Paramecium tetraurelia, differentiation of the somatic nucleus from the zygotic nucleus is characterized by massive and reproducible deletion of transposable elements and of 45,000 short, dispersed, single-copy sequences. A specific class of small RNAs produced by the germline during meiosis, the scnRNAs, are involved in the epigenetic regulation of DNA deletion but the underlying mechanisms are poorly understood. Here, we show that trimethylation of histone H3 (H3K27me3 and H3K9me3) displays a dynamic nuclear localization that is altered when the endonuclease required for DNA elimination is depleted. We identified the putative histone methyltransferase Ezl1 necessary for H3K27me3 and H3K9me3 establishment and show that it is required for correct genome rearrangements. Genome-wide analyses show that scnRNA-mediated H3 trimethylation is necessary for the elimination of long, repeated germline DNA, while single copy sequences display differential sensitivity to depletion of proteins involved in the scnRNA pathway, Ezl1- a putative histone methyltransferase and Dcl5- a protein required for iesRNA biogenesis. Our study reveals cis-acting determinants, such as DNA length, also contribute to the definition of germline sequences to delete. We further show that precise excision of single copy DNA elements, as short as 26 bp, requires Ezl1, suggesting that development specific H3K27me3 and H3K9me3 ensure specific demarcation of very short germline sequences from the adjacent somatic sequences.In the ciliate  Paramecium tetraurelia provides an extraordinary model for studying the mechanisms involved in zygotic genome rearrangements. At each sexual cycle, differentiation of the somatic nucleus from the zygotic nucleus is characterized by extensive remodeling of the entire somatic genome, which includes the precise excision of 45,000 short noncoding germline DNA segments to reconstitute functional open reading frames. Exploiting the unique properties of the Paramecium genome, we show that the enhancer of zeste like protein Ezl1 is necessary for histone H3 trimethylation on lysines 27 and 9 and is required for the precise excision of 31,000 of these single copy, dispersed germline DNA segments that can be as short as 26 bp in length. This implies that histone marks usually associated with heterochromatin may contribute to the precise demarcation of segments that are even shorter than the length of DNA wrapped around a single nucleosome. A quantitative analysis of high throughput sequencing datasets further shows that the underlying genetic properties of the germline DNA segments might act in concert with epigenetic signals to define germline specific sequences.The unicellular eukaryote  Paramecium tetraurelia, as in all ciliates, germline and somatic functions are separated between two distinct nuclei that coexist in the same cytoplasm. During vegetative growth, the diploid germline micronucleus (MIC) remains transcriptionally silent, while the highly polyploid somatic macronucleus (MAC) supports gene expression. During sexual events, the MAC is fragmented and eventually lost, whereas the MIC undergoes meiosis and transmits the germline genome to the new MIC and MAC of the next generation. During the differentiation of the zygotic MAC, germline-specific regions up to several kbp in length, often containing repetitive sequences, are imprecisely eliminated leading to germline chromosome fragmentation or intra-chromosomal deletions. Moreover, and this is a critical point for the present study, 45,000 single-copy, short and non-coding Internal Eliminated Sequences (IES) are excised precisely from intergenic and genic regions. These « DNA introns » are found throughout the germline genome, appear to be remnants of ancestral insertions of transposable elements (TEs) Ciliates provide extraordinary model organisms with which to gain understanding into the organization of eukaryotic genomes. The differentiation of the somatic nucleus from the zygotic nucleus is characterized by massive and reproducible rearrangements at the DNA level Tetrahymena thermophila, where scnRNA-mediated tri-methylation of histone H3 on lysine 9 and lysine 27 (H3K9me3 and H3K27me3) TetrahymenaParamecium sRNAs, called iesRNAs, was reported Precision of IES excision is critical for the assembly of functional genes in the somatic genome and the survival of sexual progeny. Yet the weak consensus found at IES ends is not sufficient to determine the excision pattern across the whole genome Paramecium. The vast majority of IESs are shorter than 150 bp and some are as short as 26 bp; they are thus not even as long as the DNA wrapped around a single nucleosome. Excision of these 45,000 DNA segments must require a marking mechanism of considerable precision, allowing the demarcation of these very short, numerous, interspersed germline sequences from adjacent retained somatic sequences. The present study was designed to test the involvement of histone H3 methylation in the DNA elimination process, with a special interest for its role on IES excision. We show here that the putative histone methyltransferase (HMT) Ezl1 is required for the accumulation of H3K27me3 and H3K9me3 in the developing somatic macronucleus. Re-sequencing the genome following Ezl1 depletion showed that EZL1 is required for correct genome rearrangements. We found that scnRNA-mediated H3K27me3 and H3K9me3 is necessary for the elimination of a fraction of germline DNA, including transposable elements and long IESs. Strikingly, the putative HMT Ezl1 is also required for the precise excision of about 70% of the 45,000 short, unique copy IESs, providing evidence that it may contribute to the precise demarcation of short germline sequences. Our genome wide study shows that IESs display differential sensitivity to depletion of the scnRNA pathway, Dcl5 or Ezl1 proteins and identifies cis acting determinants, such as DNA length that might act in concert with epigenetic signals to define germline specific sequences.The chromatin modifications that may guide the Pgm endonuclease to specific germline sequences are not yet characterized in in situ localization of H3K27me3 and H3K9me3 during various stages of the Paramecium life cycle  the domesticated transposase Pgm that is required for the introduction of DNA-double strand breaks at the boundaries of germline-limited segments We noticed that the H3K27me3 and H3K9me3 signals remained diffuse as development proceeds in Pgm-depleted cells and no foci could be detected . The endThe scanning model posits that MIC-specific scnRNAs guide the loading of histone marks specifically on DNA segments that are eliminated in the developing MAC. We therefore expected that the co-silencing of the two Dicer-like genes, DCL2 and DCL3, that results in failure to generate scnRNAs We then investigated the effects of silencing DCL5, a gene required for iesRNA biogenesis, on H3K27me3 and H3K9me3. In contrast to what is observed in DCL2/3 KD, H3K27me3 and H3K9me3 signals were not altered in DCL5 KD, as assessed by immunofluorescence staining  and thisWe conclude that the generation of scnRNAs, but not iesRNAs, is required for establishment and accumulation of these chromatin modifications in the developing MAC. These results suggest that scnRNAs and K9 and K27 methylation participate in the same pathway leading to genome rearrangements. To support this hypothesis, it is necessary to demonstrate that K27 and K9 methylation is required for DNA elimination.P. tetraurelia MAC genome To eliminate K9 and K27 methylation, we sought to identify the gene(s) responsible for these modifications The expression patterns of EZL genes during the life cycle were examined using microarray data To test the function of EZL genes, we knocked down their expression by RNAi during autogamy. After EZL1 KD, 97% of post-autogamous progeny were unable to resume vegetative growth, whereas no lethality was observed after KD of any other EZL gene . The traIn an EZL1 KD, the transcription of the PGM gene and of all EZL genes is switched on normally during autogamy, indicating that these genes are not induced in response to EZL1 induction but more likely as part of a general transcription program during MAC development. In contrast to control cells, the levels of these mRNAs do not decrease at later time-points in an EZL1 silencing experiment , suggestTo gain further insight into the role of EZL1, we examined the subcellular localization of Ezl1. A GFP fusion was constructed by inserting the GFP coding sequence into the EZL1 gene, downstream of the start codon. Expression of the fusion gene was under the control of the natural EZL1 up- and downstream sequences. After microinjection of the construct into the MAC, no fluorescence could be detected during vegetative growth of transformed clones . When auWe therefore investigated the effects of EZL1 KD on H3K27me3 and H3K9me3. Immunofluorescence staining with H3K27me3- and H3K9me3- specific antibodies showed little or no signal in Ezl1-depleted cells, whereas in control cells H3K27me3 and H3K9me3 increased as development proceeded and completely disappeared at the latest time point . For morEZL1 KD led to phenotypes consistent with an essential function for Ezl1 during MAC development since: (1) no viable sexual progeny were isolated from Ezl1-depleted cells, a phenotype described in KDs defective in DNA elimination Different assays were used to monitor genome rearrangements in autogamy time course experiments, after EZL1 KD. We first tested the role of the EZL1 gene in the imprecise DNA elimination mechanism that is responsible for the deletion of MIC transposable elements during MAC development. We analyzed by Southern blot hybridization the germline region located downstream of the surface antigen G gene, which contains a Sardine transposon that is eliminated imprecisely during MAC development, leading to chromosome fragmentation We further tested the role of Ezl1p in the imprecise elimination mechanism also responsible for maternally inherited deletions of non-essential cellular genes, which can be induced experimentally To expand these results genome wide, we sequenced DNA isolated from newly developed MACs following EZL1 silencing. DNA isolated from newly developed MACs at the same developmental stage from a cell culture grown without RNAi was also sequenced as a control . We compIf we compare the complexity of regions not covered by the control sample, which correspond to the part of the MIC genome that is not collinear with MAC chromosomes, PGM and EZL1 datasets again show a similar complexity. We also performed a qualitative evaluation of Sardine retention by mapping reads from each dataset to the known cloned copies of this transposable element We then investigated the role of the EZL1 gene in IES excision. Excision was first analyzed by PCR on genomic DNA, extracted after EZL1 or control silencing at a time when IES excision is normally finished. In control RNAi experiments, the 10 IESs analyzed were completely excised from the new developing MACs as expected . In contBased on our PCR analyses, not all IESs are affected following EZL1 KD. To observe the effects of Ezl1p depletion genome-wide, we used the EZL1 DNA-seq dataset. A retention score (RS) was calculated for each IES in the reference set −16). Based on these data, we can define two classes of IESs: those that are significantly retained after EZL1 KD and those that are not. Importantly, our PCR analyses are completely coherent with our genome-wide analysis , the median does not change much, which indicates that IESs of these sizes are similarly affected by Ezl1p depletion. For the largest IESs (>1 kb), the retention score distribution is significantly shifted to higher values. Among those, there is one group composed of 28 IESs, which have been shown to derive from Tc1/mariner TEs named Anchois We then wondered what distinguishes the two classes of IESs. Our PCR analyses indicated that long IESs were retained in the new developing MAC following EZL1 KD . To confWe searched for features other than size that could be associated