Escherichia coli reprograms host regulation of an anaerobic respiratory system, thereby inhibiting a bet hedging strategy. The phage exerts this effect by upregulating a host-encoded signal transduction protein through transcription initiated from a phage-encoded promoter. We further show that this phenomenon occurs not only in a laboratory strain of E. coli, but also in a natural isolate that contains a prophage at this site.Temperate bacteriophages are viruses that can incorporate their genomes into their bacterial hosts, existing there as prophages that refrain from killing the host cell until induced. Prophages are largely quiescent, but they can alter host phenotype through factors encoded in their genomes (often virulence factors) or by disrupting host genes as a result of integration. Here we describe another mechanism by which a prophage can modulate host phenotype. We show that a temperate phage that integrates in  Animals and plants can all fall prey to viruses – and so can bacteria. The viruses that infect bacteria are called bacteriophages (or phages for short), and they are found everywhere bacteria live and probably outnumber bacteria by at least ten to one.While some phages quickly kill every bacterial cell they infect, others enter a dormant state by inserting their DNA into the DNA of their host cell. Here they lie in wait for a signal that reactivates them, triggering the production of more phages and the death of the host cell. While the phage lies dormant its DNA may harm the host by interfering with nearby bacterial genes, or it may actually provide new genes that benefit the host. In most cases the effects of dormant phages are unknown.Escherichia coli is commonly found in the intestines of humans and other mammals. It can use a nutrient called trimethylamine oxide (TMAO) to help it survive rapid decreases in oxygen levels that can occur in its environment. When a phage called HK022 infects E. coli, the phage enters a dormant state by inserting its DNA between two genes that are critical for E. coli to use TMAO. However, it is not clear what effect, if any, HK022 has on E. coli’s behavior.A bacterium known as E. coli cells carrying dormant HK022 phages. The experiments showed that the bacteria lost the ability to use TMAO to survive rapid decreases in oxygen because the dormant phages switched on one of the neighboring E. coli genes. Unexpectedly, the phage achieved this by neatly replacing the gene’s own promoter – the stretch of DNA that contains information about when the gene should be switched on, and how strongly – with a substitute promoter carried in the phage’s DNA. This substitute promoter is stronger than the normal version – meaning that the gene is more active than it should be.To address this question, Carey et al. used genetic approaches to study Phages are key players in every natural population of microbes and are therefore entwined in the health of humans and the environment. The findings of Carey et al. show a new mechanism through which phages modify their hosts. In the future it may be possible to develop this mechanism into a tool to manipulate bacteria in complex environments like infection sites, for example by introducing phages that block the mechanisms that allow bacteria to tolerate antibiotics. Bacteria and the phages that infect them have a generally antagonistic relationship, with evolution arming each side to defeat the other. Sometimes, though, a bacterium and a temperate phage can form an uneasy truce through lysogeny, wherein the integrated prophage confers some beneficial attribute to its host cell that provides a fitness advantage; after all, unless the host cell dies on the phage’s own terms, the phage dies too. Prophage alteration of host phenotype, known as lysogenic conversion , can benEscherichia coli BW25113 increased the strain’s susceptibility to exogenous stresses and decreased its growth rate through mechanisms yet to be understood (pckA (phosphoenolpyruvate carboxykinase), repressing its expression and producing a slow growth phenotype in some conditions  to prevent any infection of the non-lysogen by phage particles produced by spontaneous prophage induction in the lysogen. The results of this experiment, shown in torCAD expression at the time of transition. From this we conclude that the HK022 prophage deactivates TMAO-dependent bet hedging on rapid oxygen depletion.We previously showed that high cell-to-cell variability in aerobic robiosis . Only cetorCAD in the absence of oxygen indicates that the prophage does not simply eradicate the torS promoter. To investigate the effect of the prophage on torS transcription, we measured β-galactosidase activity produced from an operon fusion of lacZ to torS in the HK022 lysogen and non-lysogen, both with and without oxygen. We found that torS expression was substantially elevated in the HK022 lysogen  near its attP site that, upon integration, affect torS