Hepatitis B virus infection (HBV) is a major risk factor for the development of hepatocellular carcinoma. HBV replicates from a covalently closed circular DNA (cccDNA) that remains as an episome within the nucleus of infected cells and serves as a template for the transcription of HBV RNAs. The regulatory protein HBx has been shown to be essential for cccDNA transcription in the context of infection. Here we identified Spindlin1, a cellular Tudor-domain protein, as an HBx interacting partner. We further demonstrated that Spindlin1 is recruited to the cccDNA and inhibits its transcription in the context of infection. Spindlin1 knockdown induced an increase in HBV transcription and in histone H4K4 trimethylation at the cccDNA, suggesting that Spindlin1 impacts on epigenetic regulation. Spindlin1-induced transcriptional inhibition was greater for the HBV virus deficient for the expression of HBx than for the HBV WT virus, suggesting that HBx counteracts Spindlin1 repression. Importantly, we showed that the repressive role of Spindlin1 is not limited to HBV transcription but also extends to other DNA virus that replicate within the nucleus such as Herpes Simplex Virus type 1 (HSV-1). Taken together our results identify Spindlin1 as a critical component of the intrinsic antiviral defense and shed new light on the function of HBx in HBV infection. Hepatitis B virus (HBV) represents a major risk factor for the development of hepatocarcinoma. Within the nucleus, HBV transcription is activated by both cellular and viral factors but is also repressed by cellular proteins that could be part of cellular antiviral defense mechanisms. Recently it has been shown that the regulatory protein HBx is essential to initiate and maintain HBV transcription in the setting of infection. Here we identify Spindlin1, a cellular protein involved in transcriptional regulation, as an HBx interacting partner. We show that Spindlin1 is recruited to the HBV DNA and inhibits its transcription in the context of infection. A virus deficient for the expression of HBx is more severely repressed by Spindlin1 than the wild type virus, suggesting that HBx counteracts Spindlin1 repression. We found that Spindlin1 also represses the transcription of Herpes Simplex Virus type 1 in the setting of infection. Our study not only provides new insights into the mechanisms regulating HBV transcription and the role of HBx in this process, but also reveals a new function of Spindlin1 as a component of the intrinsic antiviral defense. Despite the existence of an effective preventive vaccine, hepatitis B virus infection remains a major health problem. Chronic HBV infection affects 350 million people worldwide who are at high risk of developing liver diseases including cirrhosis and hepatocellular carcinoma (HCC) cccDNA is organized into a chromatin like structure and viral genes transcription is directed by four promoters and two enhancers. cccDNA transcription is likely regulated as cellular DNA by the activity of transcription factors, histone modifiers and chromatin remodelers Beside cellular factors, the regulatory protein HBx that is essential for virus replication plays a crucial role in HBV transcription Spindlin1 was initially identified in mice as a highly expressed maternal protein that might play a role during meiosis In this study, we show that Spindlin1 represses HBV transcription in the context of infection. We demonstrate that Spindlin1 is recruited to the HBV cccDNA and that repression is dependent on its tudor-like domain II. Finally, Spindlin1 repression extends to HSV-1 transcription during infection of HepaRG hepatoma cells. Together our findings suggest that Spindlin1 participates in the intrinsic antiviral defense against at least two DNA viruses, HBV and HSV-1.Wild type HBx (adw subtype) and deletion mutants carrying an N-terminal HA tag have been described previously 5′GCGGCCGCCACTGTGCTGGATATCTGCAGA3′ containing the restriction site EcoRV and anti-sense 5′TGAATCATTGGAATCAGGTTTGCCAATCATTGTGTCTGC3′ and PCR2 was performed using the following primers: sense 5′ TTCGGGCCCAAGCTTGGTACCGAGCTCGGA3′and anti-sense 5′GCAGACACAATGATTGGCAAACCTGATTCCAATGATTCA3′ containing the restriction site HindIII. The products of PCR1 and PCR2 were then mixed and amplified by PCR using the following primers: sense 5′GCGGCCGCCACTGTGCTGGATATCTGCAGA3′ and anti-sense 5′GCAGACACAATGATTGGCAAACCTGATTCCAATGATTCA3′ containing the restriction site EcoRV and HindIII respectively. The EcoRV-HindIII fragment containing the cDNA Spin1mut was cloned in His6-Myc-Spindlin1 digested by EcoRV-HindIII. All the constructions have been verified by sequencing.His-Myc-Spin1mut plasmid encoding Spindlin1 deleted of the second Tudor like domain, was generated by several PCR amplifications using the His-Myc-Spindlin1 expression vector Control siRNAs and siGENOME SMART pool siRNAs directed against Spindlin1 were purchased from ThermoScientific:GCAUGCCAGUGAAGCGUUASPIN1 D-019983-17: CCACAUGUAAUGACGCUAUSPIN1 D-019983-18: GUUGCGACAUCUCGAAUCASPIN1 D-019983-04: GGACUAGAACUUAAUAAAGSPIN1 D-019983-02: −7 M hydrocortisone hemisuccinate, and 5 µg/ml insulin. HepAD38 cell line is derived from HepG2 cells and contains the HBV genome under tetracycline control −5 M hydrocortisone hemisuccinate, 5 µg/ml insulin and 2% DMSO (Sigma-Cell Culture reagent). HepaRG differentiation has been described previously HEK293 and HEK293T cells were maintained in Dulbecco's modified Eagle's medium (DMEM) (Gibco) with 10% fetal bovine serum (FBS). Huh7.25.CD81 has been described previously and was maintained in DMEM (Gibco) with 10% FBS 5′CCGGGGATCCATGATGAAGAAGAGGACAT3′ and antisense 5′CCGGCTCGAGTCAATGATGATGATGATGATGGC3′ containing the restriction sites BamHI and XhoI. pTRIP-His-Myc-Spindlin1 plasmid was generated by cloning the BamHI-XhoI fragment containing His-Myc-Spindlin1 cDNA in the BamHI-XhoI sites of the lentiviral vector pTRIPΔU3. pTRIP Flag-HA (Mock) and pTRIP His-Myc (Mock) contain, respectively the Flag-HA and the His-Myc Tag cloned into the lentiviral vector pTRIPΔU3. Virions were produced by calcium phosphate transfection of HEK293T cells as previously described pTRIP-Flag-HA-HBx plasmid has been previously described pALPS-shSpindlin1.1, pALPS-shSpindlin1.2 and pALPS-shSpindlin1.3 plasmids were generated by PCR amplification from the following template:shSpindlin1.1:5′TGCTGTTGACAGTGAGCGATGGAACTATGAAATGTATTATTAGTGAAGCCACAGATGTAATAATACATTTCATAGTTCCACTGCCTACTGCCTCGGA3′shSpindlin1.2:5′TGCTGTTGACAGTGAGCGCGCCCTCCGTCTATTTCATCAATAGTGAAGCCACAGATGTATTGATGAAATAGACGGAGGGCTTGCCTACTGCCTCGGA3′shSpindlin1.3:5′TGCTGTTGACAGTGAGCGACCAAATTTGTGGAACTATGAATAGTGAAGCCACAGATGTATTCATAGTTCCACAAATTTGGCTGCCTACTGCCTCGGA3′with the following primers:sense5′GATGGCTGCTCGAGAAGGTATATTGCTGTTGACAGTGAGCG3′antisense5′GTCTAGAGGAATTCCGAGGCAGTAGGCA3′ containing the restriction sites XhoI and EcoRI. The XhoI-EcoRI fragment containing the shRNAs against Spindlin1 were cloned in the XhoI-EcoRI sites of the lentiviral vector pALPs-mir30. Virions were produced as a pool as described previously. HepAD38 or HepaRG cells stably expressing shSpindlin1 or the control shCtrl were established by transducing cells twice with virus normalized for RT activity. After 48 h of incubation with fresh medium, clones have been selected with puromycine (4 µg/ml). The pool of selected clones was analyzed for Spindlin1 expression by Western blot and quantitative RT-PCR (RT-qPCR).ActinomycinD was purchased from Sigma-Aldrich and used at a concentration of 5 µg/ml. The protease inhibitors were obtained from Roche (Complete EDTA free) and from Sigma-Aldrich (Pefabloc). TURBO DNAse was purchased from Ambion (Life technologies). Antibodies are described in 2, 0,1% Triton X-100, 0,5 mM EDTA, 10% glycerol, 10 mM de β-mercaptoethanol, 0.5 mM PMSF) containing EDTA-free protease inhibitors cocktail (Roche). After lysis, the extracts were cleared by centrifugation . Supernatant was then incubated with appropriate antibodies and beads for 2 h. Proteins complexes bound to the beads were washed 3 times in lysis buffer and then eluted from the beads by boiling in Laemmli buffer for 10 min. Samples were resolved by SDS-PAGE and electro-transferred to nitrocellulose membranes. Blots were incubated with the indicated primary antibodies and then with alkaline phosphatase conjugated secondary antibodies. Proteins were visualized by chemiluminescence . When using Odyssey procedure, after incubation with the primary antibody, blots were probed with Dye-conjugated secondary antibodies. Fluorescent immunoblot images were acquired and quantified by using an Odyssey scanner and Odyssey 3.1 software (Li-CorBiosciences).For immunoprecipitation and Western blot experiments, cells were lysed at 4°C in lysis buffer  or HepG2 H1.3Δx (HBV X-) cells were cultivated in Williams E medium containing 5% fetal calf serum 7×10nd RC 3′  [4]. Onl6 undifferentiated HepaRG cells were incubated at 37°C for 1 h with virus at the indicated MOI. After 1 hour of incubation, cells were washed with DMEM, centrifuged for 5 min at 1500 rpm and then cultured in 6-well plates for the indicated time.HSV-1 strain KOS was propagated in U373MG cells and was kindly provided by M. Lafon HCV JFH1 was propagated in Huh7.25/CD81 cells was kindly provided by E. Meurs. The preparation of JFH1 stocks have been described previously cccDNA was purified from differentiated HepaRG cells 8 days after infection with HBV wild-type or HBV X- viruses. Cells were harvested and lysed in buffer 1 . After 15 min at 4°C, NP40 1% was added. Supernatant (cytosol) and pellet (nuclei) were separated by centrifugation . Nuclear content was extracted in a volume/volume mixture of buffer A  and buffer B . Proteinase K (Fermentas) digestion was then carried out for 1 h at 45°C. After DNA purification, cccDNA was quantified by qPCR using specific primers . Level oGenomic DNA of HSV1 was extracted from HepaRG cells 2 h post infection. Cells were incubated 15 min at 37°C in lysis buffer . KCl 0.625 M was added and the supernatant was recovered after centrifugation at 13,000 rpm for 15 min. DNA was purified by phenol-chloroform extraction and genomic HSV1 DNA was quantified by qPCR using specific primers . The levChIP experiments were carried out on infected HepaRG cells 8 days post infection as described previously with minor modifications Total RNA was prepared from transfected HepAD38 cells or infected HepaRG cells using TRIzol reagent (Invitrogen) and TURBO DNA-free reagent (Ambion). RNA (500 ng) was retrotranscribed using random primers and RevertAid H Minus M-MuLV reverse transcriptase (Fermentas). cDNA was analyzed by quantitative PCR (qPCR) using SybrGreen PCR Master mix (Applied Biosystems) on ABI PRISM 7900HT Sequence Detection System (Applied Biosystems) using standard PCR protocol , and a final dissociation step to ensure amplicon-specific detection. The primers used in RT-qPCR are listed in All synthetic oligonucleotides were ordered from Eurofins MWG Operon .32P-labeled full-length HBV genome. Relative intensities of DNA RC and SS DNA were quantified using the Storm840 PhosphorImager (Molecular Dynamics).HBV replication intermediates were extracted from HepAD38 cells control or expressing His-Myc-Spindlin1. 