with EZL1 retained IESs. We compared IESs of the same size (26-32 bp) and did not find any meaningful correlation for a large number of criteria, including the consensus present at IES ends and the scnRNA density on IESs. We did observe that EZL1-retained IESs have: (i) a slightly higher GC content, (ii) a more frequent location within gene coding sequences . It is iSince DCL2 and DCL3 genes, like EZL1, are required for establishing H3K27me3 and H3K9me3 in the developing MAC  and 4, w−16) despite use of different silencing constructs. Furthermore, the RS measured for the DCL2/3 dataset are in agreement with our PCR analyses . Deposition of H3K27me3 and H3K9me3 would allow the recruitment, or activation of the excision machinery, followed by elimination of marked DNA segments , consistWhile all IESs are ultimately excised by the Pgm endonuclease More importantly, our data indicate that most IESs are correctly excised in the absence of scnRNAs. IESs, even those that are not under maternal control, normally do produce scnRNAs during MIC meiosis In addition to these two classes of IESs, genome wide analysis of the effects of Ezl1 depletion provided evidence for additional classes of IESs, showing differential sensitivity to the different factors studied here. In order to group IESs into functionally similar classes, we have quantitated the requirement of each of the 45,000 IESs for each of the factors analyzed . Our datEZL1 encodes a putative histone methyltransferase necessary for H3K27me3 and H3K9me3 and for excision of about 70% of IESs, suggesting that H3K27me3 and H3K9me3 are required for their excision, as discussed below. DCL2/3 KD also leads to diminution of H3K27me3 and H3K9me3 signals at early stages of MAC development  and yet,We now understand that IES excision involves partially overlapping pathways given our observations of different classes of IESs. This led us rethink the simple model according to which scnRNAs -produced by the Dcl2/3 proteins- lead to the loading of chromatin modifications- H3K27me3/HK9me3 through the action of the putative histone methyltransferase Ezl1- and recruitment of the Pgm endonuclease. Indeed, a small subset of IESs require DCL2/3 and EZL1 (7%), while the majority of IESs require only EZL1 (63%), and some IESs require neither EZL1 nor DCL2/3 for complete excision (30%) . The relDrosophila and in mammals, Enhancer of zeste proteins are catalytic subunits of Polycomb complexes, which target H3K27me3 and maintain repression of numerous developmental genes. Domains enriched in H3K27me3 cover large regions of the genome, usually exceeding 10 kb S. cerevisiae, the chromatin remodeler SWR1 binds in vitro long nucleosome-free DNA and the adjoining nucleosome core particle, allowing discrimination of gene promoters over gene bodies. SWR1 binding is enhanced on acetylated nucleosomes, but recognition of nucleosome-free and nucleosomal DNA is dominant over interaction with acetylated histones In P. tetraurelia. Cells were grown in wheat grass powder (WGP)  infusion medium bacterized the day before use with Klebsiella pneumoniae, unless otherwise stated, and supplemented with 0.8 mg/mL β-sitosterol (Merck). Cultivation and autogamy were carried out at 27°C as described Unless otherwise stated, all experiments were carried out with the entirely homozygous strain 51 of http://www.atgc-montpellier.fr/phyml/). PHYML analyses were performed using the Le and Gascuel (LG) amino-acid substitution model SET domain proteins were retrieved using Pfam Escherichia coli strain HT115 DE3, as previously described Plasmids used for T7Pol-driven dsRNA production in silencing experiments were obtained by cloning PCR products from each gene using plasmid L4440 and Paramecium codon usage For the construction of in-frame GFP-EZL1 fusion, a GFP-coding fragment adapted to Plasmids carrying the GFP-EZL1 or PGM-GFP DNA samples were typically extracted from 200-400-ml cultures of exponentially growing cells at <1,000 cells/ml or of autogamous cells at 2,000–4,000 cells/ml as previously described RNA samples were typically extracted from 200–400-ml cultures of exponentially growing cells at <1,000 cells/ml or of autogamous cells at 2,000–4,000 cells/ml as previously described PCR amplifications were performed in a final volume of 25 µL, with 10 pmol of each primer, 10 nmol of each dNTP and 1.9 U of Expand Long Template Enzyme mix . PCR products were analyzed on 0.8% agarose gels . For PCR2, 10 mM Tris pH 6.8, 0.2% Nonidet P-40) with a Potter-Elvehjem homogenizer. Following the addition of 2.5 volumes of washing solution , the nuclei-containing pellet was collected by centrifugation at 1000 g for 1 min and acid extraction of histones was performed as previously described Cell pellets were mechanically lysed in three volumes of lysis solution ; paraformaldehyde 1%, Triton X-100 2.5%, Sucrose 4%) and for 10 minutes in solution II . The primary antibodies used were rabbit polyclonal α-H3K27me3  and α-H3K9me3  at 1∶500. After incubation with the primary antibodies, cells were washed in 1X phosphate-buffered saline (PBS), incubated with the secondary antibodies  at 1∶500 for 1h, stained with 1 µg/mL Hoechst, washed in 1X PBS, centrifuged on microscope slides with the CytoSpin™ 4 Cytocentrifuge (Thermo Scientific) and finally mounted in Citifluor AF2 glycerol solution . Images were acquired using a Zeiss LSM 710 laser-scanning confocal microscope and a Plan-Apochromat 63x/1.40 oil DIC M27 objective. Z-series were performed with Z-steps of 0.5 µm.To quantify the H3K27me3 and H3K9me3 signals, the Imaris 3D visualization software (Bitplane) was used. For each time point, the fluorescence intensities of H3K27me3/H3K9me3 in the developing MACs  and in the corresponding volume of the cytoplasm (noise) were measured. The mean value and standard deviation of the signal to noise ratios were calculated using ten individual cells at each time point.DNA for deep-sequencing was isolated from post-autogamous cells as previously described The following reference genomes MAC reference (strain 51):http://paramecium.cgm.cnrs-gif.fr/download/fasta/ptetraurelia_mac_51.faMAC+IES reference (strain 51):http://paramecium.cgm.cnrs-gif.fr/download/fasta/ptetraurelia_mac_51_with_ies.faPGM contigs:http://paramecium.cgm.cnrs-gif.fr/download/fasta/assemblies/ptetraurelia_PGM_k51_ctg.faMacronuclear DNA reads for PiggyMac P. tetraurelia MAC reference genome and MAC+IES reference genome) using BWA After quality filtering and removal of adapters, Illumina reads were aligned to the reference genomes (+) and the number of reads that contain only the macronuclear IES junction consisting of a TA dinucleotide (IES−) were determined. Only reads with unambiguous alignments were counted. Each read was counted only once to avoid over-counting owing to paralogous matches. Reads were only counted at IES ends, to avoid length biases resulting from IES length variation. The retention score (RS) of an IES is then given by the following equation:For each sample, IES retention scores were determined as follows, for each IES in the genome previously identified in +) / (IES+ + IES−)RS  =  .The EZL1 KD, DCL2/3 KD and control DNA-seq datasets have been deposited in the European Nucleotide Archive (Accession number ERA309409). All IES retention scores may be obtained via ParameciumDB  N terminal tails (1-31) of human and Paramecium H3 are aligned. H3K9 epitope (in red) is present in Paramecium H3. Considering the fact that the N terminal tail of Paramecium histone H3 displays some amino acid differences around K27 with respect to mammalian histone H3 (H3K27 epitope in orange), we checked crossreactivity of the H3K27me3 antibodies and verified the specificity by dot blot and competition assays. B) Dot blot assay using H3 peptides. 100 pmol of each of the indicated peptides was spotted on the membrane, and probed with the H3K27me3 antibodies. Polyclonal antibodies raised against tri-methyl K27 showed specific reactivity with human (HsH3K27me3: SKAARK(Me3)SAP) or Paramecium tri-methyl K27 (PtH3K27me3: TKAARK(Me3)TAP) but not against no-methyl-, mono-methyl, di-methyl Paramecium K27, di-mehyl, tri-methyl Paramecium K9 (QTARK(Me3)STAGN). H3K27me3 antibodies are specific for H3K27me3 peptides (Paramecium and human) and do not cross react with Paramecium H3K9me3 peptide. C) Competition assay. 0 to 100 pmol of Paramecium H3K27me3 peptides were spotted and probed with the H3K27me3 antibodies alone or in presence of a 50-fold molar excess of the indicated peptides. Competition with the Paramecium (PtK27me3: TKAARK(Me3)TAP) or human (HsK27me3: SKAARK(Me3)SAP) tri-methyl K27 peptides completely eliminates the signal, while competition with the un-methylated peptide (PtK27me0: TKAARKTAP) does not.Alignment of (EPS)Click here for additional data file.Figure S2Paramecium life cycle, co-localization of Pgm-GFP fusion protein and H3K27me3. A) Immunostaining with H3K27me3 antibodies during Paramecium life cycle. Schematic representations of key nuclear events in Paramecium autogamy are depicted on the left: (a) vegetative growth, (b-c-d) meiosis I, (e) meiosis II, (f-i) MAC development. The time points refer to hours after T =  0 hr that is defined as the time when cells begin fragmentation of the maternal MAC, as evaluated by cytological observation. See Immunostaining and Western blot analysis with H3K27me3 antibodies during (TIFF)Click here for additional data file.Figure S3Paramecium life cycle, co-localization of Pgm-GFP fusion protein and H3K9me3. A) Immunostaining with H3K9me3 antibodies during Paramecium life cycle. Schematic representations of key nuclear events in Paramecium autogamy are depicted on the left: (a) vegetative growth, (b-c-d) meiosis I, (e) meiosis II, (f-i) MAC development. The time points refer to hours after T =  0 hr that is defined as the time when cells begin fragmentation of the maternal MAC, as evaluated by cytological observation. See Immunostaining with H3K9me3 antibodies during (TIFF)Click here for additional data file.Figure S4Paramecium autogamy are depicted. B) and C) Progression of autogamy was followed by cytology with Hoechst staining in time course experiments after (B) ND7 (control), or PiggyMac (PGM) or EZL1 silencing and (C) ND7 (control) silencing or DCL2 and DCL3 co-silencing. Linear charts show quantifications of positive signals in the developing MAC after immunolabeling with H3K27me3- or H3K9me3- antibodies at each time point. The time-points refer to hours after T = 0 hr that is defined as the time when cells begin fragmentation of the maternal MAC, as evaluated by cytological observation. Because cells enter autogamy from a fixed point of the cell cycle Progression of autogamy. A) Schematic representations of key nuclear events in (EPS)Click here for additional data file.Figure S5P. tetraurelia, T.thermophila and O. trifallax. An unrooted Maximum-likelihood (ML) tree is shown. This tree has been constructed using the SET domains encoded by the genome of a large number of species. The full list of used sequences and studied species can be found in Drosophila proteins that are included in the group. We tried to use as much as possible the nomenclatures used in previous phylogenetic analyses of SET domain proteins Arabidopsis protein(s) to name these groups (ATXR3 and ATXR5/6). We listed in the figure the proteins from Paramecium tetraurelia (Ptet), Oxytricha trifallax (Otri), and Tetrahymena thermophila (Tthe), which belong to the different monophyletic groups. The robustness of the nodes that define the different monophyletic groups was assessed by evaluating their statistical support  in the ML analysis and by performing phylogenetic analyses (ML and Bayesian inference) using a smaller sampling of species (ciliates + Human + Drosophila + yeasts). This is represented on the tree by the presence close to the name of the group of *** , ** , or * . We obtained strong support for the existence of ciliate members of the EZH, ASH/SET2/NSD and SET1 groups that also include proteins from several other species including animals and Arabidopsis. No member of the