indicated pluripotency-associated or early lineage-associated markers is shown in the right-hand panel. Quantitative data from three independent experiments is presented as mean ± SD (error bars). P-values were determined by two-tailed two-sample t-tests (*P < 0.05; **P < 0.01; ***P < 0.001)CC BY · PubMed Central
Fig. 7 ESRP1 promotes the biogenesis of circBIRC6 in hESCs. a RT-qPCR analysis of the splicing factors (as indicated) in H9 hESCs. b RT-qPCR analysis of the expression of circBIRC6 (exon 9–2), linear BIRC6 (linBIRC6 exon 9–10 and exon 54–55), NANOG, and OCT4 in ESRP1-knockdown hESCs (shESRP-1 and shESRP-2). c Schematic illustrating the putative ESRP1-binding sites on the flanking introns in the circB-s minigene. The 5′ terminus of the circular exons of circBIRC6 was defined as position 0. Putative ESRP1-binding sites A, B, and C are located in the intron at the 5′ terminus of the circBIRC6 exon (position: −1282 to −1287), within the circBIRC6 exon (position: 84–89), and on the intron at the 3′ terminus of the circBIRC6 exon (position: 1733–1738). d RIP analysis of ESRP1-binding to circB-s and circB-s-Em minigenes in hESCs. Bound complexes were pulled-down using an antibody against ESRP1. RT-qPCR was then used to measure circB-s binding to ESRP1. Values were normalized to the level of background RIP, as detected by an IgG isotype control. Inserted panel: Immunoblotting (IB) of immunoprecipitated (IP) ESRP1 protein. e RT-qPCR analysis of the expression of circBIRC6 relative to GAPDH in hESCs. Cells were co-transfected with shRNA that target ESRP1 and a circBIRC6 minigene (circB-s), or circBIRC6 minigene containing deleted ESRP1-binding sites (circB-s-Em). f Schematic showing sites of insertion of ESRP1-binding sequence and the location of primers used for detecting the two minigenes (EGFR and ZEB1). g RT-PCR analysis using circRNA-specific or precursor specific primers on RNA from hESCs that were transfected with the indicated minigenes, with or without shRNA-mediated disruption of ESRP1. Quantitative data from three independent experiments is presented as mean ± SD (error bars). P-values were determined by two-tailed two-sample t-tests (*P < 0.05; **P < 0.01; ***P < 0.001)
Fig. 7 ESRP1 promotes the biogenesis of circBIRC6 in hESCs. a RT-qPCR analysis of the splicing factors (as indicated) in H9 hESCs. b RT-qPCR analysis of the expression of circBIRC6 (exon 9–2), linear BIRC6 (linBIRC6 exon 9–10 and exon 54–55), NANOG, and OCT4 in ESRP1-knockdown hESCs (shESRP-1 and shESRP-2). c Schematic illustrating the putative ESRP1-binding sites on the flanking introns in the circB-s minigene. The 5′ terminus of the circular exons of circBIRC6 was defined as position 0. Putative ESRP1-binding sites A, B, and C are located in the intron at the 5′ terminus of the circBIRC6 exon (position: −1282 to −1287), within the circBIRC6 exon (position: 84–89), and on the intron at the 3′ terminus of the circBIRC6 exon (position: 1733–1738). d RIP analysis of ESRP1-binding to circB-s and circB-s-Em minigenes in hESCs. Bound complexes were pulled-down using an antibody against ESRP1. RT-qPCR was then used to measure circB-s binding to ESRP1. Values were normalized to the level of background RIP, as detected by an IgG isotype control. Inserted panel: Immunoblotting (IB) of immunoprecipitated (IP) ESRP1 protein. e RT-qPCR analysis of the expression of circBIRC6 relative to GAPDH in hESCs. Cells were co-transfected with shRNA that target ESRP1 and a circBIRC6 minigene (circB-s), or circBIRC6 minigene containing deleted ESRP1-binding sites (circB-s-Em). f Schematic showing sites of insertion of ESRP1-binding sequence and the location of primers used for detecting the two minigenes (EGFR and ZEB1). g RT-PCR analysis using circRNA-specific or precursor specific primers on RNA from hESCs that were transfected with the indicated minigenes, with or without shRNA-mediated disruption of ESRP1. Quantitative data from three independent experiments is presented as mean ± SD (error bars). P-values were determined by two-tailed two-sample t-tests (*P < 0.05; **P < 0.01; ***P < 0.001)CC BY · PubMed Central
Fig. 8 NANOG and OCT4 regulate ESRP1 expression in hESCs. a ENCODE ChIP-Seq data for NANOG and OCT4 occupancy, and H3K27Ac modifications were aligned to the ESRP1 promoter, defined as −1 kb to +1 kb relative to the transcription start site. The y axis represents the intensity of ChIP-Seq reads. The highest NANOG and OCT4 binding peaks on the ESRP1 promoter are labeled as P0 (red bars; Chr8: 95653024–95653560), and four flanking regions within −1 kb to +1 kb (yellow bars) of P0 are labeled P−1, P−2, P+1, and P+2. b ChIP fragments containing the NANOG and OCT4 binding peak, or its four flanking regions, were quantified in hESCs and embryoid bodies (EBs) by qPCR using primers specific for P0, P+1, P+2, P−1, and P−2, respectively (Supplementary Table 1), and normalized to the input genome used in ChIP. c Schematic illustrating luciferase reporters containing an ESRP1 promoter (ESRP1-luc), ESRP1-luc with deleted NANOG binding sites (ESRP1-m1-luc and ESRP1-m2-luc), and ESRP1-luc with a deleted OCT4 binding site (ESRP1-m3-luc). d Luciferase reporter analysis showing the luciferase activity of ESRP1-luc, ESRP1-m1-luc, ESRP1-m2-luc, and ESRP1-m3-luc in 293 T cells ectopically expressing NANOG (left panel) or OCT4 (right panel). Quantitative data from three independent experiments is presented as mean ± SD (error bars). P-values were determined by two-tailed two-sample t-tests (*P < 0.05; **P < 0.01; ***P < 0.001)
Fig. 8 NANOG and OCT4 regulate ESRP1 expression in hESCs. a ENCODE ChIP-Seq data for NANOG and OCT4 occupancy, and H3K27Ac modifications were aligned to the ESRP1 promoter, defined as −1 kb to +1 kb relative to the transcription start site. The y axis represents the intensity of ChIP-Seq reads. The highest NANOG and OCT4 binding peaks on the ESRP1 promoter are labeled as P0 (red bars; Chr8: 95653024–95653560), and four flanking regions within −1 kb to +1 kb (yellow bars) of P0 are labeled P−1, P−2, P+1, and P+2. b ChIP fragments containing the NANOG and OCT4 binding peak, or its four flanking regions, were quantified in hESCs and embryoid bodies (EBs) by qPCR using primers specific for P0, P+1, P+2, P−1, and P−2, respectively (Supplementary Table 1), and normalized to the input genome used in ChIP. c Schematic illustrating luciferase reporters containing an ESRP1 promoter (ESRP1-luc), ESRP1-luc with deleted NANOG binding sites (ESRP1-m1-luc and ESRP1-m2-luc), and ESRP1-luc with a deleted OCT4 binding site (ESRP1-m3-luc). d Luciferase reporter analysis showing the luciferase activity of ESRP1-luc, ESRP1-m1-luc, ESRP1-m2-luc, and ESRP1-m3-luc in 293 T cells ectopically expressing NANOG (left panel) or OCT4 (right panel). Quantitative data from three independent experiments is presented as mean ± SD (error bars). P-values were determined by two-tailed two-sample t-tests (*P < 0.05; **P < 0.01; ***P < 0.001)CC BY · PubMed Central