48 h after transduction, cells were harvested and lysed in lysis buffer . After 1 min of centrifugation at 13,000 rpm, the supernatant was recovered, and 3.3 mM MgOAc was added. The samples were then digested with 100 µg DNAse I and 50 µg RNAse A (Roche) for 1 h at 37°C. After 1 min of centrifugation at 13,000 rpm, the supernatant was collected and supplemented with 6.25 mM EDTA, 0.5% SDS, 62.5 mM NaCl and 150 µg of proteinase K. After 1 h at 55°C, the DNA was purified. After 1 h at 55°C, HBV replication intermediates were recovered by phenol-chloroform extraction and isopropanol precipitation. DNA was analyzed by electrophoresis through 1% agarose gel and transferred to nylon membranes (Amersham Hybond-XL). Blots were hybridized with 32P-labelled full-length HBV genome.For analysis of cccDNA by Southern blot, total DNA was extracted as described previously Total RNA was extracted using TRIzol reagent as recommended by the manufacturer (Invitrogen). RNA samples (20 µg) were resolved on 1% formaldehyde-agarose gel and transferred to a Hybond N+ nylon membrane (Amersham). Blots were hybridized with full-length 3.2 kb HBV DNA or 18S rDNA probes labeled by random priming.Nuclei were isolated from Hepa38 cells and the reaction was performed as previously described It has been shown that HBx is required for the establishment of a transcriptionally active cccDNA during infection The Tudor-like domain II of Spindlin1 has been shown to be important for binding to H3K4me3 and to SERBP1 32P-full-length HBV DNA probe  promoter under tetracycline control, were transduced with a lentiviral vector coding for His-Myc-Spindlin1 or with a control lentiviral vector. After 48 h, HBV RNA expression level and HBV replicative intermediates were respectively analyzed by quantitative RT-PCR (qRT-PCR), northern blotting and southern blotting using NA probe . As showNA probe . BecauseNA probe . To nextNA probe . The relNA probe . The lacNA probe .Finally, to confirm the role of Spindlin1 on HBV transcription we silenced the expression of Spindlin1 in HepAD38 cells and analyzed HBV transcription using RT-qPCR . As contTogether these results show that Spindlin1 represses HBV replication by acting at the level of transcription.We next studied whether Spindlin1 represses HBV transcription in the context of infection. Moreover, since HBx interacts with Spindlin1, we examined whether Spindlin1 differentially affects the transcription of wild type HBV (HBV wt) and HBx deficient HBV (HBV X-) viruses. HepaRG cells stably expressing the shRNA Spindlin1 (shSpin1) or the shRNA control (shCtrl) were then infected with normalized amount of HBV wt or HBV X- viruses and both HBV and ribosomic transcription were analyzed 8 days later using RT-qPCR. In line with previous report, depletion of Spindlin1 resulted in a decrease of rRNA transcription; in parallel it induced HBV wt and HBV X- transcription . InteresTo investigate the role of Spindlin1 on HBV transcription, we studied its recruitment to the cccDNA. Eight days after infection of differentiated HepaRG cells with normalized amount of HBV wt or HBV X- viruses, chromatin was prepared and subjected to quantitative Chromatin immunoprecipitation (ChIP-qPCR) assays using the indicated antibodies. As shown in We then studied whether the aromatic cage of the Tudor-like domain II of Spindlin1 needed for methyllysine binding is required for the transcriptional repression of HBV. To this aim we tested the activity of previously described Spindlin1 constructs containing mutation in their Tudor-like domain II. Mutation at F141 residue (Spindlin1 F141A) and mutation at residue Y170 (Spindlin1 Y170A) both lead to a decrease in H3K4me3 binding and in the activation of pre-rRNA transcription A previous study has shown that the recruitment of Spindlin1 to the rDNA correlates with the increase of H3K4me3 marks Together our data suggest that cccDNA transcriptional repression by Spindlin1 correlates with its recruitment to the cccDNA and with the decrease of H3K4me3; a process that required Tudor-like domain II integrity.Having shown that Spindlin1 represses the transcription of HBV, we asked whether it could also repress the transcription of different DNA viruses that replicate in the nucleus. To this aim, we tested the role of Spindlin1 on the transcription of Herpes simplex virus type 1 (HSV-1). Upon HSV-1 infection, the viral nucleocapsid is transported to the nuclear pores and the viral genome is released into the nucleus. HSV-1 transcription that takes place after the circularization of the linear viral DNA is temporally organized with transcription of immediate early (IE), early and late gene products To study whether this negative regulation is specific to DNA viruses, we tested the role of Spindlin1 overexpression on the replication of the Hepatitis C virus (HCV), a positive strand RNA virus. Huh7.25.CD81 cells were first transfected with increasing amount of His-Myc-Spindlin1 vector and 24 h later, the cells were infected with normalized amount of HCV at MOI 0.3. HCV replication was assessed by RT-qPCR 48 h after infection. As shown in All together our data suggest that Spindlin1 is involved in the negative regulation of the transcription of the DNA viruses in the nucleus.A major finding in this manuscript is that Spindlin1, a protein that we isolated as an HBx interacting partner, represses the transcription of not only HBV but also HSV-1 through epigenetic regulation, suggesting that Spindlin1 could participate in the recognition and the silencing of these two viral nuclear DNAs. Indeed, we observed that suppression or overexpression of Spindlin1 respectively increases or inhibits the transcription of both HBV and HSV-1 in the context of HepaRG cells infection. Using nuclear run-on assays, we showed that Spindlin1 acts at the level of transcription. Finally, using ChIP experiments, we demonstrated that Spindlin1 inhibits transcription through its recruitment to the viral DNA and that its depletion correlates with an increase of active chromatin marks. Importantly, we observed that HBV transcriptional repression by Spindlin1 is higher for the HBV X- virus than for the HBV wt virus, suggesting that HBx via its interaction with Spindlin1 counteracts its activity. This hypothesis is further supported by the fact that Spindlin1 is recruited at a higher level to the HBV X- cccDNA than to the HBV wt cccDNA.Previous studies have shown that HBx is essential for the establishment of productive infection by acting mainly at the level of transcription. Indeed Lucifora and colleagues have observed that HBV X - infection leads to a very low level of HBV RNA that correlates with the decrease of H3 acetylation onto the cccDNA Transcriptional activation of rRNA by Spindlin1 requires its interaction with H3K4me3 Spindlin1 has been to date identified in different complexes such as the β-arrestin complex, Argonaute 3 complex and to interact with different proteins such as H3K4me3, Tcf4, SERBP1 and hyaluronan binding protein 4 (HABP4), suggesting that Spindlin1 is involved in diverse cellular processes While to date, Spindlin1 has been identified as a transcriptional activator, we observed here that Spindlin1 represses the transcription of HBV and HSV-1 in the context of infection. The molecular mechanisms of both transcriptional activation and transcriptional repression by Spindlin1 is yet unknown. The switch from a repressor function to that of an activator can be dependent on the partner associated with Spindlin1 and/or on post-translational modifications. It has been shown that lysine-specific demethylase 1 (LSD1) behave as a repressor through the demethylation of H3K4me1/2 when associated with CO-REST a chromatin-associated transcriptional repressor but as an activator when associated with androgen receptor through the demethylation of H3K9 We also showed that Spindlin1 represses the transcription of HSV-1, as its depletion in HepaRG increases the transcription of early ICP27 transcripts used as a marker of viral transcription. Upon entry into the nucleus, HSV-1 gene expression is rapidly repressed by constitutively expressed proteins such as components of the PML nuclear body : hDaxx, ATRX, Sp100 and PML, as well as proteins of the DNA repair machinery such as RNF8, RNF168 and 53BP1 Spindlin1 was identified as a partner of HBx. Using HBx deletion mutants and HBx alanine mutants; we observed that the N-terminal domain of HBx to aa 50, as well as domains spanning aa 59 to 65 and aa 84 to 140 are needed for a maximal interaction with Spindlin1. A former study using HBx alanine mutants has shown that amino acids 52 to 65 and 88 to 154 are critical for the transcriptional activation and the replication of an HBV X- vector transfected into HepG2 cells Owing to the development of cell lines that support infection such as HepaRG, it is now possible to assess the function of cellular and viral proteins on HBV replication in the setting of infection. Here we have identified Spindlin1 as a cellular factor that represses the transcription of DNA viruses such as HSV-1 and HBV. Taken together our results suggest that Spindlin1 contributes to the intrinsic antiviral defense against at least two DNA viruses and that in the case of HBV, its repressive activity is counteracted by HBx.Figure S1Determination of the region of HBx interacting with Spindlin1. Left panel, schematic representation of full-length HBx protein and in frame HBx deletion mutants. Right panel: HEK293 cells were co-transfected with wild type HA-tagged HBx (HA-X0) construct or HA-tagged HBx deletion mutants  and the His-myc-Spin1 plasmid. Cellular extracts were immunoprecipitated with anti-HA antibodies and analyzed by anti-Myc and anti-HA immunoblot. The expression of tubulin was used as loading control.(TIF)Click here for additional data file.Figure S2A) HepAD38 cells cultured without tetracycline were transduced with an empty lentiviral vector or a lentiviral vector encoding His-myc-Spindlin1 (Myc-Spin1). 48 h after transduction cells were harvested for total RNA extraction. Transcription was analyzed by Northern blotting. (B) HepAD38 cells cultured and transduced as in (A) were harvested 48 h after transduction for total RNA extraction and Cyclin A2 transcription was analyzed by RT-qPCR. Transcript level in cells transduced with the empty lentiviral vector was set to 1. Error bars represent SD of three independent experiments. (C) HepAD38 cells grown without tetracycline for several days, were treated with tetracycline for 16 h before transduction with an empty lentiviral vector or a lentiviral vector encoding His-myc-Spindlin1 (Myc-Spin1). Total RNA were prepared as in (B) and HBV transcription was analyzed by RT-qPCR. Error bars represent SD of two independent experiments. (D) HepaAD38 cells were transduced as in (B) and nuclear run-on assays were performed on isolated nuclei. Transcripts generated during run-on were purified using anti-BrdU beads and Cyclin A2 RNAs were quantified by RT-qPCR. Transcript level in cells transduced with the empty lentiviral vector was set to 1. Error bars represent SD of three independent experiments. (E) HepAD38 shCtrl cells or HepAD38 shSpindlin1 were transfected with 25 nM of control siRNA (siCtrl) or directed against Spindlin1 (siSpin1) respectively. 48 h post-transfection, cells were harvested for total RNA extraction and Cyclin A2 transcription was analyzed by RT-qPCR. Transcript level in HepAD38 shCtrl+ siCtrl cells was set to 1. Error bars represent SD of four independent experiments.((TIF)Click here for additional data file.Figure S3A) Culture supernatants of shSpin1 or shCtrl HepaRG cells were collected 8 days after infection with normalized amount of HBV wt or HBV X- viruses. Secreted HBsAg was measured by ELISA. Secreted HBsAg level in shCtrl cells infected with HBV wt was set at 1. (B) Differentiated shSpin1 or shCtrl HepaRG cells were infected with normalized amount of HBV wt HBV X- viruses at MOI 1000. 8 days after infection, cells were harvested for total DNA extraction. Viral DNA was analyzed by Southern blot hybridization using 32P labeled HBV-DNA probe. 30 µg of total DNA extracted from HepaD38 cells were used as control (C) Differentiated HepaRG cells were transduced with an empty lentiviral vector (Ctrl) or a lentiviral vector encoding His-myc-Spindlin1 (Myc-Spin1). 24 h post-transduction, cells were infected at MOI 200 with HBV wt virus. 8 days after infection, cells were harvested and total RNA was isolated. Viral RNAs were analyzed by RT-qPCR. The level of transcription in the cells transduced with the control lentiviral vector was set at 1. The expression of His-myc-Spindlin1 was analyzed by anti-Myc immunoblot (*: none specific band).((TIF)Click here for additional data file.Figure S4Huh7.25.CD81 cells transfected with 1 or 2 µg of plasmid coding for His-myc-Spindlin1 or with a control plasmid, were infected with HCV at MOI 0.3. 48 h after infection, cells were collected for RNA extraction. Viral RNA was quantified by RT-qPCR. Spindlin1 expression was analyzed by immunoblotting with anti-Myc antibodies.(TIF)Click here for additional data file.