Suvar39/EHMT/SETDB8/SETMAR group could be identified in the ciliate genomes. Several ciliate SET domains cluster with either Arabidopsis ATXR3 or Arabidopsis ATXR5/6 in groups that only include sequences from a small number of species and none from Human or Drosophila. These groups may therefore correspond to ancestral SET domain proteins that have been lost in some lineages such as animals or to divergent members of some other groups. This latter possibility is supported by the fact that the two groups are associated to the ASH/SET2/NSD group in the phylogenetic analyses made on the ciliates + Human + Drosophila + yeasts dataset. The inclusion of T. thermophila and O. trifallax proteins in the SMYD and SETD7 groups, as well as the belonging of a large number of ciliate proteins to the SETD6 group, has to be taken with caution, given the poor support of these groups, and may correspond to the artefactual grouping of highly divergent sequences.Phylogenetic analysis of SET domain proteins from (EPS)Click here for additional data file.Figure S6P. tetraurelia, T. thermophila, O. trifallax and other organisms. Sequences were aligned using the Muscle v3.8 software. The white text on a black background denotes invariant residues; white text on a gray background indicates conserved residues. Highly conserved residues are highlighted with different colors according to Allomyces macrogynus (Fungi); Atha  =  Arabidopsis thaliana (Viridiplanta); Aque  =  Amphimedon queenslandica (Metazoa); Dmel  =  Drosophila melanogaster (Metazoa); Hsap  =  Homo sapiens (Metazoa); Lgig  =  Lottia gigantea (Metazoa); Mbre  =  Monosiga brevicolis (Choanoflagellata); Nvec  =  Nematostella vectensis (Metazoa); Otri  =  Oxytricha trifallax Ptet  =  Paramecium tetraurelia (Ciliata); Spun  =  Spizellomyces punctatus (Fungi); Tetrahymena thermophila (Ciliata); Ttra  =  Thecamonas trahens (Apusozoa).Alignment of SET domains from EZH-EZL proteins from (DOCX)Click here for additional data file.Figure S7Expression patterns of EZL genes after EZL1 knockdown. A) Progression of autogamy in time course experiments, in which ICL7 (control) or EZL1 genes have been knocked down by RNAi. See (EPS)Click here for additional data file.Figure S8Localization of a GFP-EZL1 fusion protein. A) Localization of a GFP-EZL1 fusion protein during vegetative growth (a), meiosis I and II (b-e) and MAC development (f-j). Panels (g-j) are the entire images of the magnified views presented in (TIFF)Click here for additional data file.Figure S9Sardine coverage. The bar plots represent read coverage of 8 individual copies of the Sardine transposon (GenBank Accession No. HE774468-HE774475). The coverage was determined by mapping reads using BWA with default parameters, for the control (KLEB) dataset (grey), PGM silencing (red), EZL1 silencing (green), DCL2/3 co-silencing (yellow), and DCL5 silencing (blue) datasets. The normalized units (RPKM) are reads per kilobase of the transposon sequence per million library read mapped against the MAC reference genome.(EPS)Click here for additional data file.Figure S10PCR analysis of IES retention after EZL1, DCL2/3 or PGM silencing. PCR analysis of IES retention with primers Click here for additional data file.Figure S11IES retention score and size in control, EZL1, DCL2/3 or PGM datasets. IESs were grouped by size (cf. legend of (EPS)Click here for additional data file.Figure S12IES properties. IESs are grouped according to sample: those with significant retention scores , those that do not have significant retention scores  and ALL IESs. The left column  concerns only the IESs in the first peak of the size distribution (< 32 bp) and the right column  considers all IESs whatever their size. These IES groups were examined with respect to GC content  and with respect to the proportion of IESs that are within genes . The number of IESs in each group is indicated in boldface type inside the boxplots in A and B. Significant differences in GC content  and genomic distribution  for sensitive versus insensitive IESs are designated by asterisks (p<2.2 10 -16) and were determined using a Mann-Whitney test  or a Chi2 test .(EPS)Click here for additional data file.Figure S13EZL1 retention score for DCL2/3 and DCL5 retained IESs. Boxplots show the distribution of EZL1 retention scores for EZL1-retained IESs (green), DCL2/3-retained IESs (yellow) and DCL5-retained IESs (blue), showing that the significantly retained IESs in the DCL2/3 and the DCL5 datasets are among the most retained IESs in the EZL1 dataset. Significant differences are designated by asterisks (p<2.2 10-16) and were determined using a Mann-Whitney test.(EPS)Click here for additional data file.Table S1P. tetraurelia. Accession numbers , names and conserved domains are indicated.SET domain proteins identified in the somatic MAC genome of (DOCX)Click here for additional data file.Table S2Sequencing and mapping statistics. Read statistics are provided for the samples sequenced for this study, with the exception of the PGM and DCL5 silencing samples, which were previously published (DOCX)Click here for additional data file.Table S3Sequence complexity of control, PGM, EZL1, DCL2/3 and DCL5 datasets. The previously published contigs assembled from a PGM dataset (DOCX)Click here for additional data file.Table S41: S. Duharcourt and E. Meyer, personal communicationIES retention analyzed by PCR and deep-sequencing after control, DCL2/3, EZL1 and PGM silencing. Maternally (mcIES) or non-maternally controlled (non-mcIES) is indicated as “+” or “–“ respectively, based on previous studies (DOCX)Click here for additional data file.Table S5Anchois retention score. The 28 IESs that were used to identify the Anchois Tc1/mariner transposon (DOCX)Click here for additional data file.Table S6Oligonucleotides used in this study.(DOCX)Click here for additional data file.Text S1Accession numbers and sequences of SET domain protein used in (DOCX)Click here for additional data file.


The locations of transcriptional enhancers and promoters were recently mapped in many mammalian cell types. Proteins that bind those regulatory regions can determine cell identity but have not been systematically identified. Here we purify native enhancers, promoters or heterochromatin from embryonic stem cells by chromatin immunoprecipitations (ChIP) for characteristic histone modifications and identify associated proteins using mass spectrometry (MS). 239 factors are identified and predicted to bind enhancers or promoters with different levels of activity, or heterochromatin. Published genome-wide data indicate a high accuracy of location prediction by ChIP-MS. A quarter of the identified factors are important for pluripotency and includes Oct4, Esrrb, Klf5, Mycn and Dppa2, factors that drive reprogramming to pluripotent stem cells. We determined the genome-wide binding sites of Dppa2 and find that Dppa2 operates outside the classical pluripotency network. Our ChIP-MS method provides a detailed read-out of the transcriptional landscape representative of the investigated cell type. The protein factors that bind to regulatory regions in the genome have not been systematically mapped. Here the authors performed chromatin immunoprecipitations for histone modifications associated with promoters, enhancers or heterochromatin in mouse embryonic stem cells and assigned a genome location to many factors important for pluripotency. The ENCODE project has generated a wealth of data on the genome-wide chromatin landscape of many different mouse and human cell types1345A mammalian genome supports the generation of the hundreds of different cell types in an organism. These cell types display distinct gene expression profiles as a direct consequence of differences in the activation state of their gene promoters and distal Our ChIP-MS experiments identified 239 factors that we could predict to bind to promoters, enhancers or heterochromatin. Among these factors are subunits of several chromatin-modifying complexes and proteins that play a role in different aspects of transcriptional regulation. We also find key ESC transcription factors such as Oct4, Esrrb, Dppa2 and Klf5 that are not only important for maintaining ESC self-renewal but also facilitate the reprogramming of somatic cells to induced pluripotent stem cells (iPSCs)478For many of the detected factors, the genome-wide localization has not yet been determined and our ChIP-MS results provide the first evidence of their binding preference for a particular type of regulatory DNA. To illustrate that ChIP-MS can identify factors with an interesting genome-wide location, we determined the genome-wide binding sites of pluripotency marker and reprogramming factor Dppa2. We show that Dppa2 is not part of the classical pluripotency transcriptional network and that Dppa2 target genes reach full activation only later in development.Transcriptional enhancers and promoters can be recognized by the chemical modifications of their associated histones, especially histone H3. Promoters of transcribed genes were found to contain histone H3 tri-methylated at lysine 4 (H3K4me3)101215We anticipated that ChIP for H3K4me3 would precipitate active promoters and ChIP for H3K4me1 would precipitate enhancers. ChIP for H3K27ac would preferentially precipitate the most active promoters and enhancers, whereas ChIP for H3K9me3 would precipitate heterochromatin. Accordingly, we performed large-scale ChIPs in biological duplicate for H3K4me3, H3K4me1, H3K27ac or H3K9me3, and for green fluorescent protein (GFP) as a control, in mouse ESCs . Crossli181920Tcfcp2l1  and mapped to the genome. DNA precipitated by modified ChIP correlated well with the corresponding published conventional ChIP-seq for all four histone modifications . ModifieTcfcp2l1  and Nano1  score, a measure for the amount of protein presentper se. Small emPAI values for H3K4me3 or H3K4me1 increase the uncertainty of the H3K27ac ratio value. We used the H3K27ac ratio as a predictor for the level of H3K27ac, and thereby the activity, of the promoters or enhancers bound by the factor . Dppa2 is a member of family that also contains Dppa3 (Stella) and Dppa4, which all harbour a SAP DNA-binding domain. The genome-wide binding sites of members of this family have not been determined so far. Dppa2 is exclusively expressed in the inner cell mass of the early embryo and later in the developing germ line and in cells derived from these tissues, such as ESCs and primordial germ cellsd Nkx2–5 , the onld Nkx2–5  is lowerd Nkx2–5 . Subsequd Nkx2–5 . Interesd Nkx2–5 , a bindid Nkx2–5 . These aAs expected, Dppa2 binds the promoters of genes with a lower median expression than other H3K4me3-marked promoters and Oct4-bound genes in ESCs . The genWe describe here ChIP-MS, a method to predict the binding of factors to enhancers or promoters. Our experimental setup is straightforward and does not rely on metabolic labelling for quantification by MS. Nevertheless, using emPAI1920in vitro assembled modified nucleosomesA number of studies have been performed to screen for protein factors that bind to individual histone modifications by using modified histone peptides3738The list of proteins for which we predict genome localization by ChIP-MS  contains34per se, as it compensates for the considerable differences in ChIP-MS detection levels for different factors. Perhaps the clearest indication that the H3K27ac ratio performs well in differentiating factors by the activity of their bound regions is that out of the nearly 240 factors detected by ChIP-MS, 3 members of the family of BET proteins, including Brd4, are among the proteins with the highest H3K27ac ratios. Brd4 was recently identified as a functional component and marker of ‘super enhancers'25We performed our ChIP-MS experiments in ESCs. The establishment and maintenance of pluripotency, as well as the exit from pluripotency, is intensely studied in ESCs and several large data sets of relevant factors for the above processes are available. We find that a quarter (63 factors) of the ChIP-MS-detected factors contributes to maintaining pluripotency. ChIP-MS detected Oct4, Esrrb, Klf5, Dppa2 and Mycn, factors which, as part of a 3–4 factor mix, reprogramme somatic cells to iPSCs47We found that Dppa2 had a different ChIP-MS profile compared with other pluripotency factors and accordingly we determined its genome-wide binding sites by ChIP-seq. Indeed, Dppa2 turned out to be an unusual pluripotency factor. Dppa2-binding sites and target genes do not overlap with Oct4, suggesting that Dppa2 is not part of the classical pluripotency circuit. Furthermore, Dppa2 target genes were found to be much lower expressed than Oct4 target genes in ESCs and higher expressed later in development. It is an intriguing question how Dppa2 can be an early marker and factor for reprogramming to iPSCs, as was recently shownIn conclusion, we established here a method to annotate factors to enhancers and promoters with different activities. ChIP-MS is straightforward in its setup, which should facilitate its application to other cell types and growth conditions, provided sufficient cell quantities can be obtained. We showed that ChIP-MS data add to our knowledge and understanding of the transcriptional circuitry that determines cell identity.