Mariner Transposons Contain a Silencer: Possible Role of the Polycomb Repressive Complex 2
[['Almost all eukaryotic genomes contain transposable elements ( TEs ) .', 'Some of these , known as DNA transposons , move by a simple ‘cut-and-paste’ mechanism removing DNA from one site and inserting it into a new target site .', 'Others , called retrotransposons , move via an RNA intermediate that is copied into DNA and integrated into the genome .', 'The overall fraction of TEs that make up currently described genomes remains difficult to estimate due to the accumulation of several layers of such elements .', 'These layers originate from TE amplification bursts at different periods during the evolution of the element , followed by ageing of the DNA sequence .', 'Recent improvements in sequence analysis methods have showed that the human genome likely consists of at least 66–69% of repeated or repeat-derived sequences [1] , which is much higher than the 45–50% that had been reported when this genome was first sequenced .', 'This suggests that the extent to which genomes have been shaped by TEs has probably been underestimated for many eukaryotic species .', 'Mobility , distribution and exaptation of certain TE sequences have been considered as important sources for expansion and diversification of transcriptional regulatory networks as well as for genetic innovations [2 , 3] .', 'Today , DNA segments derived from TEs that were exapted or inactivated over time by accumulation of mutations appear as remnants of repeated sequences of various ages .', 'While they are rare , active TEs are still present in the genome of extant species in which de novo insertions can generate genetic variations .', 'In multicellular eukaryotes TE insertions must occur within the germinal lineage or during early development in order to be transmitted to the following generations .', 'This leads to the suggestion that transposition into somatic cells had no value for the TEs or their host .', 'However , in the early 1980’s evidence began to accumulate showing that somatic TE activity ( i . e . single excision or excision followed by re-insertion ) occurred at high frequency in animal taxa .', 'This was first shown for a DNA transposon , Tc1 in the worm Caenorhabditis elegans [4] .', 'Recently , somatic activity was also observed for mammalian LINE-1 and dipteran R2 retrotransposons [5 , 6] .', 'Interestingly , all of these somatic transpositions occurred in primordial cells associated with neuron-related lineages during embryonic or metamorphic development .', 'Activation of TE transcription within some cell lineages requires that the factors silencing their expression be specifically switched off in these lineages .', 'The Neuron-Restrictive Silencer Factor ( NRSF ) that corresponds to the Charlatan ( Chn ) protein in arthropods [7] and to the SPR-3/SPR-4 in nematodes [8] , represses transcription of many neuronal genes in non-neuronal cell types and in neuronal stem cells prior to their differentiation .', 'NRSF binds to a 21 to 30 bp long element called the Neuron-Restrictive Silencer Element [9] ( NRSE ) .', 'NRSF has never been shown before to interfere with TE transcription , even though NRSEs were found in human retrotransposons such as LINE2 [9 , 10] and that transcription of Tc1-like DNA transposons was shown to be activated during development of the Xenopus nervous system [11] .', 'We report the existence of a silencer element located in the last 300 bp of the Mos1 transposase ( MOS1 ) ORF that is functional in both vertebrate and arthropod cells .', 'This silencer is able to interfere with the transposon promoter as well as with promoters of genes located downstream of the silencer sequence .', 'We show that the presence and location of this silencer element is conserved in mariner-like elements ( MLEs ) , even though their DNA sequences have significantly diverged .', 'Our data reveal that YY1 , NFAT-5 , NRSF , Alx1 , GAF and GTF proteins have binding sites within these silencer elements .', 'Furthermore , our results are consistent with the hypothesis that these silencers function with the Polycomb Repressive Complexes ( PRC ) .', 'Together , mariner silencers might not only regulate the transcription of active MLEs , but might also modify the expression pattern of genes in which active or remnant MLEs are inserted .'], ['Although it was originally used for another purpose ( negative controls of transposition done in absence of a transposase source ) , a stable expression assay was used to investigate whether Mos1 was able to interfere with the expression of neighbouring