Epigenetic marks critically control gene expression and thus the cellular activity state. The functions of many epigenetic modifiers in the vascular system have not yet been studied. We screened for histone modifiers in endothelial cells and observed a fairly high expression of the histone plant homeodomain finger protein 8 (PHF8). Given its high expression, we hypothesize that this histone demethylase is important for endothelial cell function. Overexpression of PHF8 catalyzed the removal of methyl-groups from histone 3 lysine 9 (H3K9) and H4K20, whereas knockdown of the enzyme increased H3K9 methylation. Knockdown of PHF8 by RNAi also attenuated endothelial proliferation and survival. As a functional readout endothelial migration and tube formation was studied. PHF8 siRNA attenuated the capacity for migration and developing of capillary-like structures. Given the impact of PHF8 on cell cycle genes, endothelial E2F transcription factors were screened, which led to the identification of the gene repressor E2F4 to be controlled by PHF8. Importantly, PHF8 maintains E2F4 but not E2F1 expression in endothelial cells. Consistently, chromatin immunoprecipitation revealed that PHF8 reduces the H3K9me2 level at the E2F4 transcriptional start site, demonstrating a direct function of PHF8 in endothelial E2F4 gene regulation. Conclusion: PHF8 by controlling E2F4 expression maintains endothelial function. An intact endothelial barrier is essential for vascular function. It prevents vessel occlusion and controls vascular permeability. After vascular injury, endothelial cells locally proliferate, enlarge and migrate to restore an intact vascular surface. Indeed, endothelial cell death (apoptosis), dysfunction or senescence has been implicated in the pathogenesis of numerous vascular diseases such as atherosclerosis, thrombosis and vascular leakage –3.Epigenetic control of gene expression by histone modification is a central mechanism determining cell-fate and cell-phenotype maintenance . CompareIn this study we focused on the enzyme plant homeodomain finger protein 8 (PHF8). The biology of this histone demethylase is inadequately understood but its relevance for one human disease has already been demonstrated: Mutations of PHF8 are a cause for the X-linked intellectual disability but vascular phenotypes have not been reported ,11. The As we observed significant mRNA expression of PHF8 in endothelial cells and based on its broad impact on gene regulation, we hypothesize that PHF8 also impacts on endothelial cell function.Human recombinant TNFα (#300-01A) was purchased from PeproTech , cycloheximide from Sigma-Aldrich . Anti-PHF8 (#ab36068), anti-H4K20me1 (#ab9051) and anti-H4 (#ab10158) were purchased from abcam. The second anti-PHF8 was from bethyl (#A301-772A). Anti-H3K9me1 (#pAB065-050), anit-H3K9me2 (#C15410060), anit-H3K27me1 (#pAb-045-050), anti-H3K27me2 (#pAb-046-050), H3K4me3 (#pAb-003-050) and anti-H3 (#C15200011) were from diagenode. Topoisomerase 1 (#sc5342) and anti-Tubulin beta (#sc-9104) antibodies were acquired from Santa Cruz . Anti-ßActin (#A1978) was purchased from Sigma-Aldrich .Human umbilical vein endothelial cells (HUVECs) were purchased from Lonza  and PELOBiotech . Cells were cultured on fibronectin-coated  dishes in endothelial growth medium (EGM), consisting of endothelial basal medium (EBM) supplemented with human recombinant epidermal growth factor (EGF), EndoCGS-Heparin, , 8% fetal calf serum (FCS) , penicillin (50 U/ml) and streptomycin (50 μg/ml)  in a humidified atmosphere of 5% CO2 at 37°C. For each experiment at least three different batches of HUVEC from passage 3 were used.Immortalized human microvascular endothelial cells (HMEC-1) were provided by CDC . Cells were cultured on fibronectin-coated dishes in endothelial growth medium (EGM). For experiments cells from passage 6 were used.Human embryonic kidney (HEK) 293T/17 cells (#CRL-11268) were purchased from ATCC . Cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM), high glucose, GlutaMAX from Gibco, Lifetechnologies , supplemented with 8% fetal calf serum (FCS), penicillin (50 U/ml), and streptomycin (50 μg/ml) in a humidified atmosphere of 5% CO2 at 37°C.Human aortic smooth muscle cells (HAoSMC)(#354-05a) were purchased from PELOBiotech . Cells were cultured in Smooth Muscle Cell Medium (#PB-MH-200-2190) supplemented with 8% fetal calf serum (FCS), penicillin (50 U/ml), streptomycin (50 μg/ml), EGF, FGF, glutamin and insulin from singlequots . Cells were cultured in a humidified atmosphere of 5% CO2 at 37°C.Human foreskin fibroblasts were cultured in DMEM/F12 (#11039–021) from Gibco  supplemented with 8% fetal calf serum (FCS), penicillin (50 U/ml), and streptomycin (50 μg/ml) in a humidified atmosphere of 5% CO2 at 37°C.http://www.addgene.org/tools/protocols/plko/). Cells were selected with puromycin (0.5 μg/ml). The PHF8 target sequence was: 5’- CCGGCCCAACTGTGAAGTCTTGCATCTCGAGATGCAAGACTTCACAGTTGGGTTTTTG -3’. Control shRNA against green fluorescent protein (shGFP) and shScrambled (shScr) were purchased from Addgene. For siRNA treatment, endothelial cells (80–90% confluent) were transfected with GeneTrans II according to the instructions provided by MoBiTec . siRNAs for PHF8 were purchased from Sigma . siE2F4 was purchased from ThermoFisher Scientific (#114193 siE2F4-1 and #114194 siE2F4-2). Control siRNAs (siScrambled) were from Invitrogen . Plasmid overexpression was achieved with the Neon electroporation system (Invitrogen). The plasmid PHF8 (vector: pcDNA3) was kindly provided by Shi Yang  [For shRNA treatment, endothelial cells were infected with lentiviral particles according to Addgene “plKO.1 Protocol” . cDNA was prepared with SuperScript III reverse transcriptase (Invitrogen) and random hexamer together with oligo(dT) primers (Sigma #O4387). Quantitative real-time PCR was performed with Eva Green Master Mix and ROX as reference dye (Bio&Sell #76.580.5000) in a Mx3005 cycler (Stratagene). Relative expression of target genes were normalized to (RNA)II (DNA-directed) polypeptide A (POLR2A) or ß-Actin and analyzed by the delta-delta Ct method with the MxPro software .Cells were lysed with Triton X-100 lysis buffer , 10 nM okadaic Acid, protein-inhibitor mix (PIM), 40 μg/ml phenylmethylsulfonylfluorid). Cells were centrifuged for 10 min at 16,000 g. Supernatant and pellet were used and after determination of protein concentration by the Bradford assay, equal amounts of proteins were boiled in Laemmli buffer and separated by SDS-PAGE gel electrophoresis. Infrared-fluorescent-dye-conjugated secondary antibodies were purchased from Licor  and detected with an infrared-based laser scanning detection system . For nucleus protein extraction cells were lysed in a buffer containing . After incubation on ice for 15 min, Nonidet was added (0.75%) and cells were centrifuged for 1 min at 16,000 g. Pellet was lysed in a buffer  containing 0.033 u Benzonase (Sigma) and 2 u DNase (Promega) and rotated with 8 rpm by 4°C for 30–60 min. Subsequently, proteins were boiled in Laemmli buffer.6 HUVECs), crosslinking and nuclei isolation were performed with the truCHIP™ Chromatin Shearing Kit  according to the manufacturers protocol. Afterwards, the lysates were sonified with the Bioruptur Plus  at 4°C. Cell debris were removed by centrifugation and the lysates were diluted 1:3 in dilution buffer . After preclearing with 20 μL DiaMag protein A coated magnetic beads  for 30 minutes at 4°C, samples were incubated over night at 4°C with 3 μg of anti-H3K9me1, anit-H3K9me2, anti-H4K20me1 and anti-H4 . 5% of the samples served as input. The antibody complexes were collected with 35 μL DiaMag protein A coated magnetic beads  for 3 hours at 4°C, subsequently washed twice for 5 minutes with each of the wash buffers 1–3  and finally washed with TE-buffer pH 8.0. Elution of the beads was done with elution buffer  containing 1x proteinase K  and shaking at 600 rpm for 1 hour at 55°C, 1 hour at 62°C and 10 minutes at 95°C. After removal of the beads, the eluate was purified with the QiaQuick PCR purification kit  and subjected to qPCR analysis. Chip data were calculated relative to input.Cell preparation  containing FCS (8%). Endothelial cell migration was monitored by live cell imaging (Zeiss TIRF System LASOS77). The distance of migration was calculated using ImageJ software.Scratch wound-healing assays were performed in 24-well plates. 