−1 puromycin (Sigma) and stable expression of V5-tagged Dppa2 tested by western blot analysis with anti-V5 antibody .Mouse embryonic stem cell line CGR8 was grown on gelatin-coated dishes without feeders in Glasgow Minimum Essential Medium (GMEM) supplemented with leukemia inhibitory factor (LIF), 15% fetal bovine serum, 0.25% sodium bicarbonate, 1 mM glutamine, 1 mM sodium pyruvate, non-essential amino acids, 50μM β-mercaptoethanol and penicillin/streptomycin, as previously described6 ESCs were used. For chromatin preparation, cells were washed on plate three times with PBS and incubated with 2 mM DSG (Thermo Scientific) in PBS for 45 min at room temperature. Subsequently, ESCs were washed in PBS three times, 0.1 volume of 11% formaldehyde (Merck) in 50 mM HEPES-KOH (pH 7.5), 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA was added, mixed and incubated for 12 min at room temperature, washed two times in 4 °C PBS and collected by centrifugation. All subsequent steps were performed on ice with pre-cooled buffers. Cell lysis was performed as described6 ESCs yielded approximately 10 mg chromatin (as measured by DNA content).For each histone modification ChIP, 300 × 10−1 BSA (New England Biolabs) and 0.2 mg ml−1 sonicated salmon sperm DNA (Stratagene) for 1 h. The antibody–bead mixture was rotated overnight with ∼10 mg chromatin at 4 °C. Beads were transferred to 1.5 ml no stick tubes (Alpha laboratories) and washed five times for 5 min in RIPA buffer (50 mM HEPES-KOH (pH 7.6), 500 mM LiCl, 1 mM EDTA, 1% NP-40, 0.7% Na-deoxycholate). After washing, the beads were boiled for 35 min at 95 °C in 2 × SDS sample buffer (100 mM Tris-HCl (pH 6.8), 200 mM DTT, 4% SDS, 20% Glycerol, 0.2% Bromophenol blue) and supernatant was transferred to a fresh tube. ChIP-MS samples were run on 10% precast SDS–PAGE gels (NuPage Invitrogen) and stained with colloidal Coomassie stain (Invitrogen). Gel lanes were sliced, in-gel digested with trypsine to yield peptides and proteins identified by analyses on an LQT-Orbitrap mass spectrometer (Thermo), as describedhttp://www.nature.com/protocolexchange/. For western blot analyses, ChIP samples were separated on a 4–12% polyacrylamide gel (Novex) and nitrocellulose blots probed with antibodies against the used histone modifications  and Nanog . Non-cropped versions of the western blot panels in Antibodies used in the different histone modification ChIPs or GFP control ChIP are against H3K4me1 , H3K4me3 , H3K27Ac , H3K9me3  and GFP . To prevent immunoglobin elution and subsequent interference with the MS analysis, 50 μg antibodies were crosslinked to 500 μl Protein A magnetic bead solution  with Dimethyl Pimelimidate (Sigma). Crosslinked antibody–bead complexes were equilibrated in LB3 buffer and subsequently blocked with 0.5 mg mlP<0.05) in the ChIP-MS samples. Significance thresholds were H3K4me3 (Experiment 1); Mascot score 28, H3K4me3 (Experiment 2); Mascot score 28, H3K4me1 (Experiment 1); Mascot score 28, H3K4me1 (Experiment 2); Mascot score 28, H3K27ac (Experiment 1); Mascot score 28, H3K27ac (Experiment 2); Mascot score 28, H3K9me3 (Experiment 1); Mascot score 28, H3K9me3 (Experiment 2); Mascot score 29, GFP (Experiment 1); Mascot score 28, GFP (Experiment 2); Mascot score 28. For inclusion into the ChIP-MS list  within 1 kb of a transcription start site (TSS). Enhancers were defined as the regions from −1 to +1 kb of the summits of a significant P300-binding sites that were not within 1 kb of a TSS. The sequencing profiles of the conventional ChIP and modified ChIP for the used histone modifications and the Dppa2 ChIP-seq experiments  for all bins in all data sets. The input was subtracted. For each histone modification and protein factor, we selected the 4,000 bins with the highest RPM. A unified list was created for each individual protein factor, containing the selected bins of the four used histone modifications and that of the protein factor itself. The Spearman correlation coefficients of the protein factor with the different histone modifications were calculated from this list. To calculate the correlations between conventional and modified ChIP-seq experiments for the used histone modifications, we used a unified list that included the 4,000 bins with the highest RPM for each conventional and modified ChIP-seq experiment.To assign promoter regions, H3K4me3 peak summits, as determined by MACS (see above) were required to be within 1 kb range of a TSS, resulting in 12,913 promoter regions. To assign enhancer regions, H3K4me1 peak summits were filtered against the presence of H3K4me3 signal in a region from −4.1 and+4.1 kb around the H3K4me1 peak summit, resulting in 30,564 enhancer regions. Promoters were sorted for number of H3K27ac reads present in the central 2 kb of the promoter region. Enhancers were sorted for the number of H3K27ac reads in the central 8.2 kb of the enhancer region, to also include broad enhancers. For both promoters and enhancers, we displayed a region from −4.1 to +4.1 kb around the peak summits, divided into 51 bins of 160 bp each. Promoter and enhancer heatmaps for each protein factor or histone modification were normalized by calculating the RPM based on the sum of all reads found in the displayed promoter and enhancer region for that factor.P-value of ≤0.10, in a Dppa2 knockout microarray data setP-value for each probe. Multiple probes to the same gene were aggregated by taking the average fold change. Oct4-bound genes contained a MACS-called Oct4 peak (see above) within 20 kb from the TSS. Oct4 target genes were defined as Oct4-bound genes with at least twofold difference in expression between 24 and 0 h after Oct4 knockdownDppa2-bound genes contained a MACS-called Dppa2 peak (see above) within 1 kb from the TSS. Dppa2 target genes were defined as Dppa2-bound genes with at least twofold difference in expression in Dppa2 knockout ESCs, compared with wild-type ESCs and an adjusted Accession codes: ChIP sequencing data are available through the Gene Expression Omnibus (NCBI), accession code GSE58113.How to cite this article: Engelen, E. et al. Proteins that bind regulatory regions identified by histone modification chromatin immunoprecipitations and mass spectrometry. Nat. Commun. 6:7155 doi: 10.1038/ncomms8155 (2015).Supplementary Figures 1-3, Supplementary Tables 1-5 and Supplementary References.Dppa2-bound transcription start sites in mouse embryonic stem cells


Saccharomyces cerevisiae. We found that eRF3 from Pneumocystis carinii (Pc-eRF3) did not complement depletion of S. cerevisiae eRF3. The strength of Pc-eRF3 binding to Sc-eRF1 depends on the GTP-binding domain, suggesting that defects of the GTPase switch in the heterologous complex causes the observed lethality. We isolated mutants of Pc-eRF3 and Sc-eRF1 that restore cell growth in the presence of Pc-eRF3 as the sole source of eRF3. Mapping of these mutations onto the latest 3D-complex structure revealed that they were located in the binding-interface region between eRF1 and eRF3, as well as in the ribosomal functional sites. Intriguingly, a novel functional site was revealed adjacent to the decoding site of eRF1, on the tip domain that mimics the tRNA anticodon loop. This novel domain likely participates in codon recognition, coupled with the GTPase function.During termination of translation in eukaryotes, a GTP-binding protein, eRF3, functions within a complex with the tRNA-mimicking protein, eRF1, to decode stop codons. It remains unclear how the tRNA-mimicking protein co-operates with the GTPase and with the functional sites on the ribosome. In order to elucidate the molecular characteristics of tRNA-mimicking proteins involved in stop codon decoding, we have devised a heterologous genetic system in  Domain N is primarily responsible for codon recognition, and the amino acid motifs TASNIKS and Y-C-F within domain N have been reported to be important for codon recognition  and the C-terminal region  have been solved . This haAeropyrum pernix has been shown to play versatile roles in multiple translational steps, such as elongation and termination, and also in mRNA quality control (S. cerevisiae)  from  control , and canevisiae) ,25. FurtOn the other hand, X-ray crystal structures of archaeal aEF1α•aPelota•GTP  and aEF1The X-ray crystal structure of the 70S ribosome•EF-Tu•tRNA complex  and cryoPneumocystis carinii  cannot replace endogenous eRF3 in S. cerevisiae, despite earlier reports that mammalian eRF3 orthologs can replace that of S. cerevisiae  strain was constructed as per the method described previously (SUP45 (eRF1) gene promoter of the tet-OFF eRF3 strain with the tet-OFF promoter along with the kanMX4 selection marker. Manipulation of yeast and plasmid DNA was performed according to standard procedures  at the N-terminal, via XbaI/BamHI sites.Plasmids and primers used in this study are listed in Supplementary Tables S2 and S3, respectively. Full-length Pc-eRF3, Pc-eRF3c and Δ1 DNA fragments were amplified by polymerase chain reaction (PCR) from a Pc-eRF3 cDNA clone (DDBJ AB052894)  using prThe tet-OFF eRF3 strain was transformed with eRF3 expression plasmid vectors based on p416GPD, and the growth of transformants was monitored on SC–Ura plates containing 7.5 μg/ml doxycycline. The double tet-OFF eRF1/eRF3 strain was co-transformed with the wild type or the mutant vector plasmids of Sc-eRF1 in p414GPD, as well as with eRF3s in p416GPD, and the growth of transformants was monitored on SC–Ura–Trp plates containing 7.5 μg/ml doxycycline.Protein expression levels of Pc-eRF3 variants in the tet-OFF eRF3 strain was monitored by western blot analysis using an anti-FLAG antibody, Monoclonal ANTI-FLAG M2 (F3165)  against the cell extracts from p416GPD-FLAG-Pc-eRF3s transformants. Expression levels of eRF3 variants, fused to the GAL4 binding domain via the yeast two-hybrid analysis vector, pGBT9, were monitored using an anti-GAL4BD antibody, GAL4 antibody (DBD) (sc-577)  against the cell lysates from AH109 transformants, as follows. Transformants were grown in the appropriate liquid media to mid-log phase. The yeast cells were collected and precipitated with 10% trichloroacetic acid, suspended in sodium dodecyl sulphate (SDS) sample buffer, neutralized with 5 N NaOH solution, and vigorously mixed with glass beads on the FastPrep 24 instrument . Proteins in the cell extracts were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene difluoride (PVDF) membranes and detected using the appropriate antibodies and an ECL Western Blotting Detection System .Wild-type or mutant eRF1 and eRF3 DNA fragments were cloned in-frame into the activation domain vector pGAD424 and the DNA-binding domain vector pGBT9, respectively, for expression as fusion proteins. The AH109 yeast two-hybrid reporter strain was transformed with both plasmids and transformants were then examined for growth on SC–Leu–Trp–His plates .The p416GPD-Pc-eRF3c DNA was mutagenized by