genes .', 'This assay consisted in transfecting HeLa cells with plasmids containing a Neomycin Resistance ( NeoR ) marker gene and one or two Mos1 DNA segments cloned upstream or downstream of the NeoR gene ( Fig 1A ) .', 'After two weeks of selection with G418 , resistant colonies were stained and counted .', 'The first evidence that Mos1 could decrease the expression of a marker gene located within its neighbourhood was obtained with the Δ1[NeoR]Δ2 construct which corresponded to a complete Mos1 element containing the NeoR gene inserted in its middle .', 'Colony numbers were at least 20-fold lower with the Δ1[NeoR]Δ2 construct than with those obtained with the [NeoR] reference ( Fig 1B ) .', 'Further constructs were tested in an effort to locate the region responsible for the observed decrease in marker expression within Mos1 , a region we refer to hereafter as the silencer element .', 'Results obtained with the Δ3[NeoR]Δ4 and Δ5[NeoR] constructs were not different from those of [NeoR] ( Fig 1B ) .', 'These observations supported two explanations:', 'i ) the silencer element was not in the non-coding terminal regions of Mos1 ,', 'ii ) the optimal activity of the silencer element was position-dependent and had an effect only when located downstream of the marker .', 'The first explanation was supported by observations based on the [NeoR]Δ6 and [NeoR]Δ7 constructs which gave results similar to those obtained with the Δ1[NeoR]Δ2 , indicating that the silencer was located within the 3’ half of the MOS1 open reading frame ( ORF ) corresponding to the Δ7 DNA segment ( Fig 1B through 1D ) .', 'The role of the position and orientation of the Mos1 silencer element was confirmed using four constructs in which the Δ7 DNA segment was cloned upstream or downstream of the NeoR gene , in positive ( i . e . with the piece of MOS1 ORF on the same strand as the NeoR ORF ) or negative orientations ( Fig 1E ) .', 'Only the [NeoR]Δ7+ construct showed a strong silencer effect .', 'Hence , the Δ7 DNA segment had a silencer effect only when located downstream of the marker gene , in the positive orientation with respect to the NeoR marker .', 'In addition , complementary experiments demonstrated that an intragenic Δ7 DNA segment in frame with a marker gene had a silencer effect on its expression since Δ7-GFP and MOS1-GFP fusions expression is similar and significantly lower ( ~5-fold ) than the GFP control ( Fig 1F ) .', 'These data support that Δ7-MOS1 segment has a silencer effect when it is fused in frame within a gene and that the silencer effect could be operating with the pCMV promoter .', 'These results were confirmed by RT-qPCR using total RNA extracted from transiently transfected cells and GFP specific primers .', 'To confirm that the Δ7-MOS1 segment contains a real silencer element , we used a transient luciferase expression assay that was previously validated to characterize silencer elements [12] ( S1 Fig ) .', 'Our first results were confirmed with this alternative approach since the only ratio lower than 1 was obtained with the HS2_P_Luc_Δ7+ plasmid ( Fig 2A ) .', 'In addition , they revealed that the expressions of the marker gene in the [NeoR]Δ7+ and HS2_P_Luc_Δ7+ constructs are of the same order of magnitude with respect to controls , [NeoR] ( 14 . 3x; Fig 1B and 1E ) and HS2_P_Luc ( 11 . 7x; Fig 2A ) , respectively .', 'Therefore , these data confirmed that the Δ7-MOS1 segment contained a silencer element which is more efficient when it is located downstream of the maker gene in a positive orientation .', 'They also confirmed that the results obtained with our stable expression assay did not reflect an ability of the plasmid to be integrated into the genome , but the capacity of the NeoR gene to be expressed post-integration .', 'Given the above observations we decided that rather than continuing with the transient assay we should use our stable expression assay to investigate the impact of the distance separating the marker and the Δ7 DNA segment by cloning a 1 . 2 or 2 . 7 kbp spacer between them .', 'We observed that the 1 . 2 kbp spacer had little or no impact on Δ7 silencing activity while the 2 . 