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TERC promotes cellular inflammatory response independent of telomerase

TERC promotes cellular inflammatory response independent of telomerase Downloaded from https://academic.oup.com/nar/article-abstract/47/15/8084/5531183 by Ed 'DeepDyve' Gillespie user on 27 August 2019 8084–8095 Nucleic Acids Research, 2019, Vol. 47, No. 15 Published online 11 July 2019 doi: 10.1093/nar/gkz584 TERC promotes cellular inflammatory response independent of telomerase 1 1 1 2 1,* Haiying Liu , Yiding Yang , Yuanlong Ge , Juanhong Liu and Yong Zhao MOE Key Laboratory of Gene Function and Regulation, School of Life Sciences, Sun Yat-sen University, Guangzhou 510006, P.R. China and Department of Endocrinology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430030, P.R. China Received March 27, 2019; Revised June 09, 2019; Editorial Decision June 23, 2019; Accepted July 05, 2019 ABSTRACT positive cells, including human stem cells and most cancer cells (2,3). However, TERC is also ubiquitously expressed TERC is an RNA component of telomerase. However, in terminally differentiated human somatic cells, which do TERC is also ubiquitously expressed in most hu- not express TERT and therefore have no telomerase activ- man terminally differentiated cells, which don’t have ity (4). The function of TERC in these cells remains largely telomerase activity. The function of TERC in these unknown. In telomerase positive cells, the level of TERC cells is largely unknown. Here, we report that TERC is often higher than that required to form telomerase with TERT (5,6). For telomerase negative cancers, termed alter- enhances the expression and secretion of inflam- native lengthening of telomeres (ALT) cancers, TERC is of- matory cytokines by stimulating NK-Bpathway in ten detected, but TERT is lacking (7). TERC has been con- a telomerase-independent manner. The ectopic ex- sidered for a long time as a nonfunctional RNA waiting pression of TERC in telomerase-negative cells alters for TERT to form active telomerase. However, recent dis- the expression of 431 genes with high enrichment coveries hypothesized that TERC may play a role beyond of those involved in cellular immunity. We perform telomerase. For instance, Gazzaniga et al. found that TERC genome-wide screening using a previously identified has telomerase independent anti-apoptotic functions in hu- ‘binding motif’ of TERC and identify 14 genes that are man T cells (8). In addition, it has been reported that TERC transcriptionally regulated by TERC. Among them, is involved in regulating ATR-mediated DNA damage sig- four genes (LIN37, TPRG1L, TYROBP and USP16) nals and in activation of DNA-PKcs that phosphorylates are demonstrated to stimulate the activation of NK- hnRNP A1 in a telomerase independent manner (9,10). LncRNAs may regulate gene expression in different ways. B pathway. Mechanistically, TERC associates with For instance, lncRNAs can modulate the epigenetic status the promoter of these genes through forming RNA– of target genes, influencing their transcription ( 11). In addi- DNA triplexes, thereby enhancing their transcription. tion, lncRNA may up- or downregulate gene transcription In vivo, expression levels of TERC and TERC target in cis by associating with their promoters (12). In this sce- genes (TYROBP, TPRG1L and USP16) are upregu- nario, recent studies found that lncRNA-chromatin interac- lated in patients with inflammation-related diseases tion is highly sequence dependent with many ‘binding mo- such as type II diabetes and multiple sclerosis. Col- tifs’ exiting in lncRNA (13). It is thus proposed that using its lectively, these results reveal an unknown function binding motif, lncRNA may hybridize with targeted genes of TERC on stimulating inflammatory response and and regulate their transcription. Indeed, it has been demon- highlight a new mechanism by which TERC modu- strated that many lncRNA–DNA interactions are mediated lates gene transcription. TERC may be a new target by formation of RNA–DNA triplexes (14–16). Although for the development of anti-inflammation therapeu- the ‘binding motif’ of TERC was identified years ago and it is hypothesized that as a typical lncRNA, TERC may par- tics. ticipate in the regulation of gene transcription, whether and how many genes are transcriptionally regulated by TERC INTRODUCTION still remained elusive. Here, we reported a new function of TERC as an Human telomerase RNA component (TERC) is a 451 nt lncRNA. It stimulates the NF-B pathway and increases long, noncoding RNA (lncRNA) that is an essential com- the expression and secretion of inflammation cytokines. By ponent of telomerase. TERC serves as a template for re- screening the genome for potential TERC binding sites in verse transcriptase TERT, which adds GGTTAG repeats to promoters, we identified 30 genes, of which four (LIN37, chromosome ends (1). TERC is expressed in all telomerase To whom correspondence should be addressed. Tel: +86 20399 43401; Fax: +86 20399 43778. Email: zhaoy82@mail.sysu.edu.cn C The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Downloaded from https://academic.oup.com/nar/article-abstract/47/15/8084/5531183 by Ed 'DeepDyve' Gillespie user on 27 August 2019 Nucleic Acids Research, 2019, Vol. 47, No. 15 8085 TPRG1L, TYROBP and USP16) were validated to be tran- Gene Ontology and KEGG pathway analyses were per- scriptionally regulated by TERC. Interestingly, these four formed for upregulated genes in TERC-U2OS cells using genes, including three first defined, were related to activa- the online tool DAVID (18,19). tion of NF-B pathway and cellular inflammation. In ad- dition, in vivo study showed that expression level of TERC Potential TERC binding site search in the promoter database as well as its downstream genes TYROBP, TPRG1L and USP16 were upregulated in patients with chronic inflam- Promoter sequences of Homo sapiens were downloaded mation disease. from Eukaryotic Promoter Database (https://epd.vital-it. ch/index.php)(20). Promoters containing at least 10 con- tinuous nucleotides of ‘GGCCACCACCCC’ or its reverse MATERIALS AND METHODS complement sequence were defined as potential binding sites of TERC. Reagents Antibodies to phosphorylated STAT3, STAT3, phosphory- ChIRP-PCR assay lated p65 and p65 were purchased from Beyotime (Shang- hai, China). The AKT inhibitor perifosine, NF-B cofac- The ChIRP pull down assay was performed exactly as tor IB inhibitor bay11-7082, and STAT3 inhibitor Stat- previously reported (13,21) using odd probes of TERC and tic were from EFEbio (Shanghai, China). LPS was from lacZ. The product was detected by PCR with primers tar- Sigma. TNF- was from PeproTech. ELISA kits for cy- geting promoters containing potential TERC binding sites. tokines were purchased from 4A Biotech (Beijing China). The potential target genes and PCR primer sequences are as follows: Lin37: F 5’-TTGGTCAGGATGCGAGATT- 3’, R 5’-TCCTCCGCCTTTGGTTGT-3’; TPRG1L: Cell culture, vectors, and transfections F 5’-GCAAGGCGGAGCCAATCG-3’, R 5’- Cells were cultured in Dulbecco’s modified Eagle’s medium ACCCCTTACCGACCCCGAC-3’; TYROBP: F (Gibco) with 10% fetal bovine serum, 100 U/ml penicillin 5’-CAAGTGAAGGAGGAAGTCTGA-3’, R 5’- and 100 g/ml streptomycin. To generate TERC stably CCTGATTCTTTCTTGGGTTTT -3’; USP16: F 5’- overexpressing cell line, 293T cells were transfected with TCAGAGCCGATGGTCCCG -3’, R 5’- CTCCGTCTTC- pBabe-TERC or empty pBabe plasmid and the retrovi- CTCCTGGTGA -3’. ral packaging plasmids pCMV-VSV.G and pCMV-Gag-Pol (Addgene) using calcium phosphate precipitation. The vi- Electrophoretic mobility shift assay ral supernatants were collected 72 h after transfection, ul- tracentrifuged at 40 000 rpm for 2h at 4 C, andthenusedto Oligonucleotides were synthesized (Generay, China) and infect U2OS cells. Forty-eight hours later, cells were selected annealed into dsDNA in binding buffer (10 mM Tris–HCl with 2 g/ml puromycin for 3 days. The retained cells were pH7.4, 125 mM NaCl, 6 mM MgCl ). Oligonucleotides ◦ ◦ cultured in 1 g/ml puromycin to produce a polyclonal cell were heated at 95 C for 3 min and cooled down to 50 C ◦ ◦ population. The NF-B luciferase plasmid was a gift from with every 5 C interval (90, 85, 80 C...) helding for 3 min. In Prof. Jun Cui at Sun Yat-sen University. Plasmid contains vitro TERC transcription was performed using High Yield a fireyfl luciferase gene driven by minimal TATA promoter Transcription Kit (Ambion), 450 nt Scramble sequence was with NF-B response elements. used as a control. 1pmol of dsDNA was incubated with dif- SiRNAs were used to knock down genes. SiRNA trans- ferent amounts of TERC in binding buffer for 2 h at 42 C. fection was carried out with Lipofectamine RNAiMAX The samples were analyzed on 2% agarose gel at 4 C. (Invitrogen) according to the manufacturer’s instructions. Experiments were performed 72 h after siRNA transfection. Melting profiles The sequences of siRNAs are as follows: TERC si1: GUCU AACCCUAACUGAGAAGG; TERC si2: CCGUUCAU The ds-promoter segments of LIN37, TPRG1L, TYROBP UCUAGAGCAAAC; Lin37 si1: GCAGCGAUCCAACA and USP16 (in Table 1) were formed and mixed with or CAUAU; Lin37 si2: CCAACACAUAUGUGAUCAA; SL without TERC or TERC1–49nt at the ratio of 1:1 (mole). C26A1 si1: GCAACACCCAUGGCAAUUA; SLC26A1 The melting profile was obtained in q-PCR working buffer si2: GCCUCUAUACGUCCUUCUU; TPRG1L si1: CC containing SYSB green I in LightCycler 480 (Roche). Sam- AUUUCCUACGGAGAAUU; TPRG1L si2: GGAAUC ples were incubated at 60 C for 1 min, and then increased ◦ ◦ CCUGGUCUACCAA; TYROBP si1: GGUGCUGACA to 95 C at the rate of 2.5 C/s. Fluorescence intensity was GUGCUCAUU; TYROBP si2: UCCUUCACUUGCCU detected at 0.2 C interval. GGACG; USP16 si1: CCAUGAGCCAGUUUCUUAA; USP16 si2: GCAGAUGCUAAUUUCUCUU; ELISA and Western blotting For detection of cytokine secretion, U2OS cells were treated RNA sequencing and data analysis with 10 ng/ml TNF-, whereas B2-17 cells were treated The RNA sample preparation, sequencing and data anal- with 1 g/ml LPS for 6 h. The culture medium was collected ysis were performed as previously reported (17). The genes and cytokines were detected using corresponding ELISA with log -fold change ≥2or ≤–2 as well as FDR<0.01 were kits. For Western blotting, U2OS cells were treated with considered up- or downregulated genes, respectively. The or without TNF- for indicated times. To inhibit NF-B Downloaded from https://academic.oup.com/nar/article-abstract/47/15/8084/5531183 by Ed 'DeepDyve' Gillespie user on 27 August 2019 8086 Nucleic Acids Research, 2019, Vol. 47, No. 15 or STAT3 pathway, U2OS cells were treated with indicated RESULTS inhibitors for 6 h. The culture medium was collected for TERC promotes cellular inflammatory response ELISA analysis, and the cells were collected for Western blotting. To explore the biologic functions of TERC beyond serving as a template for telomerase