1.5 x 105 cells were seeded on membrane inserts  in the presence of EBM. The lower chamber contained EBM supplemented with 1% FCS. After 20 hours, the cells on the upper surface of the filter were removed mechanically. Then cells that had migrated into the lower compartment were fixed , stained with DAPI and counted.Endothelial cell migration in Boyden chamber assays were investigated in a modified transwell chamber system. 2x104 HUVECs in EBM + 1%FCS for 7 hours. Subsequently fixed with 4% paraformaldehyde and images were acquired on an Axiovert135 microscope (Zeiss). Cumulative tube length was analyzed with the AxioVision software (Zeiss).For the investigation of endothelial cells to form capillary-like structures, a tube formation assay was performed. Matrigel Growth Factor Reduced (BD) was prepared and incubated with 75x106 HUVECs according to the protocol from chemometec “Annexin V Assay” (application note No. 3017 rev. 1.4). NucleoCounter NC-3000 (chemometec) was used for quantification.Measurements were performed with 1x105 cells were resolved in 200 μl propidium iodide solution  with 50 μl RNase (50 μg/ml) and quantified by flow cytometry. Cells were analyzed by the FACS Calibur machine .HUVECs were fixed with -20°C 70% ethanol and 5x106/ml) was treated with 0.5 μl 10 mM CFSE solution  and incubated for 10 min placed in the dark. Cells were washed 3 times with 4–5 volumes of ice cold Dulbecco's Modified Eagle's Medium with 8% fetal calf serum. Cells were analyzed by the FACS Calibur machine .Cell suspension . Calculations were performed with Graphpad PRISM 5.0. In case of multiple testing, Bonferroni correction was applied. For multiple group comparisons ANOVA followed by post hoc with Fisher LSD test was performed. Individual statistics of samples were performed by T-test. P values of <0.05 was considered as significant. Unless otherwise indicated, N indicates the number of individual experiments.. Western blot analysis confirmed the expression of PHF8 protein in HUVECs and HMECs . A comparison between the cytoplasmic and nuclear cellular fraction revealed that the expression of PHF8 is restricted to the nucleus, which is suggestive for a potential function as histone demethylase in endothelial cells . As the antibody used (abcam #ab36068) exhibited significant unspecific reactivity in the cytosolic fraction as well as in the nucleus, the signal was validated by siRNA technique. Whereas the signal in the cytosolic fraction was siRNA-resistant, two of the numerous bands obtained in the nuclear fraction were attenuated by siRNA. This result could be validated with a more specific antibody (bethyl #A301-772A). With this antibody, only two bands were detected in the nuclear fraction which both decreased in response to siRNA . Whether the two bands represent splice variants or post translational modifications of the protein is unclear.To uncover a potential function of PHF8 in the endothelium, we first analyzed its expression in different cell types. qRT-PCR revealed that the expression of PHF8 mRNA in human umbilical vein endothelial cells (HUVECs) and microvascular endothelial cells (HMECs) is higher than that of the closest family member JHDM1D . Importantly, reduction of PHF8 expression by RNA interference in HUVECs resulted in a global increase of H3K9me1. In contrast to the overexpression studies, however, this approach did not change global H3K9me2 and H4K20me1 levels . Thus, PHF8 decreases global level of H3K9me1 and if overexpressed also of H3K9me2 and H4K20me1 in endothelial cells.PHF8 has been described to demethylate several lysine residues in histone 3 and 4, such as H3K9, H3K27, and H4K20 –17 HowevFig 3A). In line with this, flow cytometry analyses with the CFSE dye revealed a reduced dilution of CFSE which is suggestive for a reduced cell division rate . Nuclear propidium iodide staining for cell cycle analysis demonstrated that the knockdown of PHF8 led to accelerated G1/S transition but to reduced progression of S- and G2/M phase .To identify a functional role for PHF8 in endothelial cells, the effect of depletion of the enzyme on endothelial proliferation and survival was determined. Total cell number increased significantly slower in PHF8 shRNA treated HUVEC as compared to scrambled shRNA cells  as well as anti-apoptotic (E2F4) functions –23. As P. To determine the relevance of PHF8 in a more endothelial related context, tube formation assays were performed. The downregulation of PHF8 attenuated endothelial tube forming capacity . Although this assay is a standard technique for angiogenesis research, the mechanisms affecting tube formation capacity are complex and not linked to endothelial cell proliferation or migration. The latter aspect was therefore studied separately, however, with a similar result: The knockdown of PHF8 decreased the migration of HUVECs in the Boyden chamber assay  as well as in the scratch-wound assay . These assays were performed with a fairly short observation period (5 hours), making an effect of proliferation on the migration readout unlikely. Besides cell cycle progression, E2Fs have indeed additional effects [. Similar as for the proliferation experiments, overexpression of E2F4 could rescue normal endothelial migration after PHF8 knockdown . These observations suggest that a significant portion of the effects of PHF8 are mediated through the maintenance of E2F4 expression.To exclude unspecific effects of the siRNA, rescue experiments for proliferation of HUVECs was performed. In PHF8-siRNA treated cells, overexpression of PHF8 restored the normal proliferation rate. Importantly, overexpression of E2F4 but not of E2F1 had a similar effect suggesting that loss of E2F4 expression significantly contributes to the effects of PHF8 siRNA  effects –26 and tIn this study we determined the role of PHF8 in endothelial cells. PHF8 facilitates endothelial cell proliferation and migration and this effect was probably a consequence of a PHF8-dependent demethylation of H3K9 at the E2F4 transcription start site.The E2F family has an essential role in the control of cell cycle and there is growing evidence that PHF8 is a central regulator of cell cycle genes ,27,28. IIn this mesenchymal cell type our observations suggest that at least two different versions of PHF8 are expressed. SiRNA directed against different regions of the gene all resulted in a uniform loss of the expression of both proteins. This argues against but does not exclude that the two proteins represent splice variants of the PHF8 gene. Alternatively, the bands may reflect different post-translational modifications. Indeed, for epithelial cells it has been suggested that phosphorylation of PHF8 is required for its demethylase function .Previous ChIP analysis in HeLa cells suggested that PHF8 locally reduces the abundance of the repressive H3K9me2 mark thus allowing transcription ,29. Our Different to E2F1, E2F4 is thought to function as a gene repressor. It occupies promoters and reduces the expression of numerous genes involved in cell cycle control, DNA repair and apoptosis. This leads to a prolonged G1 progression and thus enables sufficient synthesis of proteins required for DNA replication and mitosis . During It appears rather trivial that cell cycle arrest or dysfunction is associated with apoptosis ,35. E2F4As an endothelial cell specific readout the effect of PHF8 on cell migration and tube formation was determined. Our observations are consistent with previous reports in cancer cells in which depletion of PHF8 led to reduced migration and invasion of cancer cells –40. PHF8Interestingly, the closest family member of PHF8, JHDM1D, which is also as a H3K9 demethylase, appears to have opposing effects to PHF8. It was reported that the enzyme inhibits tumor induced-angiogenesis and siRNA silencing did not affect cell proliferation or cell cycle progression . The expHumans with mutation in the catalytic active JmjC domain of PHF8 suffer of mental retardation ,11, but In conclusion, the present work establishes PHF8 as a histone demethylase involved in endothelial cell phenotype control by maintaining E2F4 expression. Inhibition of PHF8 could be a potential novel anti-angiogenic strategy.