7 kbp spacer decreased its activity approximately 5-fold ( Fig 2B ) .', 'It is interesting to notice that a Δ7 DNA segment in the negative orientation located a few kbps away from the marker gene had silencing activity comparable to the one on the Δ7 DNA segment in the positive orientation .', 'These results were verified using linearized constructs ( S2 Fig ) and were not uniform , suggesting that vector configuration is important in such experiments .', 'An orientation effect similar to that previously observed in the absence of a spacer was found with linearized vectors containing a 3 kbp spacer .', 'The activity of the Mos1 silencer element was tested using [NeoR]Δ7+ and [NeoR]Δ7- , our stable expression assay and two other cellular lineages originating from distantly related species: Speedy cells [13] from Xenopus tropicalis ( Amphibia ) and Sf21 cells from Spodoptera frugiperda ( Insecta ) .', 'Our results showed that the Δ7 DNA segment had a silencer effect in both cellular systems ( Fig 3A and 3B ) , suggesting that the protein factors with which it interferes are conserved in these two species .', 'Interestingly , the orientation effect of the silencer was recovered in Speedy cells but was absent in Sf21 cells .', 'The mariner TE family consists of five sub-families designated cecropia , elegans/briggsae , irritans , mauritiana and mellifera/capitata [14] .', 'Based on the phylogeny of its transposase Mos1 belongs to the mauritiana sub-family .', 'The presence of a silencer element was surveyed within the Δ7 DNA segments of three MLEs , Himar1 , Mcmar1 and Hsmar1 , which respectively belong to the irritans , elegans/briggsae , and cecropia sub-families .', 'Results obtained using the stable expression assay ( Fig 3C ) showed that the Δ7-MCMAR1 segment had a silencing activity with features similar to those of Δ7-MOS1 ( i . e . in terms of intensity and orientation ) .', 'The Δ7-HIMAR1 and Δ7-HSMAR1 segments also had silencing activity that was not significantly different from those of Δ7-MOS1 and Δ7-MCMAR1 , but independent of their orientation .', 'This result is important because it suggests that the presence of a silencer within the Δ7 DNA segment is a characteristic shared by all MLEs .', 'It also suggests that protein factors conserved in most animal species that interfere with the mariner silencer elements might have conserved binding site motifs in their sequences .', 'Taking into account the sequence of the active promoter in Hsmar1 [15] , a variant of transient luciferase expression assay was designed with luciferase expression plasmids containing the Mos1 and the Hsmar1 promoters ( Figs 4A and S3 ) .', 'Our results with HeLa cells ( Fig 4B and 4C ) revealed that both promoters were active .', 'pMos1 was found to be 10-fold less efficient than the early promoter for SV40 ( pSV40 ) under these experimental conditions .', 'pHsmar1 was found to be two-fold more active than the pSV40 contained in the P_Luc control .', 'When their silencer were cloned downstream of the marker gene our results revealed levels of marker expression that were lower than those of the controls ( 3 . 3-fold for pMos1 and 2-fold for pHsmar1 ) .', 'This indicated that mariner silencers were able to negatively interfere with their own promoters .', 'Because the closest transcriptional start site ( TSS ) upstream of the silencer element is that of the transposase ORF this mechanism is probably a way for MLEs to repress their transcriptional activity in their host cells and maintain active copies in a state of latency when host factors required for this repression are available .', 'In the next four sections we present the results of molecular and cellular biology investigations in which the Mos1 silencer was used as the main model to elucidate the mechanism of its activity .', 'The silencer of Hsmar1 , and in a few cases those of Himar1 and Mcmar1 , were used as complements to confirm certain results .', 'In the final section of the results pertaining to silencer activity at the scale of a eukaryotic genome , the Hsmar1 and Hsmar2 silencers were used , as they were the only models for which in silico genomic data are available .', 'NeoR expression was monitored for 24 hours both at the protein and mRNA levels using cellular extracts from cells transiently transfected with our constructs .', 'Western-blot analyses ( Fig 5A ) revealed that the amount of neomycin phosphotransferase 2 ( NeoR protein ) was ~5-fold lower in cells transfected with [NeoR]Δ7- than with [NeoR] .', 'However , few or no NeoR protein was detected in cells transfected with [NeoR]Δ7+ .', 'This was also supported by RT-qPCR experiments ( Fig 5B ) which showed that there were respectively 5 and 20-fold fewer NeoR transcripts in cells transfected with [NeoR]Δ7- and [NeoR]Δ7+ than in those transfected with [NeoR] .', 'Taken together these results confirmed that the Mos1 silencer element interferes with the expression of a gene marker located immediately upstream , that it acts at the level of RNA , and that the strength of the effect depends on