Verification

Claims the paper makes

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

The self-check needs the paper’s full text.

Citations

Cites

Show all 43 references

Cited by

Sorted by citations

Items 301–346 of 346

Related

See all

Metadata

Authors
Yu, C.-Y., Li, T.-C., Wu, Y.-Y., Yeh, C.-H., Chiang, W., Chuang, C.-Y., & Kuo, H.-C.
Affiliations
  • Institute of Cellular and Organismic Biology, Academia Sinica (TW)
  • Genomics Research Center, Academia Sinica (TW)
  • National Taiwan University (TW)
Corresponding author
Kuo, Hung-Chih
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2017-10-27
Volume
8
Issue
1
Pages
1149-1149
ISSN
2041-1723 (ISSN-L)
DOI
10.1038/s41467-017-01216-w
PMC
PMC5658440
OpenAlex
W2766512489
Cited by
336
References
43
Citations by year
2026: 10 · 2025: 20 · 2024: 26 · 2023: 23 · 2022: 63 · 2021: 72 · 2020: 50 · 2019: 43 · 2018: 26 · 2017: 2
FWCI
13.74
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Communications (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • RePEc: Research Papers in Economics (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • Academia Sinica (AS-104-TP-B09, 105-0210-01-13-01, AS‐103‐TP‐B10)
  • Ministry of Science and Technology, Taiwan (MOST103-2321-B001-065)
  • National Health Research Institutes (NHRI-EX104-10320SI, EX104)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Circular RNAs in diseases
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Research and Splicing
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Pluripotent Stem Cells Research
Keywords
circular RNA, human embryonic stem cells, miR-34a, miR-145, microRNA sponge, ESRP1, splicing factors, Oct4, Nanog, cell differentiation, circRNA biogenesis
MeSH terms
Nanog Homeobox Protein, RNA, Circular, Animals, Cell Differentiation, Exoribonucleases, Humans, Mice, Inbred ICR, Microfilament Proteins, RNA, Gene Expression Profiling, MicroRNAs, Pluripotent Stem Cells, Octamer Transcription Factor-3, Inhibitor of Apoptosis Proteins, Mice, Embryonic Stem Cells, Argonaute Proteins

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

Are you an author, or do you see a problem with this page? Request a review