extension, we ectopically ex- pressed TERC in telomerase negative U2OS cells in which endogenous TERC was in the minutest amount and TERT TERC expression in clinical samples was undetectable. Differentially expressed genes were then Processed gene expression profiles of clinical samples examined by RNA-seq. The result showed that 431 genes (GSE54350, GSE65561 and GES66988) were downloaded were up- or downregulated with log -fold change ≥2or from GEO database (Gene Expression Omnibus). Expres- ≤–2, respectively (Supplementary Table S1). Upregulated sion levels of TERC and its target genes were analyzed. genes were then subjected to Gene Oncology (GO) anal- ysis. Strikingly, 8 of 15 enriched GO terms were related to immunology, including immune response, chemokine- mediated signaling pathway, macrophage chemotaxis, and Mapping of TERC-ChIRP fragments to genome inflammatory response (Figure 1A). Alternatively, when up- TERC CHIRP-seq dataset was downloaded from published regulated genes underwent KEGG pathway analysis, 22 out article (13). The original dataset included 2198 fragments of 28 KEGG terms were related to immunology such as im- located in hg18. These fragments were re-localized in hg19. mune system, immune related diseases or inflammation re- The nearest transcription start sites (TSSs) were identified. lated pathway (Figure 1B). These results strongly suggested The distance between fragment and TSS was calculated and that TERC is involved in cellular immune in a direct or in- defined as “distance to TSS”. direct manner. To further explore this hypothesis, we stimulated TERC- U2OS and control pBabe-U2OS cells with TNF- and de- Quantitative RT-PCR tected the expression and secretion of inflammatory fac- tors. The results showed that mRNA levels of vfi e cytokines Total RNA was isolated using TRIzol (Takara). Approxi- (IL-6, IL-8, IL-32, TNF- and CSF2) were upregulated in mately 1 g RNA per sample was used to generate cDNA TERC-U2OS compared to pBabe-U2OS (Figure 1C). Ac- by reverse transcription. Real-time PCR was carried out cordingly, secreted cytokines (IL-6, IL-8 and CSF2) in cul- with q-PCR buffer containing SYBR Green I in Light- ture medium were also increased (Figure 1D). Conversely, Cycler 480 (Roche). All PCR primer sequences were from when TERC was depleted by siRNAs in human astrocy- PrimerBank (22). toma B2–17cells (siRNA with a scramble sequence was used as a control), both the expression and secretion level of cy- tokines significantly decreased (Figure 1E and F). Because Luciferase reporter assays B2–17 are telomerase positive cells, to exclude the engage- ment of telomerase in the inflammatory response, TERT HEK293T cells were plated in 96-well plates ( 1×10 was knocked down (Figure 1G) and cytokine secretion was per well) and transfected with plasmids encoding NF-B determined. Our results showed that in contrast to knock- luciferase reporter (firefly luciferase plasmid), pRL-TK- down of TERC, depletion of TERT only slightly decreased luc (renilla luciferase plasmid) and pBabe-TERC (Full- the secretion of CSF2 and had no effect on IL-6 and IL-8 length TERC). For knockdown experiment, siTERC was (Figure 1F). To further confirm the role of TERC in the in- transfected into HEK293T cells. Cells were harvested at flammatory response, TERC was knocked down in human 48 h after transfection and luciferase activity was measured normal fibroblast BJ cells, which do not express TERT and with Dual-Luciferase Assay kit (Promega) according to the therefore do not have telomerase activity. We observed that protocol provided by manufacturer. The fireyfl luciferase in- depletion of TERC significantly decreased secretion of IL-6, tensity of NF-B reporter was first normalized to renilla IL-8 and CSF2 in response to immune stimulation (Figure luciferase and then divided by corresponding control to ob- 1H). tain a relative activity of NF-B luciferase (Folds). Immunofluorescence (IF) Activation of the NF-B signaling pathway by TERC Cells were grown on coverslip, washed with PBS and fixed in The NF-B signaling pathway plays a fundamental role in 4% paraformaldehyde for 15 min at room temperature, and inflammatory response, governing the release of inflamma- then permeabilized in 0.5% Triton X-100 at room tempera- tory factors (23). We noticed that NF-B signaling path- ture for 30 min. The cells were washed thrice with 1× PBST way was enriched by KEGG pathway analysis in TERC and blocked with 5% goat serum for 1 h at room temper- overexpressing cells (Figure 1B). To explore whether TERC- ature. The cells were incubated sequentially with anti-p65 induced stimulation of inflammatory response is mediated antibody overnight at 4 C and secondary antibody conju- by NF-B pathway, inhibitors of p65 cofactors IBand gated with DyLight 488 for 1 h at room temperature. The STAT3 (Bay11-7082 and Stattic, respectively) were used to coverslip was washed with PBST, mounted with DAPI, and treat TERC-U2OS. Indeed, both Bay11-7082 and Stattic, visualized using a Zeiss microscope. but not the AKT inhibitor perifosine, suppressed the in- Downloaded from https://academic.oup.com/nar/article-abstract/47/15/8084/5531183 by Ed 'DeepDyve' Gillespie user on 27 August 2019 ** ns *** *** ** *** *** *** *** Nucleic Acids Research, 2019, Vol. 47, No. 15 8087 A Enriched GO terms of up-regulated genes after TERC overexpression GO:0048566~embryonic digestive tract development GO:0010595~positive regulation of endothelial cell migration GO:0006954~inflammatory response p value GO:0045785~positive regulation of cell adhesion 0.01 GO:0048246~macrophage chemotaxis GO:0010759~positive regulation of macrophage chemotaxis GO:0070098~chemokine-mediated signaling pathway 0.001 GO:0050918~positive chemotaxis GO:0043114~regulation of vascular permeability GO:0031663~lipopolysaccharide-mediated signaling pathway GO:0070493~thrombin receptor signaling pathway GO:0030198~extracellular matrix organization GO:0006955~immune response GO:0070374~positive regulation of ERK1 and ERK2 cascade GO:0050900~leukocyte migration 0 5 10 15 Enriched gene number Enriched KEGG pathway of up-regulated genes after TERC overexpression hsa05416:Viral myocarditis hsa05200:Pathways in cancer hsa04915:Estrogen signaling pathway Othe rs hsa04940:Type I diabetes mellitus hsa04151:PI3K-Akt signaling pathway hsa04514:Cell adhesion molecules (CAMs) hsa04064:NF-kappa B signaling pathway p value Inflammation hsa04060:Cytokine-cytokine receptor interaction re late d pathway >0.05 hsa04668:TNF signaling pathway 0.05 hsa04670:Leukocyte transendothelial migration Immune hsa04612:Antigen processing and presentation 0.01 0.001 syste m hsa04611:Platelet activation hsa04621:NOD-like receptor signaling pathway Autoimmune hsa05323:Rheumatoid arthritis dise ase hsa05330:Allograft rejection hsa05332:Graft-versus-host disease Immune hsa05310:Asthma dise ase s hsa05321:Inflammatory bowel disease (IBD) hsa05150:Staphylococcus aureus infection hsa05145:Toxoplasmosis hsa05152:Tuberculosis hsa05143:African trypanosomiasis Infe ctious dise ase s hsa05133:Pertussis hsa05168:Herpes simplex infection hsa05146:Amoebiasis hsa05142:Chagas disease hsa05144:Malaria hsa05164:Influenza A 0 5 10 15 Enriched gene number C D E pB a b e-U 2 O S TE R C -U 2 O S ELISA 1.5 NC TERC si1 TERC si2 *** 20 pBabe-U2OS *** TERC-U2OS 15 *** *** *** 10 1.0 3 *** 4 *** 0.5 2 * 0 0 0.0 F G H B2-17 ELISA ELISA 1.5 1.5 IL-6 IL-8 CSF2 1.5 IL-6 IL-8 CSF2 ns ns ** 1.0 1.0 ** *** *** ** 1.0 *** *** *** *** *** 0.5 0.5 0.5 *** *** *** *** 0.0 0.0 0.0 B2-17 BJ B2-17 Figure 1. TERC promotes inflammatory response. ( A) Enriched biological processes after TERC overexpression. Gene ontology was analyzed for upregu- lated genes in TERC-U2OS compared to pBabe-U2OS. GO terms in blue boxes were immune related biological processes. (B) Enriched signaling pathways after TERC overexpression. KEGG pathway enrichment was analyzed with upregulated genes in TERC-U2OS compared to pBabe-U2OS. Different cate- gories of pathways were boxed and labeled. (C) The mRNA levels of cytokines after TERC overexpression in U2OS cells. The stable cell lines pBabe-U2OS and TERC-U2OS were stimulated with TNF- for 1 h, and cells were collected for qPCR. (D) The secreted cytokines after TERC overexpression in U2OS cells. The stable cell lines pBabe-U2OS and TERC-U2OS were stimulated by TNF- for 6 h, and culture medium was collected for ELISA. (E) The mRNA levels of cytokines after TERC knockdown in B2–17 cells. The cells were transfected with indicated siRNAs for 72 h and treated with LPS during the last 1 h of transfection. Cells were then collected for qPCR. (F) The secreted cytokines in B2–17 cells after TERC or TERT knockdown. Cells were transfected with indicated siRNAs for 72 h and treated with LPS during the last 6 h of transfection. Culture medium was then collected for ELISA. (G) The knock- down efficiency of TERT. The B2–17 cells were transfected with TERT siRNAs for 72 h, and cells were collected for qPCR. ( H) The secreted cytokines in BJ after TERC knockdown. Cells were transfected with indicated siRNAs for 72 h and treated with TNF- during the last 6 h of transfection. Culture medium was collected for ELISA. All values are means ± SEM of more than three independent experiments (*P < 0.05, **P < 0.01,***P < 0.001). I L - 6 I L -8 IL -3 2 T N F - α C SF 2 NC TERC si1 TERC si2 NC TERC si1 TERC si2 NC TERC si1 TERC si2 NC TERC si1 TERC si2 TERT si NC TERC si1 TERC si2 TERT si NC TERC si1 TERC si2 TERT si IL-6 IL-8 CSF2 NC siTERT hTR IL-6 IL-8 IL-32 TNF-α CSF2 Re la tive f o ld of m RN A Relative fold of protein Relative fold of protein Relative fold of TERT Relative fold of protein Relative fold of mRNA Downloaded from https://academic.oup.com/nar/article-abstract/47/15/8084/5531183 by Ed 'DeepDyve' Gillespie user on 27 August 2019 8088 Nucleic Acids Research, 2019, Vol. 47, No. 15 AB C D E F G HI J Figure 2. TERC activates the NF-Bpathway.