its orientation since the amount of NeoR transcripts in cells transfected with [NeoR]Δ7- was ~4-fold higher than in those transfected with [NeoR]Δ7+ .', 'Because the silencer element had to be located within or downstream of the marker gene to be effective , we investigated whether it directly interfered with processes occurring after transcription initiation .', 'Transcript quality and RNA interference were examined .', 'Polyadenylation tails of transcripts from cells transfected with [NeoR] , [NeoR]Δ7- and [NeoR]Δ7+ were investigated [17 , 18] according to their concentration in each sample , using GAPDH transcripts as endogenous controls .', 'No difference was found , indicating that polyadenylation was unlikely to be affected .', 'To test if the miRNA pathway was involved , Co115 human cells depleted in a key protein for miRNA processing and DICER function TARBP2 [19] were used in a transient expression assay .', 'Similar silencing activity of [NeoR]Δ7+ was found in both Co115 ( Fig 5C ) and HeLa cells ( Fig 5D ) , suggesting that there was no link between the silencing activity of Δ7 and the miRNA pathway .', 'In an attempt to locate a smaller fragment that would keep silencing activity in our expression assays the Δ7-MOS1 segment was fragmented ( Fig 6A ) .', 'The Δ8-MOS1 segment , corresponding to the last 317 bp of the MOS1 ORF was found to have the same silencing activity as the Δ7-MOS1 segment ( S4 Fig ) .', 'The size of the Δ8 segment is of interest since it was close to the upper limits for a usable protein electrophoretic mobility shift assays ( EMSA ) for investigating the binding of a TF .', 'Several viruses and transposable elements [20–30] were previously found to contain segments capable of silencing their own transcriptional activity to establish their latency in their eukaryotic hosts .', 'These silencers are bound by the transcription factor Yin Yang 1 ( YY1 in vertebrates , Pho in drosophila ) .', 'In eukaryotes , YY1 and other TFs can bind a chromosomal polycomb response element ( PRE ) to mobilize the PRC1 and PRC2 and finally induce transcriptional silencing of that chromosomal region .', 'The presence of YY1 binding sites and TF binding sites involved in PRC2 in drosophila was examined in the Δ7 and Δ8 silencer segments of Mos1 ( Fig 6B ) , Himar1 , Mcmar1 , Hsmar1 , and Hsmar2 ( S5 Fig ) .', 'A set of binding sites for YY1 or Pho , the GAGA factor ( GAF ) , GTGT factor ( GTF ) , and Zeste [31 , 32] located among the Δ7 segments was found in all natural MLEs , each of which is typically repeated .', 'Together the presence of these sites suggests that PRCs might be able to bind to these silencer elements , at least in dipteran species .', 'EMSAs were carried out to verify the presence of functional YY1 binding sites in Δ8 mariner segments .', 'Our results showed that a shifted complex was present with the Δ8-MOS1 , Δ8-HIMAR1 and Δ8-HSMAR1 probes ( Fig 6C ) .', 'These complexes were super-shifted by anti-YY1 antibodies confirming that they correspond to YY1/Δ8 complexes .', 'The absence of a complex with the Δ8-MCMAR1 probe suggested that the binding site located in Δ8 was not bound under our experimental conditions .', 'Hence , the silencing element in Mcmar1 extended beyond Δ8 and might be located at the 5’ extremity of Δ7 , which contains a YY1 binding site ( S5 Fig ) .', 'Since only one shifted band was observed with the Δ8-MOS1 segment while three binding sites were predicted in its sequence , further EMSA investigations were performed with shorter probes , Δ81 to Δ85 ( Figs 6A and 6D and S4 ) .', 'These results were consistent with the sequence binding site prediction analysis , showing that there was one YY1 binding site within Δ81 and Δ83 , two in Δ84 , and none in Δ82 and Δ85 .', 'This last result suggested that the motif located in 3’ was unable to be bound by YY1 under our experimental conditions .', 'However , this was likely an artefact due to its location at the 5’ end of the Δ85 probe .', 'Indeed , when both YY1 sites are located in the middle of the Δ84 probe , two shifted bands were observed ( Fig 6D , lane 12 ) , suggesting that both sites could be bound .', 'Taken together , these data supported the conclusion that the silencer activity of the Δ8 segments was possibly mediated by one or several YY1 silencing pathways .', 'Since the definition of PREs in vertebrate genomes is an issue that has yet to be fully elucidated [31 , 32] , we searched for motifs conserved for both sequence and location using the MEME software suite with the DNA sequences of 34 mariner Δ7 segments ( S6 Fig ) .', 'A single conserved 30 bp motif was found ( p-values ranging from 3 . 