ARF-p53 pathway. pRb and p14ARF can repress ribosomal DNA (rDNA) transcription in part by targeting the Upstream Binding Factor 1 (UBF1), a key factor in the activation of RNA polymerase I machinery. We showed, through ectopic expression and siRNA silencing of p14ARF and/or E7, that E7 stimulates UBF1-mediated rDNA gene transcription, partly because of increased levels of phosphorylated UBF1, preventing the inhibitory function of p14ARF. Unexpectedly, activation of rDNA gene transcription was higher in cells co-expressing p14ARF and E7, compared to cells expressing E7 alone. We did not find a difference in P-UBF1 levels that could explain this data. However, p14ARF expression induced E7 to accumulate into the nucleolus, where rDNA transcription takes place, providing an opportunity for E7 to interact with nucleolar proteins involved in this process. GST-pull down and co-immunoprecipitation assays showed interactions between p14ARF, UBF1 and E7, although p14ARF and E7 are not able to directly interact. Co-expression of a pRb-binding-deficient mutant (E7C24G) and p14ARF resulted in EC24G nucleolar accumulation, but not in a significant higher activation of rDNA transcription, suggesting that the inactivation of pRb is involved in this phenomenon. Thus, p14ARF fails to prevent E7-mediated UBF1 phosphorylation, but could facilitate nucleolar pRb inactivation by targeting E7 to the nucleolus. While others have reported that p19ARF, the mouse homologue of p14ARF, inhibits some functions of E7, we showed that E7 inhibits a p53-independent function of p14ARF. These results point to a mutually functional interaction between p14ARF and E7 that might partly explain why the sustained p14ARF expression observed in most cervical pre-malignant lesions and malignancies may be ineffective.High-risk human papillomavirus oncoproteins E6 and E7 play a major role in HPV-related cancers. One of the main functions of E7 is the degradation of pRb, while E6 promotes the degradation of p53, inactivating the p14 INK4a that inhibits cell cycle progression by inactivating CDK4/6. However, E2F1 is released in these cells by E7-mediated pRb degradation rather than CDK4/6 phosphorylation, thereby abrogating the growth-inhibitory functions of p16INK4aINK4a is encoded by the INK4a/ARF locus on human chromosome region 9p21, that also encodes p14ARF, another tumor suppressor protein INK4a and inhibition of its functions have been widely explored in the context of high-risk HPV infection, and p16INK4a is now considered as a useful indirect marker of high-grade cervical lesions ARF, even though p14ARF expression is found in most cervical pre-malignant lesions and malignancies Human papillomaviruses (HPV) belonging to the high-risk (HR) or oncogenic group are major etiological agents for cervical cancer, other anogenital malignancies and, to a lesser extent, head and neck cancers ARF, a predominantly nucleolar protein, is considered to be one of the most important oncogenic stress sensors ARF is to arrest the cell cycle in response to oncogenic stress, in a p53-dependent manner. P14ARF inactivates the E3-ubiquitin ligase MDM2, a negative regulator of p53, leading to p53 stabilization and activation of p53-responsive genes ARF stimulates p53 acetylation through hAda3, a component of histone acetyltransferase (HAT) complexes required for the full transcriptional activity of p53 ARF-p53 pathway by targeting p53 functions through several mechanisms. The most important of these mechanisms is probably E6-induced p53 degradation, through recruitment of the cellular E3-ubiquitin ligase E6AP ARF-dependent inhibition of MDM2 is inefficient in this case, as E6 acts independently of MDM2. E6 also uses indirect strategies, involving transcriptional co-activators (HAT or components of HAT complexes), to induce loss of p53 function. Thus, E6 inhibits p300-mediated p53 acetylation, leading to repression of p53-dependent gene activation ARF-p53 pathway inactivation P14ARF also displays p53-independent biological activities that regulate not only cell growth but also apoptosis, angiogenesis, tumor cell migration and senescence I proteins), are involved in the assembly of the pre-initiation complex on the rDNA promoter ARF and p19ARF, the mouse homolog of p14ARF) inhibits ribosome biogenesis at several steps. P14ARF can repress rDNA transcription by binding the rDNA promoter, interfering with UBF1 phosphorylation Besides these p53-dependent activities, there is growing evidence that p14ARF are known to be inactivated by E6, the roles of the p53-independent activities of p14ARF in HR E6/E7-expressing cells are unclear. P14ARF is detectable in most cervical pre-malignant lesions and malignancies, in which ribosome biogenesis is thought to be upregulated, suggesting that the inhibitory function of p14ARF is inefficient. Because E7 degrades pRb, a negative regulator of ribosome biogenesis, we suspected that E7 might engage in a functional relationship with p14ARF during rDNA transcription.Whereas the p53-dependent functions of p14ARF-mediated transcriptional repression of the rRNA gene promoter. E7 expression enhanced UBF1 phosphorylation, thus repressing the control exerted by p14ARF on this fundamental RNA Pol I-transcription factor. Surprisingly, the transactivating effect of E7 was enhanced in p14ARF-expressing cells. One possible explanation is that the accumulation of E7 in the nucleolus upon p14ARF expression may result in direct interactions between E7 and nucleolar factors involved in rRNA gene promoter regulation, such as UBF1 and nucleolar pRb. These findings show for the first time that E7 can inhibit a p53-independent function of p14ARF.Here, using luciferase assays, we show that E7 stimulates UBF1-driven transcription and inhibits p14ARF-transcriptional repression of the rDNA promoter, we performed luciferase reporter gene assays in multiple independent replicate experiments. We used an rDNA promoter construct (pHrDNA-IRES-Luc) spanning the core and upstream control element (UCE) regions that function synergistically to recruit the RNA Pol I transcription complex ARF−/−) were transiently transfected with pHrDNA-IRES-Luc, together with the Renilla control vector pRLTK, E7 and/or p14ARF expression vectors, or with empty vectors (control). p14ARF and E7 expression were controlled by western blot and qRT-PCR, respectively , indicating that the effects were p53-independent (data not shown).To determine whether E7 could interfere with rDNA transcription and p14ectively . As prevtivation . In contARF were co-expressed in MCF7 (ARF inducible clone (clone 19) ARF expression, induced by doxycycline treatment, was associated with an increase in the E7-enhanced activity of the rDNA promoter . It suggests that the inactivation of pRb is involved in the increase of rDNA promoter activity observed upon E7 and p14ARF co-expression. These results indicated that pRb inactivation contributed to the E7-mediated stimulation of rDNA transcription but did not entirely account for the E7-dependent effects.As pRb modulates ribosome biogenesis by inhibiting rDNA transcription ectively . We founARF inhibit rDNA transcription by interfering with the transactivator UBF1 ARF and UBF1 expression vectors. As shown in ARF was abrogated and promoter activity was significantly enhanced. Together, these data suggested that E7 stimulated rDNA transcription and inhibits p14ARF by targeting UBF1.pRb and p14ARF is associated with the interaction of p14ARF and UBF1 and with subsequent UBF1 hypophosphorylation at serine 388 ARF , HPV16 E6 alone (LXSNE6), both HPV16 E6 and E7 (LXSNE6E7), or no insert (LXSN), and were selected for 10 days. Expression of E7 was confirmed by real time RT-PCR (data not shown) and pRb detection using western blot. Levels of Ser388-P-UBF1 were analysed by Western blot,  or RT-PCR (E7)  . DepletiPCR (E7) . This copothesis . P14ARF lone did . Moreovelone did .ARF and E7 are co-expressed endogenously, by comparison with E7 expression alone (siRNA p14ARF), a result apparently not explained by Ser388-P-UBF1 levels. Thus, it confirms the data previously observed in MCF7 and H358 cells upon exogenous co-expression of E7 and p14ARF  ARF and E7 by indirect immunofluorescence in two different cell lines. As E7 was technically difficult to detect, we used a C-terminal HA-tagged E7 expression vector (pCMV16 E7-Flag/HA). This vector was transfected into U2OS and MCF7 cells, with or without a p14ARF expression vector (pcDNA3-p14ARF). Cells expressing E7-HA alone displayed a predominantly nuclear distribution, excluding the nucleolus  or mouse IgG1 as control. As shown in ARF was immunoprecipitated by anti-E7 antibodies but not by control IgG1 .We then examined interactions in H358 and CaSki cells in order to test the physiological relevance of the interactions between E7, p14rol IgG1 . In CaSknditions . MoreoveTogether, these results suggest that p14ARF may form a complex with E7 and other proteins, thereby promoting nucleolar delocalization and UBF1-E7 interaction.ARF has been detected in cervical cancers and in most neoplastic lesions induced by high-risk HPV ARF might be induced after infection, we transduced pooled human primary cervical keratinocytes (HCK) with LXSN-based retroviruses containing no insert (LXSN) or HPV16 E6+E7 (LXSNE6E7), and monitored p14ARF expression. Expression of HPV16 E6/E7 was assessed by real-time RT-PCR (data not shown) and indirectly by Western blot (pRb). Levels of p14ARF were measured by Western blot. P14ARF induction was detectable 30 h post-transduction in HCK cells transduced with HPV16 E6+E7 and was associated with E7 expression confirmed by reduced levels of pRb . As E7 induces pRb degradation, E7 might be expected to deregulate pRb-dependent inhibition of rDNA transcription. To go further into the involvement of pRb degradation by E7, we used a pRb-binding-deficient E7 mutant (E7C24G). It appeared that the enhancing effect of E7 was not due solely to the loss of pRb, as E7C24G also enhanced rDNA promoter activity and inhibited p14ARF repression.Cells stimulated to proliferate during normal and pathological processes exhibit increased ribosome biogenesis. Production of rRNA is a major rate-limiting step in ribosome biogenesis, determining the cell's capacity to synthesize proteins that regulate its growth ARF is achieved both directly, through reduced UBF1 phosphorylation and recruitment on the transcription complex, and indirectly through p53 induction ARF. Moreover, silencing of p14ARF in CaSki cells did not affect levels of phosphorylated UBF1, but induced a decrease in rDNA promoter activity. These results demonstrate that the inhibitory action of p14ARF on UBF phosphorylation and rDNA promoter activity are inefficient in E7-expressing cells in the context of HPV-induced malignancy.Previous studies have shown that other oncogenic viruses, including SV40 and HCV, also activate rDNA transcription ARF co-expression. We cannot exclude a slight difference in E7 expression at the protein level. However, the decrease in rDNA promoter activity upon silencing of p14ARF in CaSki cells provides further evidence of this finding. This effect was not clearly observed with the E7C24G mutant, suggesting an involvement of pRb binding and degradation. We found no significant difference in levels of phosphorylated UBF1 in cells expressing E7 or E7C24G, compared to cells expressing E7 and p14ARF or cells expressing E7C24G and p14ARF. Thus, a difference of phosphorylated UBF1 levels does not account for the increased activity of the rDNA promoter observed upon E7 and p14ARF co-expression. Pan et alARF in ARF-deficient U2OS cells induced nucleolar localization of E7. Moreover, immunofluorescence and electron microscopy with immunogold staining revealed the presence of E7 within the nucleolus of the HPV16-positive cervical carcinoma cell line CaSki, that expresses endogenous p14ARFARF overexpression. The E7C24G mutant exhibited a similar nucleolar accumulation upon p14ARF expression. Together, these results suggest that the nucleolar localization of E7 facilitates its enhancement of rDNA transcription by a mechanism involving pRb degradation. In the nucleolus, Rb directly represses transcription of the rRNA genes by binding to UBF1 and inhibiting its DNA binding activity ARF. These interactions are physiologically relevant, as they were also observed in immunoprecipitation experiments in CaSki cells. However, we detected no direct interaction between E7 and p14ARF, suggesting that the interaction occurs through a protein complex, probably involving UBF1. Thus, a possible explanation of the increased E7-activation of rDNA transcription is that the nucleolar accumulation of E7 could allow E7 to inhibit the binding of pRb to UBF1 and facilitate the degradation of nucleolar pRb. SV40 LT, that shares both structural and functional features with E7, is recruited to the nucleolar rDNA promoter through direct protein-protein interactions with the SL1 initiation factor and the formation of a stable UBF1-SL1 complex ARF-induced