(A) Inhibition of NF-B or STAT3 activation by inhibitors in TERC-U2OS. TERC-U2OS cells were treated with inhibitors of AKT, IB and STAT3 (Perifosine, Bay11–7082 and Stattic, respectively) for 6h, and cells were collected for Western blotting of indicated proteins. (B-D) The TERC promotion of cytokine secretion was counteracted by inhibitors of p65 cofactors IB and STAT3. Stable cell lines pBabe- U2OS and TERC-U2OS were treated with TNF- plus indicated inhibitor for 6 h, and culture medium was collected for IL-6, IL-8 and CSF2 detection by ELISA. (E) Total and phosphorylated p65 were upregulated by TERC. PBabe-U2OS and TERC-U2OS cells were treated with TNF- for indicated times. Total and phosphorylated levels of p65 were determined by Western blotting. (F) Quantification of (E). Protein levels in (E) were quantified and normalized by actin. (G) Inhibited nuclear translocation of p65 in TERC depleted cells. B2–17 cells were transfected with TERC siRNA or NC for 72 h and then treated with TNF- (10 ng/ml) for 20 min. Immunofluorescence (IF) using p65 antibody was performed to determine the amount of p65 in nucleus and cytoplasm. (H) Quantification of (G). Nuclear p65 (Fluorescence intensity) was quantified as a percentage of overall p65 in cell. ∼100 cells were counted for each experiment. (I, J)NF-B luciferase reporter assay for analysis of activation of NF-B in TERC overexpressed (I) and depleted cells (J). HEK293T cells were transfected with TERC/pBabe (I) or siTERC/NC (J) together with NF-B-luc and pRL-TK. Luciferase activity was determined 48 h after transfection. All values are means ± SEM of more than three independent experiments (*P < 0.05, **P < 0.01, ***P < 0.001). Downloaded from https://academic.oup.com/nar/article-abstract/47/15/8084/5531183 by Ed 'DeepDyve' Gillespie user on 27 August 2019 Nucleic Acids Research, 2019, Vol. 47, No. 15 8089 A B DNA TERC TERC *** 40 pBabe-U2OS 30 TERC-U2OS *** *** *** *** **** * ** ** * ** *** *** D E F HeLa B2-17 293T NC siTERC 1.5 2.0 NC siTERC NC siTERC ns ns 1.5 *** ** *** *** ** *** 1.0 *** *** *** *** ** 1.0 * ns *** *** *** ** 0.5 0.5 0.0 0 0.0 Figure 3. TERC targets gene’s promoters using its binding motif. (A) Schematic diagram of TERC targeting to gene’s promoters that contain the sequence of TERC binding motif. (B). Distribution of TERC targeted fragments on genome. The fragment was classified according to its distance to the nearest TSS. (C) The mRNA levels of 30 genes with TERC binding motif in their promoters in TERC-U2OS vs pBabe-U2OS cells. (D–F). Determination of mRNA levels of top 6 genes in (C) that have the highest fold change (up- and downregulation) in response to TERC expression. Gene expression was tested in indicated cells with or without TERC knockdown. Each panel represents different cells as indicated. All values are means ± SEM of more than three independent experiments (*P < 0.05, **P < 0.01, ***P < 0.001). crease of IL-6, IL-8 and CSF2 induced by TERC overex- scription of target genes (24). The amount of p65 in nu- pession (Figure 2A–D). cleus is indicative of activation extent of NF-B pathway. Furthermore, we observed that NF-B subunit p65 and We observed that in response to TNF- treatment, TERC- phosphorylated p65 (p-p65) were higher in TERC-U2OS depleted cells display much less p65 in nucleus than control than in control pBabe-U2OS, suggesting the activation of cells, demonstrating that TERC positively regulates the ac- the NF-B pathway (Figure 2E, 0min). When cells were fur- tivation of NF-B pathway (Figure 2G, H). Moreover, we ther stimulated with TNF-, the level of p65 and p-p65 in also performed luciferase reporter assay, in which NF-B TERC-U2OS were continuously higher than pBabe-U2OS response element is placed in TATA promoter that drives throughout the activation procedure, suggesting that TERC the transcription of luciferase. The result showed that com- stimulates activation of NF-B pathway (Figure 2E, F). pared to control cells, overexpression of TERC increases lu- Upon activation of NF-B pathway, p65 migrates from cy- ciferase activity, whereas knockdown of TERC decreases its toplasm to nucleus where it functions to initiate the tran- ALPL LIN37 DUSP21 MSL1 SLC26A1 TPRG1L TYROBP RRAGA CIZ1 CNOT2 RPH3A CHD3 USP16 RAD17 CCR3 GBA2 CERCAM PYGO2 CEP63 LCN15 MTERFD3 ACTR8 SCRN1 EML3 ASTL SNAI1 ALG3 UNC5A APOM TMEM139 TERC LIN37 SLC26A1 TPRG1L TYROBP USP16 UNC5A TERC LIN37 SLC26A1 TPRG1L TYROBP USP16 UNC5A TERC LIN37 SLC26A1 TPRG1L TYROBP USP16 UNC5A Relativ e fold of mRNA Relative fold of mRNA Re lative mRNA level Re lativ e mRNA le v e l Downloaded from https://academic.oup.com/nar/article-abstract/47/15/8084/5531183 by Ed 'DeepDyve' Gillespie user on 27 August 2019 8090 Nucleic Acids Research, 2019, Vol. 47, No. 15 Table 1. Promoters containing potential TERC binding site Gene Name Gene Promoter Seq. from EPD ALPL actcgggccccgcggccgcctttataaggcggcgggggtggtggcccggGCCGCGTTGCG LIN37 taataggaacaagctactgccgaaggggcccgcccacagaagggtggtgGCCACGGTCCA DUSP21 agatgggagtggtgagaggagacagaaagagggtggtggccgatagctgGTCCTCTTTCT MSL1 gcggccagcgagggcagatggaagagTATGAGGAAGAGCCCTCTCGGGGGTGGTGGCGGC SLC26A1 cagagtccagggcacagaccactgcctgcaggttggcgccaccacccccACTCTCCCCGC TPRG1L ccgcggggcggggccgggggcgcggccgggtggtggcggtggctgcggcgacggcggtcG TYROBP ccctgtctcctcctcccttctgccaccacccgcctcagacttcctccttCACTTGCCTGG RRAGA gagatcgccgccggaagtgggtggtggcggggacgcagcggctccctccCGGAAAGCGAG CIZ1 ggcaaaatggcgaaatccctctctacaaaaaatacaaacattagccagggtggtggcggg CNOT2 agggagggagggggtgtgtatgggggtggtggtggACCGGACGTAAAGCGTCGCTGTACT RPH3A aggagggagagggggtggtggaggagggagagagggtgggagaaggagtgatgaagatgg CHD3 ggggaggcgggcgggcggtgggtgggggggtggtgggggggccAGAGCCACAGGATGGCT USP16 GGGAGGTGGGGGTGGGGTGGTGGTGGCCTAGCCACTTCCCATAATGCCGCGTTCCGGAAG RAD17 CGAATATTTGAGCTTAGTATTCCCTGTTCACTGTGTGGGGTGGTGGTGGGTCGGCTAGGA CCR3 aggtggtggcctgcccctccccgcaggcactctgtcccagggagaaatcagaactcttta GBA2 CACGGCCACTTCTGCATCCAGGTGGGGATGCTGGCACTGAAGGTGGTGGCCCTTCTGGGA CERCAM GGAGCCGGGGAAGCCCGGGAGGTGGTGGCCGAGTGGGCGCCGCCCCTCTGGGTCTGCGGC PYGO2 ttgctccccctccccgcagcgctcagtggtggtggccgcgacgagttccGGTTCCGGTTG CEP63 GCCTCGCAGGCCACCACCATCCGCACCGTACGACAGGCCGTCCCTCAGCTGCGGCTTCCT LCN15 caggtggtggcctgggctataaagctggccccctggggcttggggactcAGCACCAGGGG MTERFD3 ggaagcaaatgcagctggtgcaggagagggaaatgggaattagggtggtGGCAGAGCCCA ACTR8 cccctggtggggggAGTGCGGAAGCGGTCGTTCTTTTCCGGGTGGTGGCGCGCCGGGACG SCRN1 tcccactcctctccacctccactgccaccaccctgcaccaagccaccaccatctccagcc EML3 CTCGGGGTGGTGGTACGGCGCCCTTCGCGCGCGCCCCGGGGTGCTTCCCCTTCCCCTCTC ASTL GTAACCTAATTGCAGAACCGGCACCACCACCCCCTCTTAAATAGCAGCTGctccacctcc SNAI1 CCACCACCCCCCCGGAGTACTTAAGGGAGTTGGCGGCGCTGCTGCATTCATTGCGCCGCG ALG3 aagcggaacctaagtgtcgaaggttcgggtttccgggggtggtgggcccACACAAGCGGC UNC5A GCCCACCACCCCAAGCCCCTCCCTGGGGGAGCCTCAGGCATCGCCCAGAGGGATTCCCGG APOM acacacccaccaccccgcggctccgcccccgacttccccacggaccgtcACTTCCGGTCT TMEM139 acctacccgctccggcccttcccaccaccccccaccccatctactttctACAGTCTGTGG Underline indicates potential binding sites Lower case indicates upstream of TSS activity (Figure 2I and J). Altogether, these data confirmed by TERC using pBabe-U2OS and TERC-U2OS cells. 14 of that TERC promotes the activation of NF-B pathway. 30 genes exhibited up- or downregulated transcription in response to TERC expression (Figure 3C). The top six up- or downregulated genes were LIN37, SLC26A1, TPRG1L, TERC regulates gene transcription by targeting its promoter TYROBP, USP16 and UNC5A. Among them, four genes We next explored how TERC stimulates the NF-Bpath- (LIN37, TPRG1L, TYROBP and USP16) showed consis- way. As a typical lncRNA, we speculated that TERC may tent downregulation when TERC was knockdown in B2– regulate gene transcription by targeting specific sites in the 17, HeLa and 293T cells (Figure 3D–F). We thus focused genome. Indeed, using chromatin isolation by RNA pu- on these four genes. rification (ChIRP), it was previously reported that TERC targets the genome with a high preference for the se- TERC binds to gene’s promoters by forming RNA–DNA quence 5 -GGCCACCACCCC-3 (termed the binding mo- triplexes tif) (13), which is exactly complementary to the sequence 5 -GGGGUGGUGGCC-3 at site 25–36 of TERC. This To investigate whether TERC binds to promoters of LIN37, strongly suggested that TERC binds to genomic DNA using TPRG1L, TYROBP and USP16 in vivo, ChIRP-PCR was its binding motif, which may form a triplex structure with performed, in which biotin-labeled oligonucleotides were target DNA (Figure 3A). used to pull down TERC and associated chromatin (13,21). To survey the locations of TERC target sites, we down- PCR was then performed using primers that cover the loaded 2198 TERC-ChIRP fragments from published study TERC binding site in the gene’s promoter (Table 1). The (13) and mapped them to the human genome. The distance results showed that promoters of four genes were enriched to the nearest transcription start site (TSS) of each frag- by ChIRP, demonstrating association of these promoters ment was calculated. Most fragments (TERC targeted sites) with TERC (Figure 4A). were located within ±1000 bp of TSSs (Figure 3B), suggest- To further validate the interaction between TERC and ing that TERC tends to target gene’s promoters. Then, we promoters, electrophoretic mobility shift assay (EMSA) screened potential binding sequences using a TERC binding was carried out in vitro by incubating TERC and synthe- motif in Eukaryotic Promoter Database (EPD) (20), which sized 59 bp double-stranded (ds) DNA that is from in- contains all identified promoters and sequences adjacent to dicated gene promoter and consists of binding motif se- TSSs. As a result, as many as 30 sequences on gene pro- quence of TERC. Indeed, shifted bands were observed for moters were identified that were listed in Table 1. We then all four tested promoters, but not for scramble sequence verified whether the transcription of these genes was altered (Figure 4B). In addition, with increased TERC, shift bands Downloaded from https://academic.oup.com/nar/article-abstract/47/15/8084/5531183 by Ed 'DeepDyve' Gillespie user on 27 August 2019 Nucleic Acids Research, 2019, Vol. 47, No. 15 8091 A B C Figure 4. RNA–DNA triplex formation between TERC and gene’s promoters. (A) TERC binds to the promoter of indicated gene in vivo. TERC ChIRP was performed using TERC probe. LacZ probe was used as control. Promoters of the indicated genes were detected by PCR. (B) TERC binds to the promoters of indicated genes in vitro. EMSA was performed to detect the triplex formation between synthesized gene promoters containing TERC binding motif and TERC. RNA with random sequence was used as control. Products were analyzed on 2% agarose gels. (C) Triplex formation in a dose dependent manner. Two picomoles of indicated gene promoters were incubated with increasing amounts of TERC. Products were analyzed on 2% agarose gels. (D) Expression levels of indicated genes after RNH1 knockdown. U2OS cells were transfected with siRNH1 for 72 h, and corresponding mRNAs were detected by qPCR. (E) Melting temperature decreased after triplex formation at neutral pH. Melting temperatures of gene’s promoters were detected in the presence or absence of TERC or TERC1–49. All values are means ± SEM of more than three independent experiments (*P < 0.05, **P < 0.01, ***P < 0.001). (RNA–DNA) gradually increased and free promoter DNA TERC-targeted genes activate the NF-B pathway decreased accordingly (Figure 4C). Because NF-B component such as p65 or STAT3 is not TERC may form R-loop or triplex with targeted ds- direct target of TERC, we hypothesized that TERC stim- promoters. To test the possibility that TERC may form ulates the NF-B pathway in an indirect manner, poten- R-loops by hybridizing with targeted DNA, we knocked tially through promoting the expression of targeted genes. down the RNH1 gene in TERC-U2OS cells. RNase H is We thus tested identified four genes for their ability to ac- an enzyme that digests RNA in