36e-21 to 6 . 11e-14 ) that spanned the region coding one of the two signature motifs of mariner transposases , the PHxxYSPDLAPxD peptide [33] , and located in the region as a putative non-cardinal binding site for NRSF [34 , 35] and charlatan [36] .', 'In mammalian genomes , approximately 80% of the 2 000 characterized NRSEs ( called RE1 ) correspond to a 21 bp motif consisting of two conserved motifs of 9 and 10 bp separated by a 2 bp linker .', 'Approximately 12% consist of multiple rearrangements of this motif [34–36] .', 'The remaining 8% are sites with no conserved motifs .', 'The putative NRSE in the mariner silencer element described above belongs to the second category .', 'EMSAs were carried out to assay these predicted NRSEs .', 'HeLa nuclear extracts containing charlatan , human or fugu NRSF tagged with FLAG or Myc were prepared as described in previous studies [36 , 37] .', 'The activity of the nuclear extracts was validated using an NRSE probe ( RE1 ) in EMSA ( S7 Fig ) carried out with appropriate competitor and/or antibodies [36–38] .', 'The binding of NRSF to Δ8-MOS1 was then further investigated with EMSA using shorter versions of Δ8 , Δ81 to Δ85 segments as probes and HeLa nuclear extracts containing charlatan , human or fugu NRSF tagged with FLAG or Myc .', 'No shifted bands were obtained with Δ83 , Δ84 and Δ85 probes .', 'By contrast , shifted complexes sensitive to the competition by a specific competitor ( unlabelled RE1 fragment ) were obtained with the Δ82 probe for the three NRSF proteins ( Fig 7A ) .', 'Since this probe contained the motif encoding the PHxxYSPDLAPxD peptide , we concluded that it was an NRSE .', 'To verify whether these NRSE intervened in the silencer activity under our experimental conditions , transient luciferase expression assays were performed using constructs with the Δ8 , Δ8-ΔNRSF , or Δ8-mutNRSF of Mos1 or Hsmar1 ( S8A and S8B Fig ) cloned in positive orientation downstream of the marker cassette of HS2_P_Luc plasmids ( Fig 7B and 7C ) .', 'Δ8-MOS1-ΔNRSF and Δ8-HSMAR1-ΔNRSF were specified by the deletion of the NRSE motif and Δ8-MOS1-mutNRSF and Δ8-HSMAR1-mutNRSF by the mutagenesis of the NRSE by randomly shuffling its sequence .', 'Results revealed that the DNA motif encoding the PHxxYSPDLAPxD peptide , i . e . the NRSE , was not essential for the silencer activity of the Δ8 silencers of Mos1 and Hsmar1 .', 'Two shifted complexes were also obtained with the three NRSF proteins and the short Δ81 probe in which there was one YY1 binding site and no overlap with the NRSE encoding the PHxxYSPDLAPxD peptide ( Fig 7D lanes 2 , 5 and 8; S9 Fig , lanes 2 and 5 ) .', 'These two complexes were sensitive to competition by a specific competitor of the YY1 binding ( Fig 7D , lanes 3 and 6; S9 Fig , lane 3 ) , indicating that they involved YY1 .', 'Interestingly , we observed that the bigger complex ( indicated by a red star in Figs 7D and S9 ) disappeared when antibodies directed against the tag of the human or fugu NRSF were added ( Fig 7D , lane 9; S9 Fig , lane 6 ) .', 'Together these results indicated that there was a second NRSF binding site in the Δ8 silencer that required the cooperative binding of YY1 to be efficient .', 'In spite of our efforts we failed to locate an NRSE or a charlatan binding element in this region .', 'Therefore , it remains possible that NRSF only interacts with YY1 when it is bound to Δ8 .', 'In order to verify whether this second site of NRSF binding was required for the silencer activity two Δ8 variants for Mos1 ( Fig 8A ) and Hsmar1 ( S8B Fig ) were generated by PCR , cloned downstream of the marker gene into HS2_P_Luc plasmid constructs in positive orientation , and tested in transient luciferase expression assays in HeLa cells .', 'Results obtained with constructs HS2_P_Luc_Δ8-MOS1-[47–311]-ΔNRSF ( Fig 8B ) and HS2_P_Luc_ Δ8-HSMAR1-[61–311]-ΔNRSF ( S8C Fig ) revealed that the silencer activity was conserved in spite of the fact that both regions bound by NRSF proteins in Mos1 were deleted in Δ8 segments .', 'Finally , these data supported that there were either one or two sites where NRSF was able to interfere with the Δ8 segment .', 'However , the binding of NRSF to the mariner silencers of Mos1 and Hsmar1 was not essential to the silencer activity under our experimental conditions .', 'Therefore , we continued our efforts to find out the regions essential for the silencer activity of the mariner Δ8 segment .', 'In addition to the variant Δ8-MOS1-[47–311]-ΔNRSF , four other variants were made ( Fig 8A ) .', 'The first , Δ8-MOS1-[1–152] , contained the 5’ half of Δ8-MOS1 ( i . e . one YY1 binding site plus the two NRSF binding sites ) .', 'The second , Δ8-MOS1-[86–311] , contained the 3’ half plus the NRSF binding site overlapping the DNA motif encoding the PHxxYSPDLAPxD peptide ( i . e . two YY1 binding sites plus one NRSF binding site ) .', 'The third , Δ8-MOS1-[116–311] was similar to the second with the exception that its NRSF binding site was removed .', 'The fourth , Δ8-MOS1-[66–311]-ΔNRSF , was similar to Δ8-MOS1-[47–311]-ΔNRSF but its 19 residues on the 5’ end were deleted .', 'Transient luciferase expression assays in HeLa cells revealed that all of these Δ8 variants had kept their silencer activity but with variable efficiency ( Fig 8B ) .', 'For Δ8-MOS1-[1–152] , Δ8-MOS1-[86–311] and Δ8-MOS1-[116–311] , the luciferase expression is higher than the P_Luc control but , significantly , it was 2 . 5 to 3 . 5-fold lower than that of HS2_P_Luc ( e . g . in Figs 2A and 5B ) .', 'This indicated that the YY1 binding site motif within the 5’ half of Δ8 and other TF binding sites within the positions 116 to 311 were enough to trigger weak silencer activity in HeLa cells .', 'Interestingly , it also indicated that Δ8-MOS1-[47–311]-ΔNRSF and Δ8-MOS1-[66–311]-ΔNRSF had better silencer activity than Δ8 in HeLa cells .', 'Taken together , these results suggested that several combinations of TFs could bind to Δ8 and could cooperatively act with YY1 to trigger the silencer activity .', 'To verify whether this property could be recovered in another mariner silencer , Δ8 variants were also made from Δ8-HSMAR1 ( S8B Fig ) .', 'Their analysis under similar experimental conditions first revealed that the 3’ half ( positions 130 to 310 ) was enough to trigger weak silencer activity in HeLa cells , but was more efficient when the DNA motif bound by NRSF and overlapping the DNA motif encoding the PHxxYSPDLAPxD peptide was present ( positions 86 to 310 ) .', 'This indicated that this NRSF binding site favoured the silencer activity in Δ8-HSMAR1 .', 'In addition , the two variants Δ8-HSMAR1-[61–310]-ΔNRSF and Δ8-HSMAR1-[81–310]-ΔNRSF that have sequence properties similar to those of Δ8-MOS1-[47–311]-ΔNRSF and Δ8-MOS1-[66–311]-ΔNRSF have kept a strong silencer activity , but this was significantly less strong than that of Δ8 .', 'A search for TF binding sites motifs within the regions 47 to 96 in Δ8-MOS1 and 61 to 104 in Δ8-HSMAR1 was achieved using the MatInspector facilities of the GENOMATIX software suite ( Munich , Germany ) .', 'Our results revealed that there were two NFAT-5 and one Alx1 binding sites in the 50 bp MOS1 segment ( Fig 8A ) , and one NFAT-5 and one Alx1 binding sites in the 44 bp HSMAR1 segment ( S8B Fig ) .', 'Under the hypothesis that the same TFs acted on this region , results obtained with Δ8-MOS1-[47–311]-ΔNRSF and Δ8-MOS1-[66–311]-ΔNRSF on the one hand , and Δ8-HSMAR1-[61–310]-ΔNRSF and Δ8-HSMAR1-[81–310]-ΔNRSF on the other hand , suggested that both these TFs might cooperatively intervene in the silencer activity .', 'In order to further investigate this feature the HeLa , Co115 , H4 and DT40 cells used in our work were phenotyped in order to determine their expression for NRSF , YY1 , NFAT-5 , Alx1 and TARBP2 .', 'We found that HeLa cells were NRSF + , YY1 + , NFAT-5 + , Alx1 + and TARBP2 + .', 'Others cells presented differences since Co115 cells were TARBP2 - , DT40 were NFAT-5 - , and H4 were NRSF—and NFAT-5 - .', 'Taking into account these phenotypes , the effect of the Δ7-MOS1 segments in transient luciferase expression assay was analyzed ( Fig 5C and 5D and Fig 8C and 8D ) .', 'Under these experimental conditions the absence of NFAT-5 significantly weakened the silencer effect of Δ7-MOS1 in H4 and DT40 , but did not suppress it entirely .', 'In conclusion , our results suggested that TFs NFAT-5 , Alx1 and NRSF , might intervene alone or cooperatively with YY1 to bind to the silencer of Mos1 and Hsmar1 and elicit the silencer activity .', 'In addition , the weak silencer activity of the region located downstream of the DNA motif encoding the PHxxYSPDLAPxD peptide of Δ8-MOS1 and Δ8-HSMAR1 might be related to the cooperative binding of YY1 and GAF and/or GTF TFs .', 'The sequence features of the Δ8 mariner silencers described above might match those of the Polycomb Responsive Elements/Trithorax Responsive Elements ( PRE/TRE ) [31 , 39–42] that respectively silence or activate gene transcription by modifying chromatin histone marks .', 'In order to further investigate whether the silencing depended on the polycomb pathway we used a specific inhibitor of PRC2 , the 3-deazaneplanocin A ( DZNep ) , in expression assays using plasmid constructs containing different variants of the mariner silencers of Mos1 ( Fig', '9 ) and Hsmar1 ( S10 Fig ) .', 'DZNep is an analogue of 3-deazadenosine that inhibits the activity of S-adenosylhomocysteine hydrolase , leading to the indirect inhibition of various S-adenosylmethionine-dependent methylation reactions , such as those catalysed by EZH2 in animal cells , including HeLa cells [43–45] .', 'DZNep efficiently inhibits EZH2 after 8 h of treatment and can induce strong apoptotic cell death reaction in cancer cells beyond 48 h [43–47] .', 'Cells were treated overnight with 5 μM DZNep prior DNA transfection and treatment was maintained until Firefly and Renilla luciferase activity measurements .', 'Before experimenting with DZNep on mariner silencers , the impact of this chemical was verified on the Firefly luciferase expression of the P_Luc and HS2_P_Luc constructs ( Fig 9A ) .', 'Results revealed that DZNep had no effect on P_Luc .', 'However , this chemical decreased the capacity of the HS2 enhancer to boost the Firefly luciferase expression ( ~3 . 5-folds ) even if the difference between P_Luc and HS2_P_Luc constructs remained significant .', 'Because our silencer DNA segments were cloned into an HS2_P_Luc plasmid backbone , expression results obtained with HS2_P_Luc in the presence or absence of 5 μM DZNep were used as references to calculate the expression rate obtained with the mariner silencer constructs in the presence or absence of 5 μM DZNep ( Fig 9B and S10 Fig ) .', 'Results showed that DZNep significantly increased ( p<0 . 05 ) the Firefly luciferase expression from HS2_P_Luc_Δ8-MOS1 , HS2_P_Luc_Δ8-MOS1-[47–311]-ΔNRSF , HS2_P_Luc_Δ8-HSMAR1 , HS2_P_Luc_Δ8-HSMAR1-ΔNRSF and HS2_P_Luc_Δ8-HSMAR1-[61–310]-ΔNRSF .', 'This supported the hypothesis that the Mos1 and Hsmar1 might contain functioning silencers depending on the PRC2 pathway , since the silencer activity of these constructs is decreased .', 'Interestingly , five constructs responding to a DZNep treatment shared the presence of YY1 , NFAT-5 , Alx1 , GAF and GTF binding sites in their DNA sequences .', 'By contrast , the Firefly luciferase expression from constructs containing the 5’ half of the Mos1 silencer ( HS2_P_Luc_Δ8-MOS1-[1–152] ) or the 3’ half of the Mos1 or Hsmar1 silencers ( HS2_P_Luc_Δ8-MOS1-[116–311] and HS2_P_Luc_Δ8-HSMAR1-[115–310] ) were not affected by the DZNep treatment .', 'This suggested that the weak silencer effect resulting from the presence of these DNA segments might result from another silencing mechanism and is detected only when PRC2 is disrupted .', 'Such a duality between silencing pathways was previously described for ITm TEs contained in the genome of murine ES cells .', 'Indeed , these TEs can switch from heterochromatinization mediated by the HP1 ( Heterochromatic Protein 1 ) dependent pathway to a PRC2-dependent silencing when the Histone-lysine N-methyltransferase Su ( var ) 39/HP1 is disrupted [29] .', 'Here , the duality between silencing pathways might also help explain why weak residual silencer effects were observed in some cases , such as in the H4 and DT40 cells ( Fig 5C and 5D ) .', 'Since there is no available animal model with active MLEs for which high throughput chromatin data are available in public databases , we have investigated the chromatin status of two human MLEs , Hsmar1 and Hsmar2 , that appeared in the human genome approximately 50 and at least 80 million years ago respectively [48 , 49] .', 'Currently these elements have lost their ability to transpose due to the accumulation of nucleotide mutations in the ORF coding for their transposase .', 'The advantage of the human model is that it has the richest set of ChIP-Seq data for TFs and histone modifications .', 'Because the recruitment of TFs bound to DNA at the moment of the establishment of histone modifications is not subsequently required for their maintenance and transmission over cell divisions [50–53] , we have focussed our investigations on histone modifications .', 'This was carried out in order to highlight potential associations between the presence or the absence of a complete mariner silencer within each human MLE , their genomic location , and two important silencing pathways: polycomb/trithorax and Su ( var ) 39/HP1 .', 'These two pathways lead to specific signatures of histone modifications:', '( i ) H3K27me3 when the genomic loci is inactivated by PRC ,', '( ii ) H3K27me3/H3K4me3 when PRC and Trithorax complexes interfere together at level of inactive poised regions ,', '( iii ) H3K27ac/H3K4me3 when the genomic loci is activated by Trithorax complexes , and', '( iv ) H3K9me3 and H4K20me1 when it is silenced and heterochromatinized by the Su ( var ) 39/HP1 pathway [39 , 41 , 54 , 55] .', 'Since it was previously shown that human TEs carry more histone modifications when they are located within or near genes [56] , we have distinguished two categories of MLEs: those located in genes co