incubation with 0.4 M hydroxylamine at pH 6.0 for 20 h at 37°C, or by the error-prone PCR method (The p414GPD-Sc-eRF1 DNA was mutagenized by the error-prone PCR method. The mutagenized plasmid mixture was introduced into the double tet-OFF strain (Y138) together with p416-Pc-eRF3c. Then, the strain was incubated on SC−Ura−Trp plates containing 7.5 μg/ml doxycycline. Plasmids from viable colonies were isolated and sequenced. These selections were repeated until redundancy of the mutated positions among the singly or multiply mutated genes was observed.et al.  background and chromosomally integrated dual-luciferase reporter, was subsequently developed (gst1–1) background were constructed here. Assay strains harboring wild-type or mutant eRF expression plasmids were liquid-cultured in plasmid-selection medium at a permissive temperature (30°C), up to mid-log phase, and were then transferred to a non-permissive temperature, i.e. 37°C, for several hours, as indicated in the Results section. The cells were harvested, suspended in sample buffer and cell lysates were then prepared by vigorous shaking in the presence of glass beads using a FastPrep 24 instrument . Then, the lysates were applied to a dual-luciferase assay system according to the manufacturer's instructions , using a GloMax™ 96 Microplate Luminometer (Promega). Percent read-through was determined by the ratio of the firefly/renilla luciferase measurement of stop codons to that of the UGG sense codon-containing strain.The dual-luciferase-based translational read-through assay system was first reported by Grentzmann et al. , and, baeveloped . SimilarP. carinii (Pc-eRF3) contains a less-conserved N-terminal region (SUP35) defective yeast cell growth complementation . In contrast, the control transformants expressing the N-terminally truncated minimal wild type yeast eRF3 (hereafter designated as Sc-eRF3c), either under control of the GPD promoter (H), ‘H’ for high expression, or the weak CYC promoter (L), ‘L’ for low expression, complemented the defective growth  and the ribosome during translation termination.The FLAG-tagged variants of Pc-eRF3s as well as Sc-eRF3c were detected in the cell lysates by western blotting, revealing that the expression levels of Pc-eRF3s in the assay strains are sufficiently comparable to that of low expression Sc-eRF3c (L), which complements cell growth of Y40 Figure . Unlike The binding between eRF1 and eRF3 has been extensively studied as a unique feature of eukaryotic translation termination factors and has recently been shown to be common in archaea; in contrast, class I and the class II release factors do not exhibit any direct binding in prokaryotes. Structural studies have revealed that class II release factors bind to class I release factors through contact sites located in three distinctive structural domains of eRF3, domains 1, 2 and 3 ,24; thisConsidering that defective binding to eRF1 could underlie the result of the yeast complementation test, binding between Pc-eRF3 and Sc-RF1 was examined by yeast two-hybrid analysis. Pc-eRF3c as well as its Δdomain1 variant (Δ1), fused to GAL4BD in pGBT9, and Sc-eRF1, fused to the GAL4-activation domain (GAL4AD) in pGAD424, were co-introduced into the AH109 reporter strain and its growth was then evaluated on assay plates lacking histidine. Consequently, Sc-eRF1 was shown to bind to Pc-eRF3c and to Sc-eRF3c equally well Figure . HoweverRecent high-resolution X-ray structural analyses of the archaeal aRF1•aEF1α•GTP complex (and of its homolog), which should be functionally equivalent to the eRF1•eRF3•GTP complex in eukaryotes, clearly demonstrated the involvement of domain 1 in ternary complex formation, suggesting exact structural and functional mimicry between eRF1•eRF3•GTP and tRNA•EF1α•GTP complexes ,26. AccoPc-eRF3c in the p416GPD vector was randomly mutagenized by hydroxylamine treatment or by error-prone PCR, to obtain Pc-eRF3c mutant libraries. The libraries were introduced into conditional lethal eRF3 yeast strains, either YK21–02 (eRF3ts) or Y40 (tet-OFF eRF3), and viable transformants were selected under restrictive conditions to obtain Pc-eRF3c mutations that restore cell growth in presence of Pc-eRF3 as the sole source of eRF3. Consequently, 12 independent missense mutations were isolated involving 11 amino acid positions located throughout the 3 domains of Pc-eRF3c Table  and 6A. wt) and mutant Pc-eRF3cs in the cell were examined using stop codon read-through assays. The dual-luciferase constructs containing the UGA stop codon or the UGG sense codon between two different luciferase genes, encoding renilla and firefly luciferase, were stably integrated into the chromosome of the temperature-sensitive eRF3 (eRF3ts) strain . The assay strains transformed with each of the eRF3 expression vectors were liquid-cultured, transferred to a restrictive temperature (37°C), and after 4 h, the luciferase activity was measured to calculate the read-through efficiency. Read-through efficiency was shown as a ratio to the control strain containing a UGG (Trp) codon, instead of the UGA codon. As expected from the growth complementation assay, the Pc-eRF3cwt exhibited high read-through efficiency compared with wild-type Sc-eRF3c, i.e. is severely defective in translation termination, and showed only slight decrease in read-through efficiency compared with the control vector. On the other hand, most of the Pc-eRF3c mutations exhibited a significant decrease in read-through . The reduced binding of Pc-eRF3 E260K and T538I mutants to Sc-eRF1 accounts for the inefficient recovery of translation termination activity mentioned above, since inactivated eRF3ts proteins at 37°C, in the read-through assay strains, more efficiently compete with these Pc-eRF3 mutations for Sc-eRF1 binding. The better growth results for Pc-eRF3 transformants harboring E260K or T538I in the tet-OFF eRF3 strain, as compared to those of eRF3ts strains, are consistent with this idea  as the sole source of eRF3 in S. cerevisiae, in order to elucidate the co-operativity of the eRF1•eRF3 complex from the eRF1 perspective. To this end, we have constructed an assay strain named ‘double tet-OFF’ (Y138) in which the endogenous promoters of eRF1 and eRF3 genes on chromosomes were replaced with the tet-OFF promoter and the expression of endogenous eRF1 in addition to eRF3 can be repressed by addition of tetracycline . Thus, only successful combinations of heterologous or homogeneous release factors, eRF1 and eRF3, introduced via plasmids, can support the growth of such a strain under non-permissive conditions.Taking a reverse approach, we then attempted to isolate Sc-eRF1 mutations that restore cell growth in the presence of wild-type Pc-eRF3c  and cell growth was then monitored under non-permissive conditions (7.5 μg/ml doxycycline). Consistently, a homogeneous Sc-eRF pair, as well as Sc-eRF1 paired with the Pc-eRF3c mutations, suppressed cell growth, while a combination of Pc-eRF3cwt were isolated. The randomly mutagenized Sc-eRF1 library and the Pc-eRF3cwt plasmid were co-introduced into the double tet-OFF strain and the mutant Sc-eRF1 plasmids were isolated from transformants that were viable in the presence of doxycycline. Consequently, 28 independent mutations were isolated in total  on the contact regions of domain C are reported to be crucial for binding as well as for translation termination  and wild-type Sc-eRF3 (+SUP35) background  that are known to be necessary for GTPase activity ,,50,E. coy Figure .A series of mutations at T538 of Pc-eRF3 and at I291, G394, A395, F401 and G403 of Sc-eRF1 are located in the classical site 1 interface region, which is composed of domain C of eRF1 and domain 3 of eRF3 . ConsistIn addition to the site 1 and site 2 mutations, mutations in the linker helices, E266G, K276E and K282E, and mutations in the adjacent domain, N161D, N161I, N161Y and E385K, were isolated Figure  and D. AS. cerevisiae, i.e. with endogenous Sc-eRF3, in exchange for the suppression of Pc-eRF3 lethality. This suggests, at least in part, that the mutated Sc-eRF1/Pc-eRF3 complex might have bypassed its harmful effects by adjusting their binding configuration to be suitable for the subsequent reactions, such as codon recognition and catalysis of polypeptide chain release as well as GTP hydrolysis, on the ribosome. In vitro analyses of the elementary processes in translation termination should be performed to confirm this.Pc-eRF3 mutations E260K and T538I as well as Sc-eRF1 mutations N161I, D228N, D228V, G394S, F401S and G403D exhibited reduced binding activity in yeast two-hybrid assays. These results indicated that formation of a tight complex between eRF1 and eRF3 through the binding sites, ‘site 1’  and ‘site 2’ (between domain M of eRF1 and domain 1 of eRF3), was not prerequisite but rather harmful, suggesting that moderate binding might ensure efficiency in later steps of translation termination by eRFs on the ribosome. Furthermore, unexpectedly, the Sc-eRF1 mutations N167R and F401S conferred weaker translation termination activity in Mutations in domain 1 of eRF3, E247, E299, T320 and R329, are distinguished from the contact areas for binding Figure  as seen in vitro, in the absence of eRF3  at the acceptor-arm/D-stem junction  through the H167 region, although this should be examined further in future.As can be seen in the superimposed model Figure , among tjunction , as welljunction . Thus, aSince we have shown that, as compared to endogenous binding of eRFs, the binding of Sc-eRF1 to exogenous Pc-eRF3c is more dependent on the secondary binding site, although it also depends on the classical binding site, the defect(s) of Pc-eRF3 in yeast could be caused by an imbalance between the contributions of the two major interaction sites located at the interface with Sc-eRF1. Many of the mutations in both eRFs that suppress the defect(s) of Pc-eRF3 in yeast affected conserved amino acid residues , and many of these mutations did not necessarily reside within interaction sites, but were located nearby . Thus, the mutations seem to induce the conformational change necessary for GTPase activation, adjusting eRF1/eRF3 interaction in their vicinity; this links stop codon recognition to the GTPase switch.eRF1 ASL-like), has been intensively studied in order to clarify the motifs and mechanisms involved in stop codon recognition and discrimination. Almost all of the known, well-studied, mutations that affect the specificity of stop codon recognition are localized on the side of the eRF1-ASL that would consistently face the stop codon bases in the ribosomal A site, as seen in the docking model structure of the aEF1α•aRF1•GTP complexed with the ribosome, as well as in the latest cryo-EM studies of the eRF3•eRF1 dimer complexed with the ribosome. However, the novel domain N mutations identified in this study clustered in a locus opposite to that previously noted . TA. TACys•Ge Figure ,46,56, ae Figure ,57,58 ree Figure . IntriguCys•GDPNP complex is conformationally distorted upon binding to the ribosomal A-site, and that this allows the whole complex to make precise contact at multiple functional sites in the large and small ribosomal subunits during decoding , a series of charge-flip mutations of the relatively conserved residues that cluster within the ASL domains, adjacent to the decoding tripeptide motifs  were analyzed in our previous studies ,65. The utations ,65. It hutations . Interesin vitro studies using all purified components are necessary to test our hypothesis and underlying molecular mechanisms in detail, our results have shed light on the novel aspects of tRNA-mimicking protein complexes.By genetic analyses, using a heterologous eRF3, this study revealed clues for novel aspects of the manner in which the eRF1/eRF3 complex mimics the tRNA/EF1α complex. It is likely that, due to their tRNA-mimicking strategy adopted in evolution, the eukaryotic translation termination complex eRF1 and eRF3 derived from heterologous species in previous reports exhibited high complementarity ,38,44. HSupplementary Data are available at NAR Online.