nucleolar localization of E7 can facilitate its enhancement of rDNA transcription could be somewhat paradoxical, since p14ARF alone inhibits rDNA transcription and displays tumor suppressor activities. Pan et alARF can inhibit two functional properties of E7, namely pRb proteolysis and stimulation of DNA replication. They suggested that this effect could rely on p19ARF-dependent nucleolar sequestration of E7. Recent data indicate that several functions of adenovirus oncoprotein E1A, which shares certain features with E7 and SV40 LT, are inhibited by p14ARFARF and nucleolar localization of E7 may facilitate its enhancement of rDNA transcription. Although puzzling, these results are not be mutually exclusive. Indeed, such relationships have already been described between ARF and another protein, the nucleophosmine NPM. NPM is a multifunctional shuttling protein involved in ribosome biogenesis, regulation of centrosome duplication, DNA repair, RNA transcription and apoptosis et alARF may have a tumor –promoting activity that could limit the progression of some tumors, such as lymphoma. Moreover, McLaughlin-Drubin et alINK4A, a tumor suppressor highly expressed in response to E7, displays an oncogenic activity in HPV16+ cervical cancer cells depending on inhibition of CDK4/CDK6 and cellular context. Our results do not prove that p14ARF could exert an oncogenic function by itself. However, it is conceivable that the abundance of each protein may modulate the nature of their cellular partners, as well as their localization and functions.Intriguingly, we also found that the E7-induced transcriptional activation of the rDNA promoter in MCF7 and H358 cells was moderately but significantly increased upon p14ARF control of ribosome biogenesis.In addition to its negative control of rRNA synthesis, ARF can also inhibit rRNA processing and rRNA nuclear export. These functions can be achieved through ARF binding to NPM et alARF early after transduction of primary cervical keratinocytes, before their immortalization. Thus, immortalization is not essential for E7-dependent p14ARF induction. E7 could induce p14ARF during productive viral cycling in suprabasal cell layers. Our results are in agreement with those of Garcia et alARF induction in cervical keratinocytes. It suggests that E7 could stimulate rDNA transcription during the HPV replicative cycle. Nucleolar E7 accumulation can limit nuclear pRb degradation The HPV replicative cycle is unusual. Whereas HPV infects cells of the basal layer of stratified squamous epithelia, new virions can only be produced by differentiated cells of the suprabasal layers which, in physiological conditions, have exited the cell cycle ARF. Moreover, the nucleolar localization of E7 facilitates its enhancement on rDNA transcription by a mechanism independent of UBF1 phosphorylation but dependent of pRb-E7 interaction. Although stimulation of rDNA transcription is probably involved in the HPV replicative cycle, it could also contribute to the oncogenicity of high-risk HPV. Growth-stimulating signals triggered by the E7 oncoprotein and growth-suppressive signals derived from the p14ARF tumor suppressor pathway oppose one another and could influence the outcome of infection. However, the sustained p14ARF expression observed in most cervical pre-malignant lesions and malignancies suggests that p14ARF is ineffective in these cells. Additional experiments will be needed to determine whether the other p53-independent functions of p14ARF are similarly inhibited by E7.In conclusion, we show that HPV16 protein E7 stimulates rDNA transcription and inhibits a p53-independent function of p14Oral informed consent was obtained from the patients providing fresh cervical tissue by the Gynecology Unit of Poitiers University Hospital (Pr G Magnin). This study was approved by the institutional ethic committee .ARF inducible clone (H358 clone 19), kindly provided by B. Eymin, was obtained by using a modified doxycyclin-regulated inducible expression system , as previously described ARF expression was induced in the presence of 1 µg/ml doxycyclin (Sigma-Aldrich). The media were supplemented with 10% FBS and antibiotics  (Invitrogen) and the cells were cultured at 37°C with 5% CO2.MCF7 human breast adenocarcinoma cells (ARF-null) and U2OS human osteosarcoma cells (ARF-null) were grown in Dulbecco's modified Eagle's medium (DMEM). H358 human bronchoalveolar carcinoma cells  and CaSki, a HPV16-positive human cervical carcinoma cell line, were grown in RPMI 1640 medium (Invitrogen). The H358/Tet-On/p14Primary human ectocervical keratinocytes (HEK) were isolated from fresh cervical tissue obtained during hysterectomy for benign uterine diseases after informed consent. The epidermal layer was separated from the dermis with 25 U/ml dispase supplemented with 5 µl/ml gentamicin overnight at 4°C. Keratinocytes were then isolated from the epidermal layer with standard trypsination procedures and cultured in serum-free keratinocyte medium supplemented with insulin, epidermal growth factor and fibroblast growth factor . After reaching 85% confluence, the cells were trypsinized, expanded once, and frozen. Pooled keratinocytes were obtained from vials of keratinocytes derived from 5 individuals.6 cells were cultured in T25 flasks. Transfection with LXSN constructs was performed with Fugene 6 (Roche), and recombinant retroviruses were prepared following the manufacturer's instructions (Orbigen). Second-passage HFK were pooled and infected after thawing with recombinant retroviruses mixed with Polybrene (4 ug/ml). Three days after infection, cells were selected with G418 (400 µg/ml). HPV16 E6 and E7 were detected by RT-PCR or immunoprecipitation, and the concentration of G418 was then reduced by half. MCF7 and H358 clone 19 were similarly transduced.Cell lines stably expressing E7-HPV16 and/or E6-HPV16 were established by using Phoenix Ampho packaging cells (Orbigen) and LXSN amphotrophic retroviral constructs  kindly provided by Dr D. Galloway. Phoenix cells were cultured in DMEM supplemented with 10% FBS and 2 mM L-glutamine. Twenty-four hours before transfection, 1.5 to 2×10Renilla luciferase reporter driven by the HSV-TK promoter (Promega). pJ4Ω16E7 (E7) was kindly provided by Dr L Banks The pcDNA3.1-p14ARF, pcDNA-UBF and pGST-p14ARF vectors are described in detail elsewhere ARF) and/or pJ4Ω16E7 (E7), and/or pcDNA3.1-16 E7C24G, or pcDNA-UBF (0.5 or 1 µg) using the Fugene  or TFX 50 (Promega) transfection reagents, according to the manufacturers' instructions. H358 cl19 cells transduced with LXSN or LXSNE7 and selected with G418 were treated with doxycyclin (1 mg/ml) for 48 h before transfection. All transfections were adjusted with empty vector DNA to contain the same total amount of DNA per well. Cells were harvested for dual luciferase assays, western blotting, immunoprecipitation or qPCR at 24 and/or 48 h after transfection.MCF7 and H358 cells were subcultured and seeded in six-well plates. After 24 h, cells were transfected with empty pcDNA3.1 (control), pcDNA3.1 expressing ARF (p14ARF (5′-GAACAUGGUGCGCAGGUU-CTT-3′) and for E7  ARF or inhibition was checked by immunoblot analysis. Expression of E7 or inhibition was checked by detection of pRb by western blot or qRT-PCR.CaSki cells were transfected using Lipofectamine 2000 (Invitrogen). Si RNA for p14Renilla luciferase activities were determined using the Dual Luciferase Assay System (Promega) in a TD20/20 luminometer (Promega). Reporter activity was calculated as the ratio of firefly luciferase activity to reference Renilla luciferase activity, and normalized so that luciferase activity in control vector-transfected cells equaled 1. Within each experiment, transfections were done in triplicate, and the results reported are from three independent experiments.For luciferase assays, cells were transfected together with pHrDNA-IRES-Luc (200 ng), the pRLTK reference plasmid (10 ng) and the plasmids described above using the Fugene  or TFX 50 (Promega) transfection reagents, according to the manufacturers' instructions. Forty-eight hours after transfection, cells were lysed with Passive Lysis Buffer and firefly and Cells were washed in phosphate-buffered saline (PBS), resuspended in lysis buffer  0.1%, phenylmethylsulphonyl fluoride (PMSF) 1 mM, supplemented with a protease and phosphatase inhibitor cocktail (Sigma). Protein extracts were normalized for total protein concentration by BCA kit , resolved by SDS-PAGE (12 or 15%) and transferred to nitrocellulose. The membrane was blocked with 5% non-fat milk in Tris-buffered saline containing 0.1% Tween 20 (TBS-T) for 1 h and then incubated overnight at 4°C with primary antibodies diluted in blocking solution. The primary antibodies included antibodies against UBF , phosphorylated UBF , p14ARF , pRb  and actin  or GAPDH (Sigma) for loading controls for cell lysates. The membrane was then washed three times with TBS-Tween and incubated with appropriate HRP-conjugated secondary antibodies for 1 h at room temperature. After washing five times with TBS-T, the membrane was developed with an enhanced chemiluminescence kit  according to the manufacturer's instructions. Quantification of western blots was done by measuring the relative intensity of the bands compared to internal controls (Actin or GAPDH), the values given are in arbitrary units.ARF (Santa Cruz Biotechnology), anti-HPV16-E7  or mouse IgG1 as control. Immune complexes were purified by using Protein-A/G-agarose beads, then resolved by SDS-polyacrylamide gel electrophoresis and analyzed by immunoblotting, as described above.Protein extracts were cleared by centrifugation  for 30 min at 4°C. UBF1, p14ARF, and HPV16 E7 were detected by western blot as described above.GST and GST-E7 were expressed in BL21DE 5′-CGGACAGAGCCCAATACAAT-3′E7  (forward), 5′-ACGTGTGTGCTTTGTACGCAC-3′ (reverse), 5′-TGTTGCAAGTGTGACTCTACGCTTCG-3′ (probe), 5′-GAAGGTGAAGGTCGGAGT-3′GAPDH  (forward), 5′-GAAGATGGTGATGGGATTTC-3′ (reverse), 5′-CAAGCTTCCCGTTCTCAGCC-3′ (probe). The relative quantities of E7 mRNA were calculated using the ΔΔCt method with GAPDH as the calibrator gene. Fold changes are relative to level of E7 mRNA in cells expressing E7 alone (set up to 1). No-RT controls were included to check the absence of contaminating DNA.Total RNA was extracted using RNeasy mini kit (Qiagen) according to the instuctions of the manufacturer and treated with DNAse (Ambion). RNA (1 µg) was reverse-transcribed using random hexamers . The resulting cDNA (200 ng) was subjected to real-time RT-PCR using TaqMan probes with Abiprism7500 (Applied Biosystems). Primers and probes sequences were as follows: MCF7 and U2OS cells were seeded in 24-well plates containing coverslips (5×104 cells/well). One day later, cells were transfected with 200 ng of pCMV16 E7-Flag/HA or pCMV16E7C24G-Flag/HA and/or 200 ng of pcDNA3.1-p14ARF plus Fugene . Twenty-four hours later the cells were fixed with 3% paraformaldehyde and permeabilized with 0.1% Triton X-100 in PBS. Primary antibodies, diluted 1/50 in PBS for p14ARF  and 1/10 for E7-HA , were incubated for 90 min at 37°C, followed by extensive washing in PBS and incubation for 30 min at 37°C with secondary antibodies diluted 1/500 . Confocal microscopy was done at the UMR-CNRS 6187 facility. Samples were examined with a spectral confocal FV-1000 station installed on an inverted IX-81 microscope  equipped with an Olympus UplanSapo ×60, 1.2 NA, water objective. Multiple fluorescence signals were acquired sequentially to avoid cross-talk between image channels. Fluorophores were excited with the 488-nm line of an argon laser , the 543 nm line of an HeNe laser  and the 633 line of an HeNe laser (for topro-3). Emitted fluorescence was detected through spectral detection channels between 500–530 nm and 550–625 nm, for green and red fluorescence, respectively, and through a 650-nm long-pass filter for far red fluorescence.p values of 0.05 or less were considered significant. All experiments were done at least three times.The nonparametric Mann-Whitney and the two-tailed T tests were used for statistical analysis. Results are report as mean ± Standard Deviation (SD). Figure S1HPV16 E7C24G expression stimulates UBF1 phosphorylation. (A), Western blot analysis of H358 WT cells transiently transfected with the pcDNA3.1 (Control), pJ4Ω16E7 (E7), pCDNA-E7C24G (E7C24G) or pcDNA3.1-p14ARF (p14ARF) expression vector, or both and probed with antibodies to UBF, phosphorylated UBF (P-UBF Ser 388), p14ARF and actin (as a loading control). (B), Western blot analysis of H358 Cl19 cells cultured with (+Dox) or without (-Dox) 1 mg/ml doxycyclin, transfected with pcDNA-E7C24G (E7C24G), and probed with the indicated antibodies. Quantification of western blots was done by measuring the relative intensity of the bands compared to internal controls (Actin), the values given are in arbitrary units. Expression of E7 was monitored by quantitative real time RT-PCR. Western blot and qRT-PCR are representative of three experiments.(TIF)Click here for additional data file.