Watson–Crick RNA–DNA tivate the NF-B pathway. Four genes (LIN37, TPRG1L, hybrids (25). It is speculated that gene expression levels TYROBP and USP16) were individually knocked down should be altered by either overexpression or knockdown in TERC-U2OS cells. We found that while knockdown of of RNase H if they are regulated by R-loop (26). In con- LIN37, TPRG1L and USP16 decreased the level of p65 trast to this, we observed no change in the expression levels and p-p65, the knockdown of LIN37, TPRG1L and TY- of four genes when RNase H was knocked down (Figure ROBP decreased the level of STAT3 and phosphorylated 4D). In addition, we observed that melting temperatures of STAT3 (p-STAT3) (Figure 5A, B). Consistently, ELISA as- ds-promoters were reduced by approximately 1 C after in- say showed that knockdown of LIN37, TPRG1L, TYROBP cubating with TERC or 49 nt TERC fragments that contain and USP16 counteracted enhanced secretion of IL-6 by the binding motif (Figure 4E). This is consistent with the TERC (Figure 5C). Altogether, these results demonstrated previous report that melting temperature decreases during that TERC modulates the inflammatory response through conformation change from double-strand DNA into RNA– regulating a group of genes such as LIN37, TPRG1L, TY- DNA triplexes (27). Altogether, these results suggested that ROBP and USP16. While TYROBP has been previously re- TERC may target gene’s promoters by forming RNA–DNA triplexes. Downloaded from https://academic.oup.com/nar/article-abstract/47/15/8084/5531183 by Ed 'DeepDyve' Gillespie user on 27 August 2019 8092 Nucleic Acids Research, 2019, Vol. 47, No. 15 Figure 5. TERC activates NF-B pathways through targeting immune-related genes. (A) The knockdown efficiency of indicated genes. The TERC-U2OS cells were transfected with indicated siRNAs for 72 h, and corresponding mRNAs were detected by qPCR. (B) P65 and STAT3 were downregulated by knockdown of TERC targeted genes. The TERC-U2OS cells were transfected with indicated siRNAs for 72 h. Total and phosphorylated p65 and STAT3 expressions were determined by western blotting. (C) The TERC promotion of IL-6 secretion was counteracted by knockdown of TERC targeted genes. PBabe-U2OS and TERC-U2OS cells were transfected with indicated siRNAs for 72 h and treated with TNF- during the last 6 h of transfection. Culture medium was collected for IL-6 detection by ELISA. All values are means ± SEM of more than three independent experiments (*P < 0.05, **P < 0.01, ***P < 0.001). ported to be involved in the activation of the inflammatory 6E and F). Therefore, the elevated inflammatory response response (28–31), the other three genes are newly identified. is positively correlated with increased expression of TERC and TERC target genes in patients. The expression of TERC and its targeted genes are upregu- lated in inflammation related disease DISCUSSION Because TERC regulates the expression of inflammation A high incidence of TERC mutation and an increased copy related genes in vitro, we then investigated its potential number of TERC genes has been reported to associate with immune-regulation function in patients. First, we analyzed the pathogenesis of the inherited disorder dyskeratosis con- TERC levels in type II diabetes and multiple sclerosis, both genital (DC), aplastic anemia (AA) (34,35) and other ge- displaying increased chronic inflammation ( 32,33). The ex- netic diseases (36–38). Interestingly, both DC and AA pa- pression levels of TERC in CD14+ cells from type II di- tients also display immune abnormalities (39,40). More- abetes and multiple sclerosis patients were significantly over, many mutations in TERC do not affect its function as higher than normal people (Figure 6A and B). We also ex- an RNA template and telomerase activity (41–43), imply- amined the expression level of LIN37, TPRG1L, TYROBP ing that TERC may have noncanonical functions beyond and USP16 in CD14+ cells from patients and normal peo- telomerase. In this study, we revealed that TERC stimulates ple. For diabetic patients, TPRG1L and TYROBP were up- cellular inflammatory response in a telomerase independent regulated, whereas TYROBP and USP16 were upregulated manner. The evidence supporting this includes: (i) RNA-seq in multiple sclerosis patients (Figure 6C and D). Consis- data indicated that expression of immune-related genes are tently, the expression of inflammatory cytokines such as regulated by TERC; (ii) overexpression of TERC in telom- IL-8 and TNF- in diabetic patients and IL-6, IL-8, CSF2 erase negative U2OS cells resulted in increased expression and TNF- in multiple sclerosis patients increased (Figure and secretion of inflammatory factors; (iii) knockdown of TERC, but not TERT, in telomerase positive cells decreased Downloaded from https://academic.oup.com/nar/article-abstract/47/15/8084/5531183 by Ed 'DeepDyve' Gillespie user on 27 August 2019 Nucleic Acids Research, 2019, Vol. 47, No. 15 8093 A C E G S E 5 43 50 & G S E 65 561 G S E 543 50 & G S E 65 561 G S E 5 43 50 & G S E 65 561 He a lth y V o lu n te e r H e alth y V oluntee r ** Dia b e tic Dia b e tic 2.5 ** * 2.5 ** 2.0 3 2.0 1.5 1.5 1.0 1.0 0.5 0.5 0.0 0.0 0 CD1 4 Ce lls CD1 4 Ce lls CD1 4 Ce lls B D F G S E 66988 G SE6 6 9 8 8 G S E 66988 H e alth y V oluntee r H e alth y V oluntee r M u lt ip le S c le r o s is M u lt ip le S c le r o s is ** 1.4 *** *** 1.5 *** ** 1.2 1.0 1.5 *** 1.0 1.0 0.5 0.8 0.5 0.6 0.0 0.0 + + CD1 4 Ce lls CD1 4 Ce lls CD1 4 Ce lls Figure 6. The expression levels of TERC and TERC targeted genes in inflammation related diseases. ( A) TERC was upregulated in CD14 cells of diabetic patients. Data were downloaded from the GEO database (GSE54350 & GSE65561), and TERC expression levels were analyzed (n ≥ 10, *P < 0.05, **P < 0.01). (B) TERC was upregulated in CD14 cells of multiple sclerosis patients. Data were downloaded from the GEO database (GSE66988), and TERC expression levels were analyzed (n ≥ 10, *P < 0.05, **P < 0.01). (C) Expression levels of TERC targeted genes in CD14 cells of diabetic patients. Data analysis is the same as (A). (D) Expression levels of TERC targeted genes in CD14 cells of multiple sclerosis patients. Data analysis is the same as (B). (E) Expression levels of cytokines in CD14 cells of diabetic patients. Data analysis is the same as (A), except that ‘n’ of IL6 is 6 because IL6 is missing in GSE65561. (F) Expression levels of cytokines in CD14 cells of multiple sclerosis patients. Data analysis is the same as (B). secretion of inflammatory factors; (iv) overexpression of modifiers to target sites ( 13–16). A previous study identified TERC led to activation of the NF-B signaling pathway in the sequence ‘GGCCACCACCCC’ as a binding motif in the absence of allothogenic stimulation; (v) TERC upreg- TERC that may associate with genomic DNA (13). Here, we ulated the expression of LIN37, TPRG1L, TYROBP and found that TERC associated sequences were largely located USP16 that is linked to the activation of NF-B signaling near the TSSs of genes (Figure 3B). Following this route, pathway, leading to increased inflammatory response and we identified 30 potential promoters TERC may bind to. (vi) high TERC levels corresponded to high inflammation Among them, 4 genes were experimentally verified to be states in patients with type II diabetes or multiple sclerosis. transcriptionally regulated by TERC (LIN37, TPRG1L, LncRNAs may regulate gene expression in different man- TYROBP and USP16). Similar to many other lncRNAs ners (11). For example, eRNA, which is transcribed from (13–16), we demonstrated that TERC formed triplexes with enhancers, promotes chromatin accessibility by remodeling promoter sequences of these genes and thereby promoted the chromatin (44), whereas lncRNA Khps1, HOTAIR and their transcription. It should be noted that identified four MEG3 regulate gene transcription by recruiting chromatin genes are representative of 14 genes, which are transcrip- No r m a l Dia be tic LIN 3 7 TP R G 1 L T YR OB P No rm a l U SP 1 6 M ult ip le Sc ler o s i s IL6 L I N 3 7 IL 8 TP R G 1 L C S F 2 TY R O B P TN F -α U S P1 6 IL6 IL 8 CS F 2 TN F-α Re la t ivefo ld o fT E R C R e la t ivefold o fT ER C Re l a tiv e fo ld o f m R N A Re la tiv e fo ld o f m R N A R e l a ti v e fo l d o f mR N A Re lativ e fo ld o f mR N A Downloaded from https://academic.oup.com/nar/article-abstract/47/15/8084/5531183 by Ed 'DeepDyve' Gillespie user on 27 August 2019 8094 Nucleic Acids Research, 2019, Vol. 47, No. 15 tionally regulated by TERC (Figure 3). Considering that di- luciferase plasmid, and for helpful suggestions on experi- rect association is one of manners by which lncRNA regu- mental design. lates gene transcription, the total number of genes regulated Author Contributions: H. Liu and Y. Zhao designed the by TERC might be much greater than 14. Further investi- study, analyzed the data and wrote the paper. H. Liu and Y. gation is thus needed to identify whole genes regulated by Yang performed the majority of the experiments. J. Liu an- TERC. alyzed the clinical samples. Y. Ge offered ideas and helped Except for TYROBP, which was previously reported to to analyze the data. Y. Zhao supervised the project and re- engage in inflammatory response, the other three genes are vised the manuscript. All authors read and approved the newly identified that could activate the NF- B signaling manuscript. pathway. Because p65 and STAT3 are not directly regulated by TERC, we hypothesized that TERC stimulates inflam- FUNDING matory response in an indirect manner, i.e., through tran- National Natural Science Foundation of China Grants scriptional activation of inflammatory related genes that [81771506, 31571410]; National Key R&D Program of then activate NF-B signaling pathway. In the absence of China [2018YFA0107000]; Guangzhou Municipal People’s TERC, cells were less sensitive to immune stimulation by TNF- or LPS (Figure 1F, H). Therefore, we defined TERC Livelihood Science and Technology Plan [201803010108]. as a positive modulator of NF-B mediated inflammatory Funding for open access charge: National Natural Science response. Foundation of China Grants [81771506, 31571410]; Na- It was previously discovered that TERC is implicated in tional Key R&D Program of China [2018YFA0107000]; angiogenesis, metastasis and proliferation of cancer cells Guangzhou Municipal People’s Livelihood Science and by regulating the global gene expression (45,46). Similarly, Technology Plan [201803010108]. we revealed that TERC promotes cellular inflammatory re- Conflict of interest statement. None declared. sponse by upregulating the expression of immune-related genes such as LIN37, TPRG1L, TYROBP and USP16 in REFERENCES human normal and cancer cells. 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Chem., 280, 23709–23717. periodontal pathogens and oral commensal bacteria. Mol. Oral Microbiol., 32, 275–287. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Nucleic Acids Research Oxford University Press