Plasmodium falciparum, a causative agent of human malaria, the nucleosome positioning profile of regulatory regions deserves particular attention because of their extreme AT-content. With the aid of a highly controlled MNase-seq procedure we reveal how positioning of nucleosomes provides a structural and regulatory framework to the transcriptional unit by demarcating landmark sites (transcription/translation start and end sites). In addition, our analysis provides strong indications for the function of positioned nucleosomes in splice site recognition. Transcription start sites (TSSs) are bordered by a small nucleosome-depleted region, but lack the stereotypic downstream nucleosome arrays, highlighting a key difference in chromatin organization compared to model organisms. Furthermore, we observe transcription-coupled eviction of nucleosomes on strong TSSs during intraerythrocytic development and demonstrate that nucleosome positioning and dynamics can be predictive for the functionality of regulatory DNA elements. Collectively, the strong nucleosome positioning over splice sites and surrounding putative transcription factor binding sites highlights the regulatory capacity of the nucleosome landscape in this deadly human pathogen.In eukaryotes, the chromatin architecture has a pivotal role in regulating all DNA-associated processes and it is central to the control of gene expression. For  Nucleosome occupancy refers to the proportion of cells in which a given region of DNA is occupied by a nucleosome, as well as the density of nucleosomes over this region . In contin vivo nucleosome landscape can also be observable in in vitro reconstituted chromatin, suggesting that the DNA sequence provides a basic blueprint for the nucleosome landscape . Mounting evidence suggests that epigenetic regulatory mechanisms are critical for parasite survival and could serve as potential antimalarial drug targets . In pariewed in ,11. Thesiewed in ,13 and ciewed in ,15. In ciewed in –18. ‘Actiewed in ,19–20. AP. falciparum is extraordinary in that it harbours the highest proportion of adenine and thymine bases of all genomes sequenced to date. The average genome-wide AT-content is 81%, often reaching 90–95% in intergenic regions; this is greater than that of those described for Dictyostelium discoideum and Tetrahymena thermophila, which have an average AT-content of 78%. Such extreme base composition challenges existing concepts on chromatin organization, which would predict that highly AT-rich sequences form rigid structures and are therefore refractory to nucleosome formation. However, the chromatin landscape of Dictyostelium, despite its high AT-content, was found to largely reflect the nucleosome structure of multicellular organisms  withre S3 in . In contlciparum ,23–24. Tlciparum ,23–24 coP. falciparum genome. With this optimized protocol, we generated high-resolution nucleosome positioning profiles at eight stages of the P. falciparum intraerythrocytic development cycle. We observed clear positioning of nucleosomes in regulatory regions and around transcriptional landmark sites . Intriguingly, we find dynamic, local depletion of nucleosome occupancy on active TSS and reveal positioning of nucleosomes around ApiAP2 transcription factor binding sites. In combination with matched, strand-specific RNA-seq data, our MNase-seq profiles portray the static and dynamic components of the nucleosome landscape of P. falciparum regulatory DNA sequences.Therefore, we extensively optimized the MNase-seq protocol to meet the challenges of the Var2csa-selected 3D7 P. falciparum blood-stage parasites were cultured under standard conditions and synchronized as in . Nuclei were mildly sonicated to free cross-linked nucleosomes from nuclear membranes. Soluble chromatin was collected and de-cross-linked directly (MNase-Seq) or used for chromatin immunoprecipitation , whereas insoluble debris was de-cross-linked as pellet control . Since our linear amplification protocol  was dif+ mRNA, RNA fragmentation and cDNA synthesis were performed as described elsewhere . To identify sites displaying local dynamics in nucleosome occupancy we applied a minimal coverage filter and used an auto-correlation approach to reduce noise . (Positioned) nucleosomes were called using the DANPOS software  . Data processing and analysis was done with BioPerl (Version 1.6.9) , BioRubyet al.  were usenor = F) .P. falciparum nuclei were collected as in . The TSS data is available via European Nucleotide Archive (ERP010191).The Gene Expression Omnibus accession number for the MNase-seq, associated controls and strand-specific RNA-seq data reported in this paper is GSE66185. This data is also available at PlasmoDB . After formaldehyde-crosslinking, chromatin was digested by a combination of micrococcal nuclease (MNase) and exonuclease III in order to reduce enzyme-mediated digestion-bias , mono- (125–174 bp), supra- (175–199 bp), inter- (200–259 bp) and di-nucleosomal (260–339 bp) size-classes and generated genome-wide coverage plots corrected using insert-size-matched gDNA controls Figure , no appa4 bp, monTo further investigate the localization of positioned nucleosomes we use the software DANPOS  to definP. falciparum MNase digested DNA in coding, but not in intergenic sequences   Figure . This miP. falciparum nucleosomes despite the extremely AT-richness of the genome.In conclusion, by analysing sub- and mono-nucleosomal MNase-seq fragments we detect translational positioning of nucleosomes primarily in 5′ regulatory regions and in multi-exon genes. Furthermore, we found that 10bp AA/TT-dinucleotide periodicity-driven rotational positioning is a common feature of all In order to investigate the involvement of the nucleosome landscape in demarcating the transcriptional unit we visualized individual profiles of nucleosome occupancy around transcriptional landmark sites in heatmaps and plotted average profiles in line graphs Figure ) is alsoP. falciparum, TSS were previously identified based on a full-length cDNA dataset . Therefore, we also investigated the nucleosome landscape in 620 promoter regions, which contain at least two prominent TSSs . The second TSS tends to have a similar, though less pronounced, chromatin organization as the primary TSS (a well-positioned nucleosome preceded by a NDR), which is most prominent when the distance between the two TSSs is about 200–300 bp. Therefore, promoter regions with structured NDR—nucleosome arrays might be more likely to give rise to multiple TSSs .In  dataset . While t dataset . Additio dataset , appear When assessing the nucleosome landscape of transcription termination sites (TTS) , we agaiInterestingly, we observe the clearest positioning pattern around splice acceptor and donor sites, which often are enclosed by small arrays of positioned nucleosomes Figure . Such poP. falciparum genome is rather compact and therefore many of these landmark features localize closely to each other. To investigate the possibility that localization of nucleosomes at any given site is not an intrinsic feature of the region, but rather the consequence of nucleosome positioning at neighbouring sites we sorted the MNase-seq profiles based on the distance to the nearest landmark site -binding motifs. We selected three DNA motifs  for which multiple lines of evidence suggest that they might be bound by DBPs ,45. Amonelopment . Therefoelopment  and D. N-binding elopment ) as wellt Figure . ConsequP. falciparum genome at eight time-points of intraerythrocytic development. Our aims were two-fold: to understand how chromatin is organized on this AT-rich genome and to explore the relevance of the nucleosome landscape in guiding the transcriptional process. However, before we could reliably analyse the nucleosome landscape of P. falciparum, we had to overcome several methodological challenges. Given the intrinsic preference of MNase to cleave next to A or T nucleotides, preferential degradation of AT-rich nucleosomal DNA has been reported , but introns are on average only 180 bp in length. Accordingly, in case of very short introns nucleosomes are mainly positioned over the GC-richer exons leaving the introns largely nucleosome-free. While in case of longer introns an intronic nucleosome can ‘sit in the AT-richer pocket’ between two exons  can give rise to arrays of positioned nucleosomes. Interestingly, it was recently elegantly demonstrated that the well-positioned +1 nucleosome in D. melanogaster slows down RNA Pol II progression (Nucleosome positioning in orted by ). Howeveorted by . Nucleosorted by ,49. In Che exons . Similarm Figure  the posim Figure  and consm Figure  in warm-s Figure . Both ofs Figure , could egression . ConsequP. falciparum genes we made use of a 5′ end enriched RNA-seq dataset to map the most prominent start site for about half of the P. falciparum genes. Analyses of these sites substantially clarified the nucleosome organization around the TSS and revealed a NDR as its most prominent feature  demonstin vivo, (iii) the observed nucleosome dynamics are not the consequence of differential TF-binding, but of recruitment of chromatin remodelling complexes at later stages of development. To investigate the second possibility we performed DNA pulldown experiments using DNA probes resembling the genomic sequence of two 5′ intergenic region localized TGCATGCA motifs that display nucleosome landscape dynamics during asexual development. Indeed we identify a second AP2 protein, PF3D7_1107800, binding this motif in both regions. As this AP2s steady-state RNA abundance peaks around T35 (Figure P. falciparum. An exciting area of research that awaits further investigation is to determine for how many other DNA motifs the nucleosome landscape will be able to distinguish between functional and inert elements.The nucleosome landscapes obtained from differently staged asexual parasites are largely similar Figure , but dis5 Figure , it seemP. falciparum genome. It revealed nucleosome positioning primarily on 5′ intergenic regions as well as splice donor and acceptor sites and the presence of a local NDR directly upstream of the TSS which is more pronounced for highly expressed genes. Dynamic, local nucleosome depletion during asexual blood-stage development is observed over TSS and correlates to temporal gene expression. Furthermore, the highly structured and dynamic nucleosome landscape over 5′ intergenic region—but not CDS—localized TGCATGCA motifs aided the identification of a second ApiAP2 transcription factor putatively binding this motif in vivo. In summary our study revealed how positioning of nucleosomes provides a structural and regulatory framework to the transcriptional process. Now, the challenge ahead lies in identification of parasite-specific DBPs, chaperones and/or chromatin remodellers that together shape this landscape and could function as potential anti-malarial drug targets.All-in-all our adapted MNase-seq procedure allowed for the first time accurate characterization of the nucleosome landscape of the highly AT-rich regulatory regions in the