Arabidopsis thaliana and Oryza sativa contain 35 and 48 mTERFs, respectively, but the biological functions of only a few of these proteins have been explored. Here, we investigated the biological role and molecular mechanism of Arabidopsis mTERF15 in plant organelle metabolism using molecular genetics, cytological and biochemical approaches. The null homozygous T-DNA mutant of mTERF15, mterf15, was found to result in substantial retardation of both vegetative and reproductive development, which was fully complemented by the wild-type genomic sequence. Surprisingly, mitochondria-localized mTERF15 lacks obvious DNA-binding activity but processes mitochondrial nad2 intron 3 splicing through its RNA-binding ability. Impairment of this splicing event not only disrupted mitochondrial structure but also abolished the activity of mitochondrial respiratory chain complex I. These effects are in agreement with the severe phenotype of the mterf15 homozygous mutant. Our study suggests that Arabidopsis mTERF15 functions as a splicing factor for nad2 intron 3 splicing in mitochondria, which is essential for normal plant growth and development.Mitochondria play a pivotal role in most eukaryotic cells, as they are responsible for the generation of energy and diverse metabolic intermediates for many cellular events. During endosymbiosis, approximately 99% of the genes encoded by the mitochondrial genome were transferred into the host nucleus, and mitochondria import more than 1000 nuclear-encoded proteins from the cytosol to maintain structural integrity and fundamental functions, including DNA replication, mRNA transcription and RNA metabolism of dozens of mitochondrial genes. In metazoans, a family of nuclear-encoded proteins called the mitochondrial transcription termination factors (mTERFs) regulates mitochondrial transcription, including transcriptional termination and initiation, via their DNA-binding activities, and the dysfunction of individual mTERF members causes severe developmental defects.  Mitochondria, which originated through the endosymbiosis of α-proteobacteria into ancestral host cells, are the cellular powerhouses and play vital roles in diverse eukaryotic cell processes through the production of ATP and various metabolic intermediates Ler(UUR) genes to terminate mitochondrial transcription mTERF2 loss-of-function mice exhibit myopathies, memory deficits, and impaired respiratory function due to a reduction in the number of mitochondrial transcripts mTERF3 is essential, and mTERF3-knockout mice die during the early stages of embryogenesis; mTERF3 non-specifically interacts with mtDNA promoter regions and mediates the repression of mitochondrial transcription initiation The mTERF proteins were first identified two decades ago as regulators of transcription termination in human mitochondria SINGLET OXYGEN-LINKED DEATH ACTIVATOR10 (SOLDAT10) BELAYA SMERT/RUGOSA2 (BSM/RUG2) MTERF DEFECTIVE IN ARABIDOPSIS 1 (MDA1) SUPPRESSOR of hot1-4 1 (SHOT1) soldat10 mutants suppressed the 1O2-mediated cell-death and growth-retardation phenotypes of the conditional fluorescent (flu) mutant, in which excess levels of the reactive oxygen species (ROS) 1O2 were generated within chloroplasts after the dark-to-light shift MDA1 mutation leads to significant defects in plant growth and chloroplast development but enhanced tolerance to salt and osmotic stress, perhaps via reduced sensitivity to abscisic acid SHOT1 suppresses the heat-hypersensitive phenotype caused by the loss of heat shock protein 101 in Arabidopsis shot1 may be caused by reduced ROS-mediated oxidative damage during heat stress. Additionally, SHOT1 plays an important role in regulating the expression of mitochondrial-encoded genes and mitochondrial genome copy number. Although it is known that many mTERF mutations cause defective development and stress responses, studies of the precise molecular functions of these mTERF proteins are limited in Arabidopsis organelles.The genome of Arabidopsis contains 35 identified mTERFs, which vary in the number and arrangement of their mTERF motifs In Arabidopsis, several protein families that participate in organellar RNA splicing, including those containing the chloroplast RNA splicing and ribosome maturation (CRM) domain mTERF15 significantly disturbed normal plant vegetative and reproductive growth, which may be the result of defective mitochondrial development and/or function. Interestingly, mTERF15 is a mitochondria-localized protein that binds to RNA but not to double-stranded DNA (dsDNA). Moreover, an analysis of mitochondrial intron splicing in mterf15 mutants revealed defective RNA splicing of nad2 intron 3, which led to a deficiency in complex I activity. The possible molecular function of mTERF15 in regulating Arabidopsis mitochondrial RNA metabolism is discussed.In the present study, we investigated another Arabidopsis mTERF protein, mTERF15, and identified its role in post-transcriptional modification of mitochondrial RNA processing. Mutating mTERF15 T-DNA insertion mutant (SALK_134099) was obtained from the Arabidopsis Biological Resource Center . Seeds from Arabidopsis thaliana ecotype Columbia-0 and mterf15/+ plants were surface-sterilized by chloride gas after stratification for 3 d at 4°C and then either germinated on half-strength Murashige and Skoog medium  containing 1% sucrose and 0.7% phytoagar (Duchefa) or grown in soil inside a growth chamber on a 16-h light/8-h dark cycle at 22°C.The At1g74120, including a 2-kb putative promoter and the 1.3-kb coding region, was amplified using Phusion polymerase  and then ligated into a modified pPZP221 vector containing a C-terminal GFP sequence followed by the Nopaline synthase (NOS) terminator. The primers used for cloning are in Agrobacterium tumefaciens strain GV3101. Arabidopsis plants heterozygous for mTERF15 were transfected with Agrobacterium by the vacuum infiltration method 1 generation were collected and grown on solid half-strength Murashige and Skoog medium (Duchefa) containing 1% sucrose, 0.7% phytoagar (Duchefa) and the antibiotic G418 at 100 µg/µl. Leaves from T1 plants were harvested for genotyping. Plants with an mterf15 homozygous background harboring mTEFR15p::mTERF15-GFP were collected for further studies.Approximately 3.3 kb of the genomic sequence corresponding to locus 4 and 0.1% UA) with a Leica EM AFS2. The samples were kept at −85°C for 3 d, at −60°C for 1 d, at −20°C for 1 d, and at 0°C for 1 d and then brought to room temperature. The samples were infiltrated and embedded in Spurr resin. Ultrathin sections (70–90 nm) were cut using a Reichert Ultracut S or Leica EM UC6  and collected on 100 mesh copper grids. After staining with 5% uranyl acetate in 50% methanol for 10 min and 0.4% lead citrate for 4 min, sections were observed under a Philips CM 100 Transmission Electron Microscope at 80 KV; images were captured using a Gatan Orius CCD camera.For transmission electron microscopy, samples were frozen in a high-pressure freezer . Freeze substitution involved the use of anhydrous acetone (containing 1% OsOhttp://www.zeiss.com). TMRM fluorescence and chlorophyll autofluorescence were induced by excitation with a 543-nm HeNe laser and a 488-nm Argon laser. The emission signals from TMRM and from chlorophyll were recorded with using 565- to 615- and 650-nm band-pass filters, respectively. The fluorescence intensity and area were quantified using LSM510 software .TMRM staining  was performed to examine the mitochondrial membrane potential in protoplasts. Isolated protoplasts were stained with 20 nM TMRM and immediately observed under a laser scanning confocal microscope  according to the manufacturer's instructions. The resins were washed extensively with incubation buffer before elution of the recombinant proteins. The mTERF15-strep recombinant protein and the MBP-strep control protein were transferred from 12% SDS-polyacrylamide gels onto a PVDF membrane and then renatured overnight in renaturation buffer  NP-40) at 4°C. After 4 washes with renaturation buffer (15 min), blots were blocked with blocking buffer [ BSA and 0.01% (v/v) Triton X-100)] at room temperature for 5 min. Full length of nad2 intron 3 was in vitro synthesized using SP6 polymerase (Promega). Total RNA and in vitro synthesized nad2 intron3 were labeled with γ32P ATP using T4 polynucleotide kinase (Fermentas). Hybridization was performed at 4°C in the presence of 5 µg labeled RNA. Blots were washed 4 times (5 min each) in a washing buffer  to remove unbound RNA. The radioisotope signals were detected via autoradiography on X-ray film (Kodak).Northwestern blot analysis was performed to test the RNA-binding ability of mTERF15 as described previously Total RNA was isolated using the Qiagen RNeasy Mini Kit (QIAGEN) and then treated with TURBO DNA-free DNase (Ambion) to remove contaminating genomic DNA. Reverse transcription of RNA was performed using the M-MLV transcriptase system (Invitrogen) and random hexamer primers.Gene expression analysis via qRT-PCR was carried out using Power SYBR Green Supermix (Applied Biosystems) running on an ABI Prism 7000 sequence detection system (Applied Biosystems). Primer sets were designed as described previously nad2 probes were labeled using the Rediprime II DNA labeling system . Probe primers are listed in A total amount of 5 µg RNA was denatured at 65°C for 10 min and then separated on 2% formaldehyde-agarose gels in MOPS buffer and transferred to a nylon membrane (PerkinElmer). The Crude mitochondria were prepared as described previously Clear native electrophoresis was performed as described previously 2, p = 0.924; mTERF15 . Developmental defects were widespread in homozygous mterf15 plants. Compared with wild-type seedlings, mterf15 seedlings were small in stature, had small cotyledons and displayed reduced growth of primary roots  . The T2 progeny of homozygotes for the mTERF15 mutation which harbors at least one copy of the transgene mTERF15p::mTERF15-GFP showed similar phenotypes as 7-d-old wild-type seedlings and adult plants  revealed that mTERF15 contains 5 typical mTERF motifs (http://www.ncbi.nlm.nih.gov) revealed mTERF15 homologs present in Vitis vinifera, Populus trichocarpa, Brachypodium distachyon, Sorghum bicolor and Zea mays. Comparing protein similarities and identities between flowering plant mTERF15 orthologs and metazoan mTERF1, Arabidopsis mTERF15 is evolutionally closer to plant mTERF15 orthologs than metazoan mTERF1. This result suggests that mTERF15 is a plant-specific mTERF protein  and Genevestigator (https://www.genevestigator.com/gv/plant.jsp) microarray databases suggested various expression levels of mTERF15 in different tissues, with the highest and lowest expression levels in mature seeds and pollen, respectively. However, in the Genevestigator microarray database, mTERF15 showed the highest expression in sperm cells and the lowest in leaf phloem