TERC promotes cellular inflammatory response independent of telomerase

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Abstract

Downloaded from https://academic.oup.com/nar/article-abstract/47/15/8084/5531183 by Ed 'DeepDyve' Gillespie user on 27 August 2019 8084–8095 Nucleic Acids Research, 2019, Vol. 47, No. 15 Published online 11 July 2019 doi: 10.1093/nar/gkz584 TERC promotes cellular inflammatory response independent of telomerase 1 1 1 2 1,* Haiying Liu , Yiding Yang , Yuanlong Ge , Juanhong Liu and Yong Zhao MOE Key Laboratory of Gene Function and Regulation, School of Life Sciences, Sun Yat-sen University, Guangzhou 510006, P.R. China and Department of Endocrinology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430030, P.R. China Received March 27, 2019; Revised June 09, 2019; Editorial Decision June 23, 2019; Accepted July 05, 2019 ABSTRACT positive cells, including human stem cells and most cancer cells (2,3). However, TERC is also ubiquitously expressed TERC is an RNA component of telomerase. However, in terminally differentiated human somatic cells, which do TERC is also ubiquitously expressed in most hu- not express TERT and therefore have no telomerase activ- man terminally differentiated cells, which don’t have ity (4). The function of TERC in these cells remains largely telomerase activity. The function of TERC in these unknown. In telomerase positive cells, the level of TERC cells is largely unknown. Here, we report that TERC is often higher than that required to form telomerase with TERT (5,6). For telomerase negative cancers, termed alter- enhances the expression and secretion of inflam- native lengthening of telomeres (ALT) cancers, TERC is of- matory cytokines by stimulating NK-Bpathway in ten detected, but TERT is lacking (7). TERC has been con- a telomerase-independent manner. The ectopic ex- sidered for a long time as a nonfunctional RNA waiting pression of TERC in telomerase-negative cells alters for TERT to form active telomerase. However, recent dis- the expression of 431 genes with high enrichment coveries hypothesized that TERC may play a role beyond of those involved in cellular immunity. We perform telomerase. For instance, Gazzaniga et al. found that TERC genome-wide screening using a previously identified has telomerase independent anti-apoptotic functions in hu- ‘binding motif’ of TERC and identify 14 genes that are man T cells (8). In addition, it has been reported that TERC transcriptionally regulated by TERC. Among them, is involved in regulating ATR-mediated DNA damage sig- four genes (LIN37, TPRG1L, TYROBP and USP16) nals and in activation of DNA-PKcs that phosphorylates are demonstrated to stimulate the activation of NK- hnRNP A1 in a telomerase independent manner (9,10). LncRNAs may regulate gene expression in different ways. B pathway. Mechanistically, TERC associates with For instance, lncRNAs can modulate the epigenetic status the promoter of these genes through forming RNA– of target genes, influencing their transcription ( 11). In addi- DNA triplexes, thereby enhancing their transcription. tion, lncRNA may up- or downregulate gene transcription In vivo, expression levels of TERC and TERC target in cis by associating with their promoters (12). In this sce- genes (TYROBP, TPRG1L and USP16) are upregu- nario, recent studies found that lncRNA-chromatin interac- lated in patients with inflammation-related diseases tion is highly sequence dependent with many ‘binding mo- such as type II diabetes and multiple sclerosis. Col- tifs’ exiting in lncRNA (13). It is thus proposed that using its lectively, these results reveal an unknown function binding motif, lncRNA may hybridize with targeted genes of TERC on stimulating inflammatory response and and regulate their transcription. Indeed, it has been demon- highlight a new mechanism by which TERC modu- strated that many lncRNA–DNA interactions are mediated lates gene transcription. TERC may be a new target by formation of RNA–DNA triplexes (14–16). Although for the development of anti-inflammation therapeu- the ‘binding motif’ of TERC was identified years ago and it is hypothesized that as a typical lncRNA, TERC may par- tics. ticipate in the regulation of gene transcription, whether and how many genes are transcriptionally regulated by TERC INTRODUCTION still remained elusive. Here, we reported a new function of TERC as an Human telomerase RNA component (TERC) is a 451 nt lncRNA. It stimulates the NF-B pathway and increases long, noncoding RNA (lncRNA) that is an essential com- the expression and secretion of inflammation cytokines. By ponent of telomerase. TERC serves as a template for re- screening the genome for potential TERC binding sites in verse transcriptase TERT, which adds GGTTAG repeats to promoters, we identified 30 genes, of which four (LIN37, chromosome ends (1). TERC is expressed in all telomerase To whom correspondence should be addressed. Tel: +86 20399 43401; Fax: +86 20399 43778. Email: zhaoy82@mail.sysu.edu.cn C The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Downloaded from https://academic.oup.com/nar/article-abstract/47/15/8084/5531183 by Ed 'DeepDyve' Gillespie user on 27 August 2019 Nucleic Acids Research, 2019, Vol. 47, No. 15 8085 TPRG1L, TYROBP and USP16) were validated to be tran- Gene Ontology and KEGG pathway analyses were per- scriptionally regulated by TERC. Interestingly, these four formed for upregulated genes in TERC-U2OS cells using genes, including three first defined, were related to activa- the online tool DAVID (18,19). tion of NF-B pathway and cellular inflammation. In ad- dition, in vivo study showed that expression level of TERC Potential TERC binding site search in the promoter database as well as its downstream genes TYROBP, TPRG1L and USP16 were upregulated in patients with chronic inflam- Promoter sequences of Homo sapiens were downloaded mation disease. from Eukaryotic Promoter Database (https://epd.vital-it. ch/index.php)(20). Promoters containing at least 10 con- tinuous nucleotides of ‘GGCCACCACCCC’ or its reverse MATERIALS AND METHODS complement sequence were defined as potential binding sites of TERC. Reagents Antibodies to phosphorylated STAT3, STAT3, phosphory- ChIRP-PCR assay lated p65 and p65 were purchased from Beyotime (Shang- hai, China). The AKT inhibitor perifosine, NF-B cofac- The ChIRP pull down assay was performed exactly as tor IB inhibitor bay11-7082, and STAT3 inhibitor Stat- previously reported (13,21) using odd probes of TERC and tic were from EFEbio (Shanghai, China). LPS was from lacZ. The product was detected by PCR with primers tar- Sigma. TNF- was from PeproTech. ELISA kits for cy- geting promoters containing potential TERC binding sites. tokines were purchased from 4A Biotech (Beijing China). The potential target genes and PCR primer sequences are as follows: Lin37: F 5’-TTGGTCAGGATGCGAGATT- 3’, R 5’-TCCTCCGCCTTTGGTTGT-3’; TPRG1L: Cell culture, vectors, and transfections F 5’-GCAAGGCGGAGCCAATCG-3’, R 5’- Cells were cultured in Dulbecco’s modified Eagle’s medium ACCCCTTACCGACCCCGAC-3’; TYROBP: F (Gibco) with 10% fetal bovine serum, 100 U/ml penicillin 5’-CAAGTGAAGGAGGAAGTCTGA-3’, R 5’- and 100 g/ml streptomycin. To generate TERC stably CCTGATTCTTTCTTGGGTTTT -3’; USP16: F 5’- overexpressing cell line, 293T cells were transfected with TCAGAGCCGATGGTCCCG -3’, R 5’- CTCCGTCTTC- pBabe-TERC or empty pBabe plasmid and the retrovi- CTCCTGGTGA -3’. ral packaging plasmids pCMV-VSV.G and pCMV-Gag-Pol (Addgene) using calcium phosphate precipitation. The vi- Electrophoretic mobility shift assay ral supernatants were collected 72 h after transfection, ul- tracentrifuged at 40 000 rpm for 2h at 4 C, andthenusedto Oligonucleotides were synthesized (Generay, China) and infect U2OS cells. Forty-eight hours later, cells were selected annealed into dsDNA in binding buffer (10 mM Tris–HCl with 2 g/ml puromycin for 3 days. The retained cells were pH7.4, 125 mM NaCl, 6 mM MgCl ). Oligonucleotides ◦ ◦ cultured in 1 g/ml puromycin to produce a polyclonal cell were heated at 95 C for 3 min and cooled down to 50 C ◦ ◦ population. The NF-B luciferase plasmid was a gift from with every 5 C interval (90, 85, 80 C...) helding for 3 min. In Prof. Jun Cui at Sun Yat-sen University. Plasmid contains vitro TERC transcription was performed using High Yield a fireyfl luciferase gene driven by minimal TATA promoter Transcription Kit (Ambion), 450 nt Scramble sequence was with NF-B response elements. used as a control. 1pmol of dsDNA was incubated with dif- SiRNAs were used to knock down genes. SiRNA trans- ferent amounts of TERC in binding buffer for 2 h at 42 C. fection was carried out with Lipofectamine RNAiMAX The samples were analyzed on 2% agarose gel at 4 C. (Invitrogen) according to the manufacturer’s instructions. Experiments were performed 72 h after siRNA transfection. Melting profiles The sequences of siRNAs are as follows: TERC si1: GUCU AACCCUAACUGAGAAGG; TERC si2: CCGUUCAU The ds-promoter segments of LIN37, TPRG1L, TYROBP UCUAGAGCAAAC; Lin37 si1: GCAGCGAUCCAACA and USP16 (in Table 1) were formed and mixed with or CAUAU; Lin37 si2: CCAACACAUAUGUGAUCAA; SL without TERC or TERC1–49nt at the ratio of 1:1 (mole). C26A1 si1: GCAACACCCAUGGCAAUUA; SLC26A1 The melting profile was obtained in q-PCR working buffer si2: GCCUCUAUACGUCCUUCUU; TPRG1L si1: CC containing SYSB green I in LightCycler 480 (Roche). Sam- AUUUCCUACGGAGAAUU; TPRG1L si2: GGAAUC ples were incubated at 60 C for 1 min, and then increased ◦ ◦ CCUGGUCUACCAA; TYROBP si1: GGUGCUGACA to 95 C at the rate of 2.5 C/s. Fluorescence intensity was GUGCUCAUU; TYROBP si2: UCCUUCACUUGCCU detected at 0.2 C interval. GGACG; USP16 si1: CCAUGAGCCAGUUUCUUAA; USP16 si2: GCAGAUGCUAAUUUCUCUU; ELISA and Western blotting For detection of cytokine secretion, U2OS cells were treated RNA sequencing and data analysis with 10 ng/ml TNF-, whereas B2-17 cells were treated The RNA sample preparation, sequencing and data anal- with 1 g/ml LPS for 6 h. The culture medium was collected ysis were performed as previously reported (17). The genes and cytokines were detected using corresponding ELISA with log -fold change ≥2or ≤–2 as well as FDR<0.01 were kits. For Western blotting, U2OS cells were treated with considered up- or downregulated genes, respectively. The or without TNF- for indicated times. To inhibit NF-B Downloaded from https://academic.oup.com/nar/article-abstract/47/15/8084/5531183 by Ed 'DeepDyve' Gillespie user on 27 August 2019 8086 Nucleic Acids Research, 2019, Vol. 47, No. 15 or STAT3 pathway, U2OS cells were treated with indicated RESULTS inhibitors for 6 h. The culture medium was collected for TERC promotes cellular inflammatory response ELISA analysis, and the cells were collected for Western blotting. To explore the biologic functions of TERC beyond