SET and MYND domain containing protein 3 (SMYD3) is a histone methyltransferase, which has been implicated in cell growth and cancer pathogenesis. Increasing evidence suggests that SMYD3 can influence distinct oncogenic processes by acting as a gene-specific transcriptional regulator. However, the mechanistic aspects of SMYD3 transactivation and whether SMYD3 acts in concert with other transcription modulators remain unclear. Here, we show that SMYD3 interacts with the human positive coactivator 4 (PC4) and that such interaction potentiates a group of genes whose expression is linked to cell proliferation and invasion. SMYD3 cooperates functionally with PC4, because PC4 depletion results in the loss of SMYD3-mediated H3K4me3 and target gene expression. Individual depletion of SMYD3 and PC4 diminishes the recruitment of both SMYD3 and PC4, indicating that SMYD3 and PC4 localize at target genes in a mutually dependent manner. Artificial tethering of a SMYD3 mutant incapable of binding to its cognate elements and interacting with PC4 to target genes is sufficient for achieving an active transcriptional state in SMYD3-deficient cells. These observations suggest that PC4 contributes to SMYD3-mediated transactivation primarily by stabilizing SMYD3 occupancy at target genes. Together, these studies define expanded roles for SMYD3 and PC4 in gene regulation and provide an unprecedented documentation of their cooperative functions in stimulating oncogenic transcription. SET and MYND domain-containing proteins (SMYD) are a special class of protein lysine methyltransferases involved in methylation of histones and non-histone proteins –3. In huThe human positive coactivator 4 (PC4) is a multifunctional protein which plays a regulatory role in diverse cellular processes, including RNA polymerase II transcription, replication, heterochromatinization and DNA repair –18. In tIn this study, we employed a combination of gene expression profiling, transcription assays, ChIP-qPCR and CRISPR/dCas9 system to investigate a possible functional interaction between SMYD3 and PC4. Our data show that SMYD3 colocalizes with PC4 at genes regulating cell proliferation and invasion and establishes transcriptional competence in bladder and colon cancer cells. SMYD3 and PC4 execute transcriptional programs in a cooperative manner, as shRNA-mediated knockdown of either SMYD3 or PC4 inhibits target gene transcription and cell proliferation/invasion. Furthermore, CRISPR/dCas9-based tethering of SMYD3 mutant deficient in PC4 interaction and DNA binding to target genes is sufficient to mediate transcriptional activation, strongly suggesting that the functional contribution of PC4 to SMYD3 transactivation is mainly through the enhancement of SMYD3 occupancy at target genes.http://www.isrec.isb-sib.ch/tagger), which is a web portal to provide tools for finding short exact matches in large sequence databases . HCT116, CaCO2, J82, MCF7, MDA-MB-231, DU145 and UROtsa cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS  or 5% FBS (UROtsa cells). LNCaP and MLC cells were maintained in RPMI culture media and T medium with 10% FBS, respectively. MCF-10–2A cells were grown in a 1:1 mixture of DMEM and Ham's F12 supplemented with 20 ng/ml epidermal growth factor, 100 ng/ml cholera toxin, 0.01 mg/ml insulin, 500 ng/ml hydrocortisone and 5% horse serum. CCD-18Co cells were cultured in Eagle's Minimum Essential Medium with 10% FBS. For mammalian expression of SMYD3 and PC4, their cDNAs were amplified by PCR and ligated into the correct reading frames of lentiviral expression vector pLenti-Hygro (Addgene) containing FLAG coding sequences. To generate mutant SMYD3/PC4 expression vectors, SMYD3 and PC4 cDNAs were mutated by the QuikChange® II site-directed mutagenesis kit (Agilent Technologies) before the construction. For bacterial expression of wild-type and mutant versions of SMYD3 and PC4, the corresponding cDNAs were amplified by PCR and inserted into pGEX-4T1 and pET15b vectors. For mammalian expression of dCas9-FLAG/dCas9-FLAG-SMYD3 wild-type and mutant, the nuclease-deficient dCas9 (D10A and H840A) cDNA (NotI-dCas9-EcoRI) was PCR-amplified, and ligated into pIRES vector digested by NotI and EcoRI. Wild-type or mutant SMYD3 coding sequences were excised from a vector and cloned into the EcoRI-BamH1-digested pIRES-dCas9 vector. For sgRNA expression vectors, oligonucleotides listed in Supplementary Table S2 were annealed and ligated into BsmBI-digested pMLM3636 vector (Addgene). Targeting specificity and the extent of potential off-target activity of dCas9-FLAG-SMYD3 were analyzed by using TagScan  and PC4 mRNA coding region (5′-GAACAGATTTCTGACATTGAT-3′) were annealed and ligated into the lentiviral expression vector pLKO.1 (Addgene). Lentivirus particles were generated in 293T cells by co-transfecting plasmids encoding VSV-G, NL-BH and the shRNAs. HCT116, CaCO2, J82 and T24 cells were infected by these viruses, and were selected with 2 μg/ml puromycin (Sigma) for two weeks.www.pathogenomics.ca/arraypipe). Genes showing detection P < 0.05 with fold change >2 were functionally analyzed in the context of gene ontology by using Ingenuity pathway software . For qRT-PCR, total RNA was isolated as for microarray and subjected to reverse transcription using the iScript cDNA Synthesis Kit (Bio-Rad) and the PerfeCta SYBR Green FastMix (Quanta biosciences) as recently described  as detailed previously . Gene expression microarray experiments were conducted using a whole-genome expression array . Differential gene expression was detected by using the ArrayPipe software  (Sigma) for 2 h at 37°C. The MTT formazan was dissolved in 200 μl of DMSO. The absorbance of the solution was quantified at 570 nm against 650 nm by a microplate reader (Bio-Rad). For cell invasion assays, cells were harvested and suspended in culture medium containing 5% FBS and then seeded to the upper chamber coated with Matrigel (BD Biosciences). Cells were allowed to invade toward 10% FBS in the lower chamber for 48 h. The invaded cells on the underside of the transwell filters were fixed with 10% formaldehyde for 15 min and stained with 1% crystal violet for 1 h. Cells were photographed and counted.HCT116 cells continuously expressing FLAG-HA-SMYD3 were generated by stable transfection of pIRES-FLAG-HA-SMYD3. Nuclear extracts were prepared as described , and ect293 cells were transfected with dCas9-FLAG plasmid or plasmids expressing dCas9-FLAG-SMYD3 wild-type or mutant for 48 h, and the proteins were purified with anti-FLAG antibody. Recombinant histone octamers (1 μg) were incubated with immunoprecipitated dCas9-FLAG or dCas9-FLAG-SMYD3 for 1 h at 30°C in HMT reaction buffer  in the presence of 50 μM S-adenosyl-methionine. Histone proteins were resolved by 15% SDS-PAGE, and analyzed by immunoblotting with anti-H3K4me3 antibody.in vitro interaction studies with SMYD3, His-tagged SMYD3 wild-type and mutants were synthesized by using TNT-Quick-coupled transcription/translation system (Promega), and incubated overnight with GST-PC4 immobilized on glutathione-Sepharose beads (Amersham Biosciences) at 4°C in binding buffer . After washing the beads three times with the binding buffer, the beads were subjected to SDS-PAGE and immunoblotting with anti-His antibody. For binding assays with PC4, GST-PC4 wild-type and mutants were prepared from E. coli and incubated with His-tagged SMYD3. For co-immunoprecipitation assays, FLAG-tagged SMYD3 proteins were expressed in HCT116 cells, and whole cell lysates were prepared from the cells with lysis buffer . The cell lysates were mixed with anti-FLAG antibody-conjugated Sepharose beads (Sigma) and incubated overnight with gentile rotation at 4°C. After removing the supernatant, the sample pellets were analyzed by immunoblot analysis using anti-PARP1, anti-SPT16, anti-Nucleolin, anti-H2B and anti-PC4 antibodies. To co-immunoprecipitate endogenous SMYD3 and PC4, HCT116 cell lysates were incubated with anti-SMYD3 antibody or normal rabbit IgG for overnight. After centrifugation, immunocomplexes in the supernatants were precipitated with Protein A/G-Sepharose (Santa Cruz Biotechnology) and separated on SDS-PAGE. Immunoblot analyses were performed with anti-PC4 and anti-SMYD3 antibodies.For For in vitro binding assays, streptavidin-coated 96well plates were incubated with biotin-conjugated oligonucleotides containing three copies of SMYD3 binding sites for 2 h at room temperature. The sequences of oligonucleotides were: 5′- CGTATTCCCTCCATACTCGCGTATTCCCTCCATACTCGCGTATTCCCTCCATACTCG -3′-biotin (sense) and 5′- CGAGTATGGAGGGAATACGCGAGTATGGAGGGAATACGCGAGTATGGAGGGAATACG -3′-biotin (antisense). The plates were washed three times with wash buffer , and incubated with SMYD3 wild-type or mutant in binding buffer  for 1 h at room temperature with gentle rotation, added PC4 wild-type or mutant, and then further incubated for 1 h at room temperature. After washing with wash buffer, the plates were incubated with FLAG/His antibodies for 1 h and then horseradish peroxidase-conjugated secondary antibody for 0.5 h. TMB substrate (Pierce) was used for color development at 450 nm, and absorbance was read by a Plate Chameleon V plate reader.As SMYD3 is often overexpressed in cancer cells, we first examined SMYD3 levels in bladder, breast, colon and prostate cell lines by immunoblotting. Three bladder  and three colon  cancer cell lines expressed significantly higher levels of SMYD3 compared to their normal counterparts (UROtsa and CCD-18Co) . In contrast, higher expression of SMYD3 was not