protoplasts. Quantitative real-time PCR spatiotemporal analysis in Arabidopsis revealed mTERF15 expression in all organs, with high levels of accumulation in seedlings, flowers and siliques , a lipophilic, cationic, red-orange fluorescent dye that accumulates within mitochondria in living cells. The intensity of fluorescence is associated with the mitochondrial membrane potential mterf15 protoplasts  showed that one mTERF protein, BSM, is required for embryogenesis and plays a role in chloroplast intron splicing. Therefore, we examined 23 intron-splicing events in mitochondria by quantitative RT-PCR (qRT-PCR). In mitochondria, 9 genes require intron splicing for the complete maturation of their respective transcripts, and 4 of these contain a single intron: rps3, cox2, ccmFc and rpl2. The others  contain multiple introns and are required for the normal function of mitochondrial complex I. Using the specific primer sets designed by Longevialle et al. (2007), we examined individual splicing events in mitochondria obtained from wild-type, mterf15 and complemented plants. All splicing events showed similar results in both the wild-type and complemented plants; however, the RNA splicing of nad2 intron 3 (nad2-3) was significantly reduced in mterf15 plants  by northwestern blot analysis  by northwestern blot analysis  was used to confirm the RNA-binding activity of mTERF15 , named Fragment 1-5 , which is required to translocate protons from the mitochondrial matrix into the intermembrane space. Complex I activity was therefore investigated in mterf15, in which the splicing of nad2 intron 3 is defective. Isolated crude mitochondria were subjected to complex separation on clear native gels to visualize protein complexes by silver staining  was significantly increased in mterf15 plants, which is consistent with a defect in NADH oxidation mterf15 plants, which may be due to an imbalance in the stoichiometry of multi-subunit complexes The steady-state levels of several mitochondrial proteins, including Nad9, Cox2, Cox3, AtpA, alternative oxidase (AOX) and the channel-forming protein Porin, were examined by immunoblot analysis of crude mitochondrial extracts from wild-type and 5 plants . The stenad2 intron 3  identified 11 and 17 mTERF proteins that localize to chloroplasts and mitochondria, respectively. However, only a few mTERFs have been well studied and are essential for vegetative growth mTERFs constitute a broad family of eukaryotic proteins that are essential for the initiation and termination of organellar transcription and the translation and replication of the organellar transcription machinery nad genes. The exons, including the flanking introns of these genes dispersed among the mtDNA, are transcribed and are the mRNAs generated by the splicing machinery. Until now, knowledge of the splicing machinery found in Arabidopsis mitochondria had been limited by the difficulty of organellar genome manipulation. However, a growing number of studies have revealed the involvement of a nuclear-encoded splicing factor in the excision of these introns nad2 intron splicing. For example, 2 proteins, ABA overly-sensitive 5 (ABO5) and RCC1/UVR8/GEF-like 3 (RUG3), were identified as splicing factors regulating the cis-splicing of nad2 intron 3 ABO5, which encodes a PPR protein, was isolated from a mutational screen for ABA sensitivity and is required for cis-splicing of nad2 intron 3 in mitochondria RUG3, which encodes an RCC1/UVR8-like protein, is responsible for the efficient splicing of nad2 introns 2 and 3 in mitochondria nad2 intron 3 RNA splicing, as demonstrated by RT-PCR and by northern blotting showing the accumulation of nad2 intron 3 in mterf15 plants , also exhibited a similar phenotype abo5, rug3 and ndufs4 plants produced viable seeds and propagated to the next generation, whereas mterf15 showed a more severe phenotype. Homozygous mterf15 plants showed a similar phenotype as these mutants in terms of complex I deficiency; however, seeds from homozygous mterf15 plants could not germinate, even in solid medium over three months. It is possible that mTERF15 in maternal tissues of heterozygotic mterf15 embryo may partially contribute to the developing homozygous mterf15 seeds, which is completely absent in the developing seeds of homozygous mterf15 plants. This suggests that the germination defect of mterf15 is not only due to embryo lethality but also caused by aberrant reproductive organs, especially maternal tissues. The defective maternal tissues may reduce the flow of nutrient into the endosperm and eventually cause seed lethality The nad2 intron 3 splicing in mitochondria. The disruption of this splicing event leads to decreased complex I activity as well as growth and developmental retardation in mterf15 mutants. Further studies of the detailed mechanism of mTERF15 are required to reveal the molecular mechanisms involved in post-transcription regulation in Arabidopsis mitochondria.Here, we identify mTERF15 as an RNA-binding protein that is required for Figure S1mTERF15p::mTERF15-GFP fully complements developmental defects found in mterf15 homozygous mutant. 7-day-old seedlings from 6 independent complementation lines, homozygotes for mTERF15 mutation and harbouring at least one copy of the transgenes mTERF15p::mTERF15-GFP. Seeds were surface-sterilized and germinated on half MS medium under 22°C with light period control (16-h light/8-h dark).(TIF)Click here for additional data file.Figure S2Protein alignment and phylogenetic tree of mTERF15 and homologs. (A) Protein sequence alignment of mTERF15 and its homologs in Vitis vinifera (Vv), Populus trichocarpa (Pt), Brachypodium distachyon (Bd), Sorghum bicolor (Sb) and Zea mays (Zm). The alignment was generated by use of CLUSTALW, which creates pairwise alignments to calculate the divergence between pairs of sequences. Five mTERF motifs present in all homologs are underlined in black. Asterisks indicate the location of highly conserved proline residues. (B) Phylogenetic tree of mTERF15 and its homologs. The phylogenetic tree was created with use of Molecular Evolutionary Genetics Analysis (MEGA) by the Unweighted Pair Group Method with Arithmatic Mean (UPGMA) method.(TIF)Click here for additional data file.Figure S3Spatial expression profile of mTERF15. qRT-PCR analysis of the expression of mTERF15 in seedlings, roots, stems, rosette leaves, flowers, siliques and pollen. Primers used in this experiment are in (TIF)Click here for additional data file.Figure S4Subcellular localization of mTERF15 protein. Confocal microscopy of the localization of the mTERF15 protein in root hairs of the mterf15 complementation line harboring mTERF15p::mTERF15-GFP. (A) Signal corresponding to the mTERF15-GFP fusion protein; (B) signal corresponding to the MitoTracker marker; and (C) merged signals from GFP and the MitoTracker marker. Scale bar  = 10 µm.(TIF)Click here for additional data file.Figure S5DNA-binding assay with mTERF15 recombinant protein. (A) In vitro binding studies of dsDNA-cellulose with mTERF15-GST and GST. mTERF15-GST and GST were incubated with dsDNA-cellulose for 2 h at room temperature. The resin was washed with washing buffer 3 times and after different salt concentrations to elute bound protein. The detailed experimental procedure was previously described . (B) Southwestern blot assay of DNA-binding ability by mTERF15-strep. The upper panel is Coomassie brilliant blue (CBB) staining of MBP-strep and mTERF15-strep without the first N-terminal 34 amino acid. The lower panel is sourthwestern blot analysis with radioisotope-labeled HindIII-digested mtDNA. Methods of  This experimental procedure has been described previously . Briefly, 1.8 µM of the mTERF15-GST fusion protein and GST control were incubated with 60 mg dsDNA-cellulose resin (Sigma) in DCBB buffer  at room temperature for 2 hr and under gentle rotation. The resin was washed 3 times with DCBB buffer, then bound protein was eluted at different salt concentrations . The samples corresponding to each step were collected and analyzed on SDS-PAGE. Southwestern blot assay. The recombinant protein were separated on 12% SDS-polyacrylamide gels and transferred onto a PVDF membrane and then renatured overnight in renaturation buffer  NP-40) at 4°C. After 4 washes with renaturation buffer (15 min), blots were blocked with blocking buffer [ BSA and 0.01% (v/v) Triton X-100)] at room temperature for 5 min. Mitochondrial DNA was isolated from crude mitochondria fraction, digested with HindIII and labeled with radioisotope using T4 polynucleotide kinase (Fermentas). Hybridization was performed at 4°C overnight in the presence of 5 µg labeled mtDNA. Blots were washed 4 times (5 min) in washing buffer  to remove unbound DNA. Radioisotope signals from PVDF membrane were detected via autoradiography on X-ray film (Kodak).(TIF)Click here for additional data file.Figure S6RNA-EMSA of mTERF15 recombinant protein with different mitochondrial transcribed intron fragments. (A) The diagram of nad2 intron 3 and the regions and sizes of 5 fragments in nad2 intron 3 used for assays. (B) Different concentration mTERF15 recombinant proteins  were incubated with 100 nM radioisotope labeled indicated RNA probes. Methods of  mTERF15 fusion protein were purified as mentioned in Northwestern blot analysis. For in vitro transcription, mitochondrial introns were amplified and cloned to pJET vector. Primer sets are nad2int3-1F “ACGCCAAGCTATTTAGGTGACACTATAGAATACGGGCGGCTGTAGGACGGAC” and nad2int3-1R “CTGTTCACCGTTGGATCTCGCC” for nad2 intron 3 Fragment 1; nad2int3-5F “ACGCCAAGCTATTTAGGTGACACTATAGAATACGCGTGTTATCTGAAGGGAGCACG” and nad2int3-5R “GGGGGAGGGGGTTTTCTTCG” for nad2 intron 3 Fragment 5; rpl2-F “ACGCCAAGCTATTTAGGTGACACTATAGAATACATGAGACCAGGGAGAGCAAGAGCAC” and rpl2-mid-intron-R “CGTTGCTAAGCCAAGGTCCC” for rpl2 intron; ccmFc-F “ACGCCAAGCTATTTAGGTGACACTATAGAATACATGGTCCAACTACATAACTTTTTC” and ccmFc-mid-intron-R “GCTTTGCCAACACAACATTAGG” for CccmF intron. Then, RNA probes were in vitro synthesized using SP6 polymerase (Promega) and labeled with γ32P ATP using T4 polynucleotide kinase (Fermentas). Different concentration of mTERF15 recombinant proteins  were incubated with 100 nM in vitro synthesized RNAs for 30 min at room temperature in binding buffer . The mixtures were separated by 5% polyacrylamide gel and gel was dried. Signals were detected via autoradiography on X-ray film (Kodak).(TIF)Click here for additional data file.Table S1Primer sequences used in mTERF125 cloning, gene expression, and probe preparation for northern blotting and RT-PCR for the nad2 exon–intron junction.(DOCX)Click here for additional data file.Table S2Primer sequences for determining RNA splicing efficiency in mitochondria.(DOCX)Click here for additional data file.Table S3Protein identity and similarity of mTERF15 with its plant homologs and mTERF1 in metazoans.(DOCX)Click here for additional data file.