serving as a template for telomerase extension, we ectopically ex- pressed TERC in telomerase negative U2OS cells in which endogenous TERC was in the minutest amount and TERT TERC expression in clinical samples was undetectable. Differentially expressed genes were then Processed gene expression profiles of clinical samples examined by RNA-seq. The result showed that 431 genes (GSE54350, GSE65561 and GES66988) were downloaded were up- or downregulated with log -fold change ≥2or from GEO database (Gene Expression Omnibus). Expres- ≤–2, respectively (Supplementary Table S1). Upregulated sion levels of TERC and its target genes were analyzed. genes were then subjected to Gene Oncology (GO) anal- ysis. Strikingly, 8 of 15 enriched GO terms were related to immunology, including immune response, chemokine- mediated signaling pathway, macrophage chemotaxis, and Mapping of TERC-ChIRP fragments to genome inflammatory response (Figure 1A). Alternatively, when up- TERC CHIRP-seq dataset was downloaded from published regulated genes underwent KEGG pathway analysis, 22 out article (13). The original dataset included 2198 fragments of 28 KEGG terms were related to immunology such as im- located in hg18. These fragments were re-localized in hg19. mune system, immune related diseases or inflammation re- The nearest transcription start sites (TSSs) were identified. lated pathway (Figure 1B). These results strongly suggested The distance between fragment and TSS was calculated and that TERC is involved in cellular immune in a direct or in- defined as “distance to TSS”. direct manner. To further explore this hypothesis, we stimulated TERC- U2OS and control pBabe-U2OS cells with TNF- and de- Quantitative RT-PCR tected the expression and secretion of inflammatory fac- tors. The results showed that mRNA levels of vfi e cytokines Total RNA was isolated using TRIzol (Takara). Approxi- (IL-6, IL-8, IL-32, TNF- and CSF2) were upregulated in mately 1 g RNA per sample was used to generate cDNA TERC-U2OS compared to pBabe-U2OS (Figure 1C). Ac- by reverse transcription. Real-time PCR was carried out cordingly, secreted cytokines (IL-6, IL-8 and CSF2) in cul- with q-PCR buffer containing SYBR Green I in Light- ture medium were also increased (Figure 1D). Conversely, Cycler 480 (Roche). All PCR primer sequences were from when TERC was depleted by siRNAs in human astrocy- PrimerBank (22). toma B2–17cells (siRNA with a scramble sequence was used as a control), both the expression and secretion level of cy- tokines significantly decreased (Figure 1E and F). Because Luciferase reporter assays B2–17 are telomerase positive cells, to exclude the engage- ment of telomerase in the inflammatory response, TERT HEK293T cells were plated in 96-well plates ( 1×10 was knocked down (Figure 1G) and cytokine secretion was per well) and transfected with plasmids encoding NF-B determined. Our results showed that in contrast to knock- luciferase reporter (firefly luciferase plasmid), pRL-TK- down of TERC, depletion of TERT only slightly decreased luc (renilla luciferase plasmid) and pBabe-TERC (Full- the secretion of CSF2 and had no effect on IL-6 and IL-8 length TERC). For knockdown experiment, siTERC was (Figure 1F). To further confirm the role of TERC in the in- transfected into HEK293T cells. Cells were harvested at flammatory response, TERC was knocked down in human 48 h after transfection and luciferase activity was measured normal fibroblast BJ cells, which do not express TERT and with Dual-Luciferase Assay kit (Promega) according to the therefore do not have telomerase activity. We observed that protocol provided by manufacturer. The fireyfl luciferase in- depletion of TERC significantly decreased secretion of IL-6, tensity of NF-B reporter was first normalized to renilla IL-8 and CSF2 in response to immune stimulation (Figure luciferase and then divided by corresponding control to ob- 1H). tain a relative activity of NF-B luciferase (Folds). Immunofluorescence (IF) Activation of the NF-B signaling pathway by TERC Cells were grown on coverslip, washed with PBS and fixed in The NF-B signaling pathway plays a fundamental role in 4% paraformaldehyde for 15 min at room temperature, and inflammatory response, governing the release of inflamma- then permeabilized in 0.5% Triton X-100 at room tempera- tory factors (23). We noticed that NF-B signaling path- ture for 30 min. The cells were washed thrice with 1× PBST way was enriched by KEGG pathway analysis in TERC and blocked with 5% goat serum for 1 h at room temper- overexpressing cells (Figure 1B). To explore whether TERC- ature. The cells were incubated sequentially with anti-p65 induced stimulation of inflammatory response is mediated antibody overnight at 4 C and secondary antibody conju- by NF-B pathway, inhibitors of p65 cofactors IBand gated with DyLight 488 for 1 h at room temperature. The STAT3 (Bay11-7082 and Stattic, respectively) were used to coverslip was washed with PBST, mounted with DAPI, and treat TERC-U2OS. Indeed, both Bay11-7082 and Stattic, visualized using a Zeiss microscope. but not the AKT inhibitor perifosine, suppressed the in- Downloaded from https://academic.oup.com/nar/article-abstract/47/15/8084/5531183 by Ed 'DeepDyve' Gillespie user on 27 August 2019 ** ns *** *** ** *** *** *** *** Nucleic Acids Research, 2019, Vol. 47, No. 15 8087 A Enriched GO terms of up-regulated genes after TERC overexpression GO:0048566~embryonic digestive tract development GO:0010595~positive regulation of endothelial cell migration GO:0006954~inflammatory response p value GO:0045785~positive regulation of cell adhesion 0.01 GO:0048246~macrophage chemotaxis GO:0010759~positive regulation of macrophage chemotaxis GO:0070098~chemokine-mediated signaling pathway 0.001 GO:0050918~positive chemotaxis GO:0043114~regulation of vascular permeability GO:0031663~lipopolysaccharide-mediated signaling pathway GO:0070493~thrombin receptor signaling pathway GO:0030198~extracellular matrix organization GO:0006955~immune response GO:0070374~positive regulation of ERK1 and ERK2 cascade GO:0050900~leukocyte migration 0 5 10 15 Enriched gene number Enriched KEGG pathway of up-regulated genes after TERC overexpression hsa05416:Viral myocarditis hsa05200:Pathways in cancer hsa04915:Estrogen signaling pathway Othe rs hsa04940:Type I diabetes mellitus hsa04151:PI3K-Akt signaling pathway hsa04514:Cell adhesion molecules (CAMs) hsa04064:NF-kappa B signaling pathway p value Inflammation hsa04060:Cytokine-cytokine receptor interaction re late d pathway >0.05 hsa04668:TNF signaling pathway 0.05 hsa04670:Leukocyte transendothelial migration Immune hsa04612:Antigen processing and presentation 0.01 0.001 syste m hsa04611:Platelet activation hsa04621:NOD-like receptor signaling pathway Autoimmune hsa05323:Rheumatoid arthritis dise ase hsa05330:Allograft rejection hsa05332:Graft-versus-host disease Immune hsa05310:Asthma dise ase s hsa05321:Inflammatory bowel disease (IBD) hsa05150:Staphylococcus aureus infection hsa05145:Toxoplasmosis hsa05152:Tuberculosis hsa05143:African trypanosomiasis Infe ctious dise ase s hsa05133:Pertussis hsa05168:Herpes simplex infection hsa05146:Amoebiasis hsa05142:Chagas disease hsa05144:Malaria hsa05164:Influenza A 0 5 10 15 Enriched gene number C D E pB a b e-U 2 O S TE R C -U 2 O S ELISA 1.5 NC TERC si1 TERC si2 *** 20 pBabe-U2OS *** TERC-U2OS 15 *** *** *** 10 1.0 3 *** 4 *** 0.5 2 * 0 0 0.0 F G H B2-17 ELISA ELISA 1.5 1.5 IL-6 IL-8 CSF2 1.5 IL-6 IL-8 CSF2 ns ns ** 1.0 1.0 ** *** *** ** 1.0 *** *** *** *** *** 0.5 0.5 0.5 *** *** *** *** 0.0 0.0 0.0 B2-17 BJ B2-17 Figure 1. TERC promotes inflammatory response. ( A) Enriched biological processes after TERC overexpression. Gene ontology was analyzed for upregu- lated genes in TERC-U2OS compared to pBabe-U2OS. GO terms in blue boxes were immune related biological processes. (B) Enriched signaling pathways after TERC overexpression. KEGG pathway enrichment was analyzed with upregulated genes in TERC-U2OS compared to pBabe-U2OS. Different cate- gories of pathways were boxed and labeled. (C) The mRNA levels of cytokines after TERC overexpression in U2OS cells. The stable cell lines pBabe-U2OS and TERC-U2OS were stimulated with TNF- for 1 h, and cells were collected for qPCR. (D) The secreted cytokines after TERC overexpression in U2OS cells. The stable cell lines pBabe-U2OS and TERC-U2OS were stimulated by TNF- for 6 h, and culture medium was collected for ELISA. (E) The mRNA levels of cytokines after TERC knockdown in B2–17 cells. The cells were transfected with indicated siRNAs for 72 h and treated with LPS during the last 1 h of transfection. Cells were then collected for qPCR. (F) The secreted cytokines in B2–17 cells after TERC or TERT knockdown. Cells were transfected with indicated siRNAs for 72 h and treated with LPS during the last 6 h of transfection. Culture medium was then collected for ELISA. (G) The knock- down efficiency of TERT. The B2–17 cells were transfected with TERT siRNAs for 72 h, and cells were collected for qPCR. ( H) The secreted cytokines in BJ after TERC knockdown. Cells were transfected with indicated siRNAs for 72 h and treated with TNF- during the last 6 h of transfection. Culture medium was collected for ELISA. All values are means ± SEM of more than three independent experiments (*P < 0.05, **P < 0.01,***P < 0.001). I L - 6 I L -8 IL -3 2 T N F - α C SF 2 NC TERC si1 TERC si2 NC TERC si1 TERC si2 NC TERC si1 TERC si2 NC TERC si1 TERC si2 TERT si NC TERC si1 TERC si2 TERT si NC TERC si1 TERC si2 TERT si IL-6 IL-8 CSF2 NC siTERT hTR IL-6 IL-8 IL-32 TNF-α CSF2 Re la tive f o ld of m RN A Relative fold of protein Relative fold of protein Relative fold of TERT Relative fold of protein Relative fold of mRNA Downloaded from https://academic.oup.com/nar/article-abstract/47/15/8084/5531183 by Ed 'DeepDyve' Gillespie user on 27 August 2019 8088 Nucleic Acids Research, 2019, Vol. 47, No. 15 AB C D E F G HI J Figure 2. TERC activates the NF-Bpathway.