['Introduction', 'Results', 'Discussion', 'Methods']
['Transposable elements are driving forces for establishing genetic innovations such as transcriptional regulatory networks in eukaryotic genomes .', 'Here , we describe a silencer situated in the last 300 bp of the Mos1 transposase open reading frame ( ORF ) which functions in vertebrate and arthropod cells .', 'Functional silencers are also found at similar locations within three other animal mariner elements , i . e . IS630-Tc1-mariner ( ITm ) DD34D elements , Himar1 , Hsmar1 and Mcmar1 .', 'These silencers are able to impact eukaryotic promoters monitoring strong , moderate or low expression as well as those of mariner elements located upstream of the transposase ORF .', 'We report that the silencing involves at least two transcription factors ( TFs ) that are conserved within animal species , NFAT-5 and Alx1 .', 'These cooperatively act with YY1 to trigger the silencing activity .', 'Four other housekeeping transcription factors ( TFs ) , neuron restrictive silencer factor ( NRSF ) , GAGA factor ( GAF ) and GTGT factor ( GTF ) , were also found to have binding sites within mariner silencers but their impact in modulating the silencer activity remains to be further specified .', 'Interestingly , an NRSF binding site was found to overlap a 30 bp motif coding a highly conserved PHxxYSPDLAPxD peptide in mariner transposases .', 'We also present experimental evidence that silencing is mainly achieved by co-opting the host Polycomb Repressive Complex 2 pathway .', 'However , we observe that when PRC2 is impaired another host silencing pathway potentially takes over to maintain weak silencer activity .', 'Mariner silencers harbour features of Polycomb Response Elements , which are probably a way for mariner elements to self-repress their transcription and mobility in somatic and germinal cells when the required TFs are expressed .', 'At the evolutionary scale , mariner elements , through their exaptation , might have been a source of silencers playing a role in the chromatin configuration in eukaryotic genomes .']
['Transposons are mobile DNA sequences that have long co-evolved with the genome of their hosts .', 'Consequently , they are involved in the generation of mutations , as well as the creation of genes and regulatory networks .', 'Controlling the transposon activity , and consequently its negative effects on both the host soma and germ line , is a challenge for the survival of both the host and the transposon .', 'To silence transposons , hosts often use defence mechanisms involving DNA methylation and RNA interference pathways .', 'Here we show that mariner transposons can self-regulate their activity by using a silencer element located in their DNA sequence .', 'The silencer element interferes with host housekeeping protein transcription factors involved in the polycomb silencing pathways .', 'As the regulation of chromatin configuration by polycomb is an important regulator of animal development , our findings open the possibility that mariner silencers might have been exapted during animal evolution to participate in certain regulation pathways of their hosts .', 'Since some of the TFs involved in mariner silencer activity play a role at different stages of nervous system development and neuron differentiation , it might be possible that mariner transposons can be active during some steps of cell differentiation .', 'Interestingly , mariner transposons ( i . e . IS630-Tc1-mariner ( ITm ) DD34D transposons ) have so far only been found in genomes of animals having a nervous system .']
['sequencing techniques', 'luciferase', 'hela cells', 'gene regulation', 'enzymes', 'biological cultures', 'enzymology', 'marker genes', 'cloning', 'genetic elements', 'cell cultures', 'sequence motif analysis', 'molecular biology techniques', 'epigenetics', 'chromatin', 'research and analysis methods', 'sequence analysis', 'chromosome biology', 'proteins', 'gene expression', 'oxidoreductases', 'cell lines', 'molecular biology', 'biochemistry', 'cell biology', 'genetics', 'transposable elements', 'biology and life sciences', 'cultured tumor cells', 'genomics', 'mobile genetic elements', 'silencer elements']