evident in breast (MCF7 and MDA-MB-231) and prostate (LNCaP and DU145) cancer cell lines compared to normal cell lines (MCF-10–2A and MLC). The observed overexpression of SMYD3 in bladder and colon cancer cell lines encourages the possibility that SMYD3 may facilitate key tumorigenic processes such as cell proliferation and invasion. To test this possibility, we depleted SMYD3 in two bladder (J82 and T24) and two colon (HCT116 and CaCO2) cancer cells expressing high levels of SMYD3. In this study, it was important that SMYD3 is depleted for prolonged periods, as this allows the study of progressive alterations of cell proliferation and invasion under identical conditions. This was achieved by using a lentiviral shRNA infection system. Immunoblot analysis confirmed that stable transfection of SMYD3 shRNA efficiently silenced the expression of SMYD3 in the cancer cells . MTT assays over a period of 4 days reproducibly showed that the bladder and colon cancer cells proliferate much more slowly following the depletion of endogenous SMYD3 . SMYD3 knockdown also led to a significant decrease in cell invasion compared with mock-depleted control cells . These observations are consistent with the hypothesis that SMYD3 is one of the key players stimulating proliferation and invasiveness of bladder and colon cancer cells.P-value cutoff of 0.005, the gene expression profiling identified 264 genes whose expression was decreased in response to SMYD3 knockdown . Representative GSEA-scoring plots and their corresponding heat maps indicating the enrichments of proliferation/invasion-related genes are shown  which were repressed after SMYD3 knockdown and contain putative SMYD3 binding elements within 300 bp upstream of the TSSs . The selected target genes are known to facilitate cell proliferation via integrin/ERK1/2 signaling . These results imply that SMYD3 is important for establishing the active H3K4me3 mark on proliferation/invasion-stimulatory genes in cancer cells.Since SMYD3 functions as a transcription regulator by binding to a specific DNA sequence (5′-CCCTCC-3′) in its target genes, we searched for this SMYD3 binding element around the transcription start sites (TSSs) of up- and down-regulated genes. We found that 176 (67%) out of 264 down-regulated genes and 49 (58%) out of 84 up-regulated genes in SMYD3-depleted cells carry the potential SMYD3 binding site (Supplementary Table S8). Thus, it is likely that the site specific binding of SMYD3 to its consensus site underlie the ability of SMYD3 to regulate these target genes. To explore this possibility, we tested whether SMYD3 was bound to the four selected target genes . The proteins co-purified with SMYD3 were resolved by SDS-PAGE, and major protein bands were excised and subjected to tandem mass spectrometric analysis in conjunction with a sequence database search. As summarized in Supplementary Table S9, we detected the stable association of 15 proteins with ectopic SMYD3 in this analysis. As predicted from recent studies, we identified HELZ and HSP90A that have been shown to interact with SMYD3 ,35. InteTo gain support for the binding results described above, we first immunoprecipitated HCT116 cell lysates with anti-SMYD3 antibody and checked the coimmunoprecipitation of endogenous PC4. In addition to SMYD3, we could confirm the presence of PC4 in our immunoprecipitates Figure . We thenWe next attempted to identify amino acid residues critical for the SMYD3–PC4 interaction. Circular dichroism spectroscopy of SMYD3, PC4 and the SMYD3–PC4 complex showed that all proteins were folded with the complex representing the superposition of the individual structures . To facilitate the search for residues in the above-outlined interaction interface, we created a structural model of the SMYD3–PC4 complex using the crystal structures of SMYD3 ,20. Our The observed interaction between SMYD3 and PC4 raised the possibility that SMYD3 functionally cooperates with PC4 to establish the active state of target genes in bladder and colon cancer cells. Congruent with these ideas, when the genes that were down-regulated in SMYD3-depleted cells were analyzed by qRT-PCR, their transcription levels were completely recovered after the ectopic expression of SMYD3 Figure . Having FNBP1 and MFGE8 genes by ChIP assays employing cross-linked chromatin isolated from control and SMYD3-depeleted cancer cells. Immunoblotting confirmed that SMYD3 knockdown had no effect on the expression levels of PC4 in HCT116 cells . The distribution patterns of PC4 across the FNBP1 and MFGE8 genes were similar to those of SMYD3 and H3K4me3, with a peak around the proximal promoter and coding regions (regions C and D) and a gradual decrease over the distal promoter and coding regions   Figure ,19,22,38) Figure  and E, tFNBP1 and MFGE8 genes . The importance of PC4 with respect to SMYD3 function was further confirmed by the finding that wild-type PC4 fully restored the proliferation rates of the cancer cells depleted of PC4, whereas SMYD3 binding-deficient PC4 was much less efficient in restoring the proliferation rates . PC4 knockdown generated a distinct repression of SMYD3 target genes, and PC4 wild-type, but not SMYD3-bindining deficient PC4 mutant, could rescue the expression of target genes Figure . ChIP ans Figure . Essentis Figure . Thus, tFNBP1 and MFGE8 genes under the control of U6 promoter , SMYD3 can neither interact with PC4  and dCas9-FLAG fused to N-terminal truncated SMYD3 (residues 101–428) wild-type (wt) or enzymatic dead mutant (F183A), and constructed their expression plasmids using pIRES mammalian expression vector containing the CMV promoter Figure . When dC4 Figure  nor bindFNBP1 and MFGE8 mRNA levels (G3 and G4), whereas G1 or G2 sgRNA transfection displayed no obvious changes in transcription (G1 and G2). Also, targeting dCas9-FLAG-SMYD3 to the proximal promoter region by G1 and G2 sgRNA pair or to proximal coding region by G3 and G4 sgRNA pair activated the target genes by ∼2–3 fold . To further support these results, expression of all four sgRNAs together with dCas9-FLAG-SMYD3 generated even higher elevation in FNBP1 and MFGE8 expression . In parallel assays, dCas9-FLAG-SMYD3 enzymatic dead mutant (F183A) failed to show any change in transcription (dCas9-FLAG-SMYD3 F183A), clearly indicating the specificity and essential role of SMYD3 HMT activity in SMYD3 function on target genes. The targeting specificity of sgRNA-guided dCas9-FLAG-SMYD3 was further confirmed by the observation that sgRNAs targeting the FNBP1 and MFGE8 genes caused no silencing effects on MFGE8 and FNBP1 gene activation, respectively  through protein-protein interaction makes it likely that other factors might be required for SMYD3-induced transactivation and tumorigenesis ,8. UsingAnother intriguing finding of our study is that the stable localization of SMYD3 at target genes is dependent upon PC4, which underscores a complex relationship between SMYD3 and PC4 in gene transcription. Further indicative of a PC4 role in enhancing SMYD3 occupancy at target genes is much lower levels of H3K4me3 in PC4-depleted cells, compared to control cells. The mechanistic basis for this reciprocal effect of PC4 on SMYD3 occupancy and function at target genes is not fully elucidated in this study. However, considering that SMYD3 binds to its cognate site through the MYND domain containing alpha helices and that PC4 beta sheet region interacts with SMYD3 MYND alpha helical motif, PC4-induced stabilization of SMYD3 alpha helical contact with target DNA sequence is likely to be part of the mechanism by which PC4 contributes to SMYD3-mediated transactivation. This finding also suggests that the presence of PC4 is critical for the stable accumulation of H3K4me3 at SMYD3 target genes, functionally linking PC4 to an active histone mark. Structural investigations of PC4-bound SMYD3 will provide information on the nature of SMYD3–PC4 interaction and how PC4 triggers a stable retention of SMYD3 at target genes. Also indicative of an apparent role of SMYD3 in early transcriptional elongation, our ChIP analysis detected higher amounts of SMYD3-mediated H3K4me3 at the proximal coding region than promoter region of target genes. Our results agree with those of previous work , which fFNBP1 and MFGE8 in 293 cells. Individual sgRNA could not effectively activate the corresponding target genes, whereas a mixture of two or four sgRNAs efficiently generated transcriptional activation. Currently, we do not have mechanistic explanation about this phenomenon, but we think that multiple sgRNAs increase the likelihood of dCas9-FLAG-SMYD3 localization at a sufficiently high level to promote transcription. In additional support of the physiological relevance of the transcription results, our ChIP analyses showed that transcriptional activation of endogenous target genes by dCas9-SMYD3 coincides with H3K4me3. Perhaps more important are observations that there is a strong correlation between the level of dCas9-SMYD3-mediated H3K4me3 and the level of dCas9-SMYD3-induced transcriptional activation and that dCas9-SMYD3 enzymatic dead mutant is unable to generate productive transcription. Furthermore, our observation that combined application of sgRNAs targeting the proximal promoter and coding regions generates a synergistic effect is supportive of the idea that SMYD3 acts to regulate both initiation and elongation events. In relation to the use of N-terminal truncated version of SMYD3 in our CRIPSR experiments, a previous study suggested that the N-terminal domain of SMYD3 is important for SMYD3 HMT activity and histone substrate specificity  and sgRNA sets targeting the proximal promoter and coding regions was sufficient to boost the transcription of two SMYD3 target genes cificity , that thIn summary, we have demonstrated that SMYD3 promotes cell proliferation and invasion by mediating H3K4me3 and PC4 recruitment at the proximal promoter and coding regions of target genes, thereby positively influencing their expression in bladder and colon cancer cells. Dynamic regulation of SMYD3-mediated H3K4me3 by PC4 also contributes to productive transcription of SMYD3 responsive genes in cancer cells. Thus, targeting therapeutic intervention to cooperative activity of SMYD3 and PC4 could provide effective strategy for cancer treatment.