(A) Inhibition of NF-B or STAT3 activation by inhibitors in TERC-U2OS. TERC-U2OS cells were treated with inhibitors of AKT, IB and STAT3 (Perifosine, Bay11–7082 and Stattic, respectively) for 6h, and cells were collected for Western blotting of indicated proteins. (B-D) The TERC promotion of cytokine secretion was counteracted by inhibitors of p65 cofactors IB and STAT3. Stable cell lines pBabe- U2OS and TERC-U2OS were treated with TNF- plus indicated inhibitor for 6 h, and culture medium was collected for IL-6, IL-8 and CSF2 detection by ELISA. (E) Total and phosphorylated p65 were upregulated by TERC. PBabe-U2OS and TERC-U2OS cells were treated with TNF- for indicated times. Total and phosphorylated levels of p65 were determined by Western blotting. (F) Quantification of (E). Protein levels in (E) were quantified and normalized by actin. (G) Inhibited nuclear translocation of p65 in TERC depleted cells. B2–17 cells were transfected with TERC siRNA or NC for 72 h and then treated with TNF- (10 ng/ml) for 20 min. Immunofluorescence (IF) using p65 antibody was performed to determine the amount of p65 in nucleus and cytoplasm. (H) Quantification of (G). Nuclear p65 (Fluorescence intensity) was quantified as a percentage of overall p65 in cell. ∼100 cells were counted for each experiment. (I, J)NF-B luciferase reporter assay for analysis of activation of NF-B in TERC overexpressed (I) and depleted cells (J). HEK293T cells were transfected with TERC/pBabe (I) or siTERC/NC (J) together with NF-B-luc and pRL-TK. Luciferase activity was determined 48 h after transfection. All values are means ± SEM of more than three independent experiments (*P < 0.05, **P < 0.01, ***P < 0.001). Downloaded from https://academic.oup.com/nar/article-abstract/47/15/8084/5531183 by Ed 'DeepDyve' Gillespie user on 27 August 2019 Nucleic Acids Research, 2019, Vol. 47, No. 15 8089 A B DNA TERC TERC *** 40 pBabe-U2OS 30 TERC-U2OS *** *** *** *** **** * ** ** * ** *** *** D E F HeLa B2-17 293T NC siTERC 1.5 2.0 NC siTERC NC siTERC ns ns 1.5 *** ** *** *** ** *** 1.0 *** *** *** *** ** 1.0 * ns *** *** *** ** 0.5 0.5 0.0 0 0.0 Figure 3. TERC targets gene’s promoters using its binding motif. (A) Schematic diagram of TERC targeting to gene’s promoters that contain the sequence of TERC binding motif. (B). Distribution of TERC targeted fragments on genome. The fragment was classified according to its distance to the nearest TSS. (C) The mRNA levels of 30 genes with TERC binding motif in their promoters in TERC-U2OS vs pBabe-U2OS cells. (D–F). Determination of mRNA levels of top 6 genes in (C) that have the highest fold change (up- and downregulation) in response to TERC expression. Gene expression was tested in indicated cells with or without TERC knockdown. Each panel represents different cells as indicated. All values are means ± SEM of more than three independent experiments (*P < 0.05, **P < 0.01, ***P < 0.001). crease of IL-6, IL-8 and CSF2 induced by TERC overex- scription of target genes (24). The amount of p65 in nu- pession (Figure 2A–D). cleus is indicative of activation extent of NF-B pathway. Furthermore, we observed that NF-B subunit p65 and We observed that in response to TNF- treatment, TERC- phosphorylated p65 (p-p65) were higher in TERC-U2OS depleted cells display much less p65 in nucleus than control than in control pBabe-U2OS, suggesting the activation of cells, demonstrating that TERC positively regulates the ac- the NF-B pathway (Figure 2E, 0min). When cells were fur- tivation of NF-B pathway (Figure 2G, H). Moreover, we ther stimulated with TNF-, the level of p65 and p-p65 in also performed luciferase reporter assay, in which NF-B TERC-U2OS were continuously higher than pBabe-U2OS response element is placed in TATA promoter that drives throughout the activation procedure, suggesting that TERC the transcription of luciferase. The result showed that com- stimulates activation of NF-B pathway (Figure 2E, F). pared to control cells, overexpression of TERC increases lu- Upon activation of NF-B pathway, p65 migrates from cy- ciferase activity, whereas knockdown of TERC decreases its toplasm to nucleus where it functions to initiate the tran- ALPL LIN37 DUSP21 MSL1 SLC26A1 TPRG1L TYROBP RRAGA CIZ1 CNOT2 RPH3A CHD3 USP16 RAD17 CCR3 GBA2 CERCAM PYGO2 CEP63 LCN15 MTERFD3 ACTR8 SCRN1 EML3 ASTL SNAI1 ALG3 UNC5A APOM TMEM139 TERC LIN37 SLC26A1 TPRG1L TYROBP USP16 UNC5A TERC LIN37 SLC26A1 TPRG1L TYROBP USP16 UNC5A TERC LIN37 SLC26A1 TPRG1L TYROBP USP16 UNC5A Relativ e fold of mRNA Relative fold of mRNA Re lative mRNA level Re lativ e mRNA le v e l Downloaded from https://academic.oup.com/nar/article-abstract/47/15/8084/5531183 by Ed 'DeepDyve' Gillespie user on 27 August 2019 8090 Nucleic Acids Research, 2019, Vol. 47, No. 15 Table 1. Promoters containing potential TERC binding site Gene Name Gene Promoter Seq. from EPD ALPL actcgggccccgcggccgcctttataaggcggcgggggtggtggcccggGCCGCGTTGCG LIN37 taataggaacaagctactgccgaaggggcccgcccacagaagggtggtgGCCACGGTCCA DUSP21 agatgggagtggtgagaggagacagaaagagggtggtggccgatagctgGTCCTCTTTCT MSL1 gcggccagcgagggcagatggaagagTATGAGGAAGAGCCCTCTCGGGGGTGGTGGCGGC SLC26A1 cagagtccagggcacagaccactgcctgcaggttggcgccaccacccccACTCTCCCCGC TPRG1L ccgcggggcggggccgggggcgcggccgggtggtggcggtggctgcggcgacggcggtcG TYROBP ccctgtctcctcctcccttctgccaccacccgcctcagacttcctccttCACTTGCCTGG RRAGA gagatcgccgccggaagtgggtggtggcggggacgcagcggctccctccCGGAAAGCGAG CIZ1 ggcaaaatggcgaaatccctctctacaaaaaatacaaacattagccagggtggtggcggg CNOT2 agggagggagggggtgtgtatgggggtggtggtggACCGGACGTAAAGCGTCGCTGTACT RPH3A aggagggagagggggtggtggaggagggagagagggtgggagaaggagtgatgaagatgg CHD3 ggggaggcgggcgggcggtgggtgggggggtggtgggggggccAGAGCCACAGGATGGCT USP16 GGGAGGTGGGGGTGGGGTGGTGGTGGCCTAGCCACTTCCCATAATGCCGCGTTCCGGAAG RAD17 CGAATATTTGAGCTTAGTATTCCCTGTTCACTGTGTGGGGTGGTGGTGGGTCGGCTAGGA CCR3 aggtggtggcctgcccctccccgcaggcactctgtcccagggagaaatcagaactcttta GBA2 CACGGCCACTTCTGCATCCAGGTGGGGATGCTGGCACTGAAGGTGGTGGCCCTTCTGGGA CERCAM GGAGCCGGGGAAGCCCGGGAGGTGGTGGCCGAGTGGGCGCCGCCCCTCTGGGTCTGCGGC PYGO2 ttgctccccctccccgcagcgctcagtggtggtggccgcgacgagttccGGTTCCGGTTG CEP63 GCCTCGCAGGCCACCACCATCCGCACCGTACGACAGGCCGTCCCTCAGCTGCGGCTTCCT LCN15 caggtggtggcctgggctataaagctggccccctggggcttggggactcAGCACCAGGGG MTERFD3 ggaagcaaatgcagctggtgcaggagagggaaatgggaattagggtggtGGCAGAGCCCA ACTR8 cccctggtggggggAGTGCGGAAGCGGTCGTTCTTTTCCGGGTGGTGGCGCGCCGGGACG SCRN1 tcccactcctctccacctccactgccaccaccctgcaccaagccaccaccatctccagcc EML3 CTCGGGGTGGTGGTACGGCGCCCTTCGCGCGCGCCCCGGGGTGCTTCCCCTTCCCCTCTC ASTL GTAACCTAATTGCAGAACCGGCACCACCACCCCCTCTTAAATAGCAGCTGctccacctcc SNAI1 CCACCACCCCCCCGGAGTACTTAAGGGAGTTGGCGGCGCTGCTGCATTCATTGCGCCGCG ALG3 aagcggaacctaagtgtcgaaggttcgggtttccgggggtggtgggcccACACAAGCGGC UNC5A GCCCACCACCCCAAGCCCCTCCCTGGGGGAGCCTCAGGCATCGCCCAGAGGGATTCCCGG APOM acacacccaccaccccgcggctccgcccccgacttccccacggaccgtcACTTCCGGTCT TMEM139 acctacccgctccggcccttcccaccaccccccaccccatctactttctACAGTCTGTGG Underline indicates potential binding sites Lower case indicates upstream of TSS activity (Figure 2I and J). Altogether, these data confirmed by TERC using pBabe-U2OS and TERC-U2OS cells. 14 of that TERC promotes the activation of NF-B pathway. 30 genes exhibited up- or downregulated transcription in response to TERC expression (Figure 3C). The top six up- or downregulated genes were LIN37, SLC26A1, TPRG1L, TERC regulates gene transcription by targeting its promoter TYROBP, USP16 and UNC5A. Among them, four genes We next explored how TERC stimulates the NF-Bpath- (LIN37, TPRG1L, TYROBP and USP16) showed consis- way. As a typical lncRNA, we speculated that TERC may tent downregulation when TERC was knockdown in B2– regulate gene transcription by targeting specific sites in the 17, HeLa and 293T cells (Figure 3D–F). We thus focused genome. Indeed, using chromatin isolation by RNA pu- on these four genes. rification (ChIRP), it was previously reported that TERC targets the genome with a high preference for the se- TERC binds to gene’s promoters by forming RNA–DNA quence 5 -GGCCACCACCCC-3 (termed the binding mo- triplexes tif) (13), which is exactly complementary to the sequence 5 -GGGGUGGUGGCC-3 at site 25–36 of TERC. This To investigate whether TERC binds to promoters of LIN37, strongly suggested that TERC binds to genomic DNA using TPRG1L, TYROBP and USP16 in vivo, ChIRP-PCR was its binding motif, which may form a triplex structure with performed, in which biotin-labeled oligonucleotides were target DNA (Figure 3A). used to pull down TERC and associated chromatin (13,21). To survey the locations of TERC target sites, we down- PCR was then performed using primers that cover the loaded 2198 TERC-ChIRP fragments from published study TERC binding site in the gene’s promoter (Table 1). The (13) and mapped them to the human genome. The distance results showed that promoters of four genes were enriched to the nearest transcription start site (TSS) of each frag- by ChIRP, demonstrating association of these promoters ment was calculated. Most fragments (TERC targeted sites) with TERC (Figure 4A). were located within ±1000 bp of TSSs (Figure 3B), suggest- To further validate the interaction between TERC and ing that TERC tends to target gene’s promoters. Then, we promoters, electrophoretic mobility shift assay (EMSA) screened potential binding sequences using a TERC binding was carried out in vitro by incubating TERC and synthe- motif in Eukaryotic Promoter Database (EPD) (20), which sized 59 bp double-stranded (ds) DNA that is from in- contains all identified promoters and sequences adjacent to dicated gene promoter and consists of binding motif se- TSSs. As a result, as many as 30 sequences on gene pro- quence of TERC. Indeed, shifted bands were observed for moters were identified that were listed in Table 1. We then all four tested promoters, but not for scramble sequence verified whether the transcription of these genes was altered (Figure 4B). In addition, with increased TERC, shift bands Downloaded from https://academic.oup.com/nar/article-abstract/47/15/8084/5531183 by Ed 'DeepDyve' Gillespie user on 27 August 2019 Nucleic Acids Research, 2019, Vol. 47, No. 15 8091 A B C Figure 4. RNA–DNA triplex formation between TERC and gene’s promoters. (A) TERC binds to the promoter of indicated gene in vivo. TERC ChIRP was performed using TERC probe. LacZ probe was used as control. Promoters of the indicated genes were detected by PCR. (B) TERC binds to the promoters of indicated genes in vitro. EMSA was performed to detect the triplex formation between synthesized gene promoters containing TERC binding motif and TERC. RNA with random sequence was used as control. Products were analyzed on 2% agarose gels. (C) Triplex formation in a dose dependent manner. Two picomoles of indicated gene promoters were incubated with increasing amounts of TERC. Products were analyzed on 2% agarose gels. (D) Expression levels of indicated genes after RNH1 knockdown. U2OS cells were transfected with siRNH1 for 72 h, and corresponding mRNAs were detected by qPCR. (E) Melting temperature decreased after triplex formation at neutral pH. Melting temperatures of gene’s promoters were detected in the presence or absence of TERC or TERC1–49. All values are means ± SEM of more than three independent experiments (*P < 0.05, **P < 0.01, ***P < 0.001). (RNA–DNA) gradually increased and free promoter DNA TERC-targeted genes activate the NF-B pathway decreased accordingly (Figure 4C). Because NF-B component such as p65 or STAT3 is not TERC may form R-loop or triplex with targeted ds- direct target of TERC, we hypothesized that TERC stim- promoters. To test the possibility that TERC may form ulates the NF-B pathway in an indirect manner, poten- R-loops by hybridizing with targeted DNA, we knocked tially through promoting the expression of targeted genes. down the RNH1 gene in TERC-U2OS cells. RNase H is We thus tested identified four genes for their ability to ac- an enzyme that digests RNA in Watson–Crick RNA–DNA tivate the NF-B pathway. Four genes (LIN37, TPRG1L, hybrids (25). It is speculated that gene expression levels TYROBP and USP16) were individually knocked down should be altered by either overexpression or knockdown in TERC-U2OS cells. We found that while knockdown of of RNase H if they are regulated by R-loop (26). In con- LIN37, TPRG1L and USP16 decreased the level of p65 trast to this, we observed no change in the expression levels and p-p65, the knockdown of LIN37, TPRG1L and TY- of four genes when RNase H was knocked down (Figure ROBP decreased the level of STAT3 and phosphorylated 4D). In addition, we observed that melting temperatures of STAT3 (p-STAT3) (Figure 5A, B). Consistently, ELISA as- ds-promoters were reduced by approximately 1 C after in- say showed that knockdown of LIN37, TPRG1L, TYROBP cubating with TERC or 49 nt TERC fragments that contain and USP16 counteracted enhanced secretion of IL-6 by the binding motif (Figure 4E). This is consistent with the TERC (Figure 5C). Altogether, these results demonstrated previous report that melting temperature decreases during that TERC modulates the inflammatory response through conformation change from double-strand DNA into RNA– regulating a group of genes such as LIN37, TPRG1L, TY- DNA triplexes (27). Altogether, these results suggested that ROBP and USP16. While TYROBP has been previously re- TERC may target gene’s promoters by forming RNA–DNA triplexes. Downloaded from https://academic.oup.com/nar/article-abstract/47/15/8084/5531183 by Ed 'DeepDyve' Gillespie user on 27 August 2019 8092 Nucleic Acids Research, 2019, Vol. 47, No. 15 Figure 5. TERC activates NF-B pathways through targeting immune-related genes. (A) The knockdown efficiency of indicated genes. The TERC-U2OS cells were transfected with indicated siRNAs for 72 h, and corresponding mRNAs were detected by qPCR. (B) P65 and STAT3 were downregulated by knockdown of TERC targeted genes. The TERC-U2OS cells were transfected with indicated siRNAs for 72 h. Total and phosphorylated p65 and STAT3 expressions were determined by western blotting. (C) The TERC promotion of IL-6 secretion was counteracted by knockdown of TERC targeted genes. PBabe-U2OS and TERC-U2OS cells were transfected with indicated siRNAs for 72 h and treated with TNF- during the last 6 h of transfection. Culture medium was collected for IL-6 detection by ELISA. All values are means ± SEM of more than three independent experiments (*P < 0.05, **P < 0.01, ***P < 0.001). ported to be involved in the activation of the inflammatory 6E and F). Therefore, the elevated inflammatory response response (28–31), the other three genes are newly identified. is positively correlated with increased expression of TERC and TERC target genes in patients. The expression of TERC and its targeted genes are upregu- lated in inflammation related disease DISCUSSION Because TERC regulates the expression of inflammation A high incidence of TERC mutation and an increased copy related genes in vitro, we then investigated its potential number of TERC genes has been reported to associate with immune-regulation function in patients. First, we analyzed the pathogenesis of the inherited disorder dyskeratosis con- TERC levels in type II diabetes and multiple sclerosis, both genital (DC), aplastic anemia (AA) (34,35) and other ge- displaying increased chronic inflammation ( 32,33). The ex- netic diseases (36–38). Interestingly, both DC and AA pa- pression levels of TERC in CD14+ cells from type II di- tients also display immune abnormalities (39,40). More- abetes and multiple sclerosis patients were significantly over, many mutations in TERC do not affect its function as higher than normal people (Figure 6A and B). We also ex- an RNA template and telomerase activity (41–43), imply- amined the expression level of LIN37, TPRG1L, TYROBP ing that TERC may have noncanonical functions beyond and USP16 in CD14+ cells from patients and normal peo- telomerase. In this study, we revealed that TERC stimulates ple. For diabetic patients, TPRG1L and TYROBP were up- cellular inflammatory response in a telomerase independent regulated, whereas TYROBP and USP16 were upregulated manner. The evidence supporting this includes: (i) RNA-seq in multiple sclerosis patients (Figure 6C and D). Consis- data indicated that expression of immune-related genes are tently, the expression of inflammatory cytokines such as regulated by TERC; (ii) overexpression of TERC in telom- IL-8 and TNF- in diabetic patients and IL-6, IL-8, CSF2 erase negative U2OS cells resulted in increased expression and TNF- in multiple sclerosis patients increased (Figure and secretion of inflammatory factors; (iii) knockdown of TERC, but not TERT, in telomerase positive cells decreased Downloaded from https://academic.oup.com/nar/article-abstract/47/15/8084/5531183 by Ed 'DeepDyve' Gillespie user on 27 August 2019 Nucleic Acids Research, 2019, Vol. 47, No. 15 8093 A C E G S E 5 43 50 & G S E 65 561 G S E 543 50 & G S E 65 561 G S E 5 43 50 & G S E 65 561 He a lth y V o lu n te e r H e alth y V oluntee r ** Dia b e tic Dia b e tic 2.5 ** * 2.5 ** 2.0 3 2.0 1.5 1.5 1.0 1.0 0.5 0.5 0.0 0.0 0 CD1 4 Ce lls CD1 4 Ce lls CD1 4 Ce lls B D F G S E 66988 G SE6 6 9 8 8 G S E 66988 H e alth y V oluntee r H e alth y V oluntee r M u lt ip le S c le r o s is M u lt ip le S c le r o s is ** 1.4 *** *** 1.5 *** ** 1.2 1.0 1.5 *** 1.0 1.0 0.5 0.8 0.5 0.6 0.0 0.0 + + CD1 4 Ce lls CD1 4 Ce lls CD1 4 Ce lls Figure 6. The expression levels of TERC and TERC targeted genes in inflammation related diseases. ( A) TERC was upregulated in CD14 cells of diabetic patients. Data were downloaded from the GEO database (GSE54350 & GSE65561), and TERC expression levels were analyzed (n ≥ 10, *P < 0.05, **P < 0.01). (B) TERC was upregulated in CD14 cells of multiple sclerosis patients. Data were downloaded from the GEO database (GSE66988), and TERC expression levels were analyzed (n ≥ 10, *P < 0.05, **P < 0.01). (C) Expression levels of TERC targeted genes in CD14 cells of diabetic patients. Data analysis is the same as (A). (D) Expression levels of TERC targeted genes in CD14 cells of multiple sclerosis patients. Data analysis is the same as (B). (E) Expression levels of cytokines in CD14 cells of diabetic patients. Data analysis is the same as (A), except that ‘n’ of IL6 is 6 because IL6 is missing in GSE65561. (F) Expression levels of cytokines in CD14 cells of multiple sclerosis patients. Data analysis is the same as (B). secretion of inflammatory factors; (iv) overexpression of modifiers to target sites ( 13–16). A previous study identified TERC led to activation of the NF-B signaling pathway in the sequence ‘GGCCACCACCCC’ as a binding motif in the absence of allothogenic stimulation; (v) TERC upreg- TERC that may associate with genomic DNA (13). Here, we ulated the expression of LIN37, TPRG1L, TYROBP and found that TERC associated sequences were largely located USP16 that is linked to the activation of NF-B signaling near the TSSs of genes (Figure 3B). Following this route, pathway, leading to increased inflammatory response and we identified 30 potential promoters TERC may bind to. (vi) high TERC levels corresponded to high inflammation Among them, 4 genes were experimentally verified to be states in patients with type II diabetes or multiple sclerosis. transcriptionally regulated by TERC (LIN37, TPRG1L, LncRNAs may regulate gene expression in different man- TYROBP and USP16). Similar to many other lncRNAs ners (11). For example, eRNA, which is transcribed from (13–16), we demonstrated that TERC formed triplexes with enhancers, promotes chromatin accessibility by remodeling promoter sequences of these genes and thereby promoted the chromatin (44), whereas lncRNA Khps1, HOTAIR and their transcription. It should be noted that identified four MEG3 regulate gene transcription by recruiting chromatin genes are representative of 14 genes, which are transcrip- No r m a l Dia be tic LIN 3 7 TP R G 1 L T YR OB P No rm a l U SP 1 6 M ult ip le Sc ler o s i s IL6 L I N 3 7 IL 8 TP R G 1 L C S F 2 TY R O B P TN F -α U S P1 6 IL6 IL 8 CS F 2 TN F-α Re la t ivefo ld o fT E R C R e la t ivefold o fT ER C Re l a tiv e fo ld o f m R N A Re la tiv e fo ld o f m R N A R e l a ti v e fo l d o f mR N A Re lativ e fo ld o f mR N A Downloaded from https://academic.oup.com/nar/article-abstract/47/15/8084/5531183 by Ed 'DeepDyve' Gillespie user on 27 August 2019 8094 Nucleic Acids Research, 2019, Vol. 47, No. 15 tionally regulated by TERC (Figure 3). Considering that di- luciferase plasmid, and for helpful suggestions on experi- rect association is one of manners by which lncRNA regu- mental design. lates gene transcription, the total number of genes regulated Author Contributions: H. Liu and Y. Zhao designed the by TERC might be much greater than 14. Further investi- study, analyzed the data and wrote the paper. H. Liu and Y. gation is thus needed to identify whole genes regulated by Yang performed the majority of the experiments. J. Liu an- TERC. alyzed the clinical samples. Y. Ge offered ideas and helped Except for TYROBP, which was previously reported to to analyze the data. Y. Zhao supervised the project and re- engage in inflammatory response, the other three genes are vised the manuscript. All authors read and approved the newly identified that could activate the NF- B signaling manuscript. pathway. Because p65 and STAT3 are not directly regulated by TERC, we hypothesized that TERC stimulates inflam- FUNDING matory response in an indirect manner, i.e., through tran- National Natural Science Foundation of China Grants scriptional activation of inflammatory related genes that [81771506, 31571410]; National Key R&D Program of then activate NF-B signaling pathway. In the absence of China [2018YFA0107000]; Guangzhou Municipal People’s TERC, cells were less sensitive to immune stimulation by TNF- or LPS (Figure 1F, H). Therefore, we defined TERC Livelihood Science and Technology Plan [201803010108]. as a positive modulator of NF-B mediated inflammatory Funding for open access charge: National Natural Science response. Foundation of China Grants [81771506, 31571410]; Na- It was previously discovered that TERC is implicated in tional Key R&D Program of China [2018YFA0107000]; angiogenesis, metastasis and proliferation of cancer cells Guangzhou Municipal People’s Livelihood Science and by regulating the global gene expression (45,46). Similarly, Technology Plan [201803010108]. we revealed that TERC promotes cellular inflammatory re- Conflict of interest statement. None declared. sponse by upregulating the expression of immune-related genes such as LIN37, TPRG1L, TYROBP and USP16 in REFERENCES human normal and cancer cells. 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Published: Sep 5, 2019

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