transcription. A simple explanation would be an outward-reading promoter that produces an mRNA transcript originating from within the prophage and reading through torS. To test this explanation, we inserted a synthetic terminator construct—an Ω element (attLHK022) (torS transcription in strains containing this terminator and found that torS expression was very low during both aerobic and anaerobic growth (torS expression is the production of torS transcripts that originate from within the prophage and are driven by a phage-encoded promoter.We next wanted to probe the mechanism by which the HK022 prophage increases  element —at the btLHK022) . We measc growth . This retorS-proximal phage/host junction encodes the viral integrase (int); there are 73 bp between the int stop codon and the junction with the E. coli chromosome. In HK022 (as in phage λ), expression of int is repressed during lysogeny  is identical to a computationally predicted start site (1) has not previously been predicted or reported. TSS2 is so close to attBHK022 that its promoter must be at least partially ablated after prophage integration; this likely explains why no TSS2 transcripts are observed in the lysogen. TSS1 transcripts, on the other hand, are likely absent in the lysogen because TSS1 lies on the far side of the attBHK022 site from the torS coding sequence; in the lysogen, the promoter and coding sequence are separated by the entire HK022 genome. It appears, then, that the only torS transcripts made in the HK022 lysogen are produced by a phage-encoded torS promoter and that the int promoter is not required for prophage-regulated torS transcription.To confirm that the transcripts produced by in vitro transcription were truly n assays . The tran assays . The trattBHK022 . The traart site , while t2 is within three base pairs of a predicted translation start site for torS and that transcripts produced from TSS2 would not have room for a ribosome binding site. This translation start site uses a GTG start codon, indicated in torS, downstream of the GTG start codon and employing an ATG start codon, has also been inferred  at the time of this analysis and calculated the torS-torT intergenic distance for each strain. For all strains with large insertions between torS and torT (relative to E. coli MG1655), we used the PHASTER web server . In most of these strains the torS-torT intergenic distance was roughly the same size as the HK022 genome, which is 40,751 bp long . When we measured torCAD transcription in NRG 857C during aerobic and anaerobic growth by fluorescence microscopy  into oxygen-dependent regulation of mean torCAD expression .We can only speculate on why HK022 shuts off aerobic egulated . The HK0E. coli strains, and these prophages are found in E. coli of diverse origins and phylogenetic groups. This suggests that HK022 is not an oddity in its integration between torS and torT. All of the prophages we identified occupying this site have, like HK022, an outward-reading integrase gene as the final identifiable gene before torS, and all share the general genomic architecture of phages belonging to the lambda supercluster  MG1655  MG1655 . These oated see .torCAD expression could provide insight into the evolutionary advantages for a phage to reconfigure the control of TMAO respiration. Prophage-mediated effects on host physiology remain largely enigmatic, and knowledge is mostly restricted to cases where the effects are readily apparent  was spotted onto the top agar and allowed to dry, and the plate was incubated at 37°C overnight. On the following day, an LB plate was spread with 100 μL HK022 lysate and allowed to dry. Selection for lysogens was carried out by streaking from the turbid zone of lysis formed on the top agar plate onto the HK022-spread LB plate and incubating at 37°C overnight. HK022-resistant colonies were patched onto LB agar, and the same colonies were tested for lysogeny by patching onto a top agar lawn containing an HK022-sensitive strain (MG1655). After overnight incubation at 37°C, candidate lysogens that produced a zone of lysis around the area of the patch (from spontaneous phage release) were nonselectively purified by streaking for single colonies from the LB plate patches and incubating at 37°C overnight. The entire patch test procedure was then repeated using the purified colonies. Candidate lysogen colonies that still produced a zone of lysis around the patch after purification were tested for the presence of the HK022 prophage by PCR. Strains produced by this method were JNC151 (HK022 lysogen of MG1655), DFE12 (HK022 lysogen of MMR8), JNC168 (HK022 lysogen of JNC163), and JNC169 (HK022 lysogen of JNC166). These strains were assayed for tandem polylysogeny by PCR essentially as in 5’-GGAATCAATGCCTGAGTG-3’), HK022-P2 (5’-GCTGATACACTACAGCAATG-3’), HK022-P3 (5’-GACAGGAGCTTGTTGACTAA-3’), and HK022-P4 (5’-GGCATCAACAGCACATTC-3’). All appeared to be tandem polylysogens (denoted (HK022)n in the Key Resources Table and Lists of all strains and plasmids used in this study are provided in Ω element strain JNC175 was constructed by recombineering (Ω element was amplified by PCR from pJB31 using primers LRpJB31_JNC169U1 (5’-CAGAGTCTTCGGGTCAGGGTTAAATTCACGGTCGGTGCACTTTAGGTGAAATCCCGAATGTGCAGTTAAC-3’) and LRpJB31_JNC169L1 (5’-TACTTACATTAATTTACTGATAATTAAAGAGATTTTAAATATACAACTTAGGCGCTGAAAGAAACCGCAA-3’). The PCR product was digested with DpnI and purified before electroporation into JNC169 carrying helper plasmid pKD46. Cultures were spread on LB agar plates containing streptomycin (20 μg/mL), spectinomycin (20 μg/mL), and kanamycin (25 μg/mL) to select for integration of the Ω element and maintenance of the torS-lacZ fusion. The strain was cured of pKD46, and correct integration of the Ω element into attLHK022 was verified by sequencing. The attLHK022::Ω construct was transduced into a clean JNC169 background to create JNC175.The ineering . The Ω etorS-lacZ translational fusions, a DNA fragment encompassing -152 to +28 bp relative to the torS ATG start codon (as determined in this study) was PCR amplified from the MG1655 chromosome and cloned into the XhoI and BamHI sites of pPK7035 upstream of lacZ’, creating pPK12792. pPK12792 served as a template for site directed mutagenesis in which bases downstream of predicted torS start codons (GTG or ATG) through the native lacZ start codon were deleted to create plasmids harboring the translational fusion constructs kan-PtorS-(GTG)lacZ’ (pPK13169) and kan-PtorS-(ATG)lacZ’ (pPK13171). pPK13169 and pPK13171 were used as templates for PCR amplification of the translational fusion constructs using primers with homology to the native Plac region. The amplicons were electroporated into PK12556, and kanamycin resistance was used to select for integrants. The kan-PtorS-(GTG)lacZ and kan-PtorS-(ATG)lacZ constructs were then moved into PK4854 using P1vir transduction to create PK13196 and PK13199, respectively.To construct strains containing chromosomally encoded PtorS, the aforementioned DNA fragment encompassing -152 to +28 bp relative to the torS ATG start codon was used, generating pPK12669. To identify the HK022-derived promoter driving torS expression, a DNA fragment encompassing -231 to +28 bp relative to the torS start codon was PCR amplified from the chromosome of JNC151 and cloned into pPK7179, generating pPK13256.For in vitro transcription and primer extension assays, promoter regions were cloned into the XhoI and BamHI sites of pPK7179. For native PtorCAD-yfp reporter was introduced into NRG 857C by conjugation. As E. coli K-12 and E. coli NRG 857C are not closely related, synteny of their chromosomes was confirmed by genomic alignment using Mauve  in the direction of conjugative transfer, so growth on minimal medium without amino acid supplementation indicated that DFE34 retained the NRG 857C iscR allele. Based on the genetic markers analyzed, the maximum amount of NRG 857C genomic sequence that could have been replaced by K-12 sequence during the construction of DFE34 is 1.1 Mbp.The Png Mauve  before p600 = 0.1–0.4 before being put on ice. Cultures were chilled on ice for 30 min at the time of streptomycin addition and then aerated on a roller drum at 37°C for 2 hr before being held at 4°C overnight. Imaging was performed the next day with no additional aeration beforehand. Microscopy was performed as described in torC control strain was included.Aerobic-to-anaerobic transition microscopy was performed as described in 600 = 0.1–0.5 before harvesting. For the β-galactosidase assays using the PtorS-lacZ translational fusion strains PK13196 and PK13199, chloramphenicol was added to the cultures at a final concentration of 20 μg/mL before placing on ice.β-Galactosidase assays were performed as in 32P]UTP, 50 μM unlabeled UTP, and 500 μM final concentrations each of ATP, CTP, and GTP for 5 min at 37°C in 40 mM Tris (pH 7.9), 30 mM KCl, 100 μg/mL bovine serum albumin, 1 mM dithiothreitol, and 10 mM MgCl2. E. coli σ70 RNA polymerase holoenzyme (50 nM) was added, and each reaction  was terminated after 10 min by adding 10 μL 95% (vol/vol) formamide, 20 mM EDTA, 0.05% (wt/vol) bromophenol blue, and 0.05% (wt/vol) xylene cyanol FF. After the mixture was heated to 90°C for 30 s, 5 μl was loaded onto an 8% polyacrylamide-7 M urea gel (0.5× TBE) and run at 1400 V for 3 hr. The gel was then dried and exposed to a PhosphorImager screen.Following purification of pPK12669 and pPK13256 with a HiSpeed Plasmid Maxi Kit (Qiagen), 2 nM supercoiled plasmid was incubated with 5 μCi of [α-32P-labeled primer (‘native torS’ or ‘HK022/torS’) by heating at 95°C for 5 min followed by slow cooling for 1 hr. Primer extension with the MMLV Reverse Transcriptase 1st-Strand cDNA Synthesis Kit (Lucigen) was carried out according to the manufacturer’s instructions. Sequencing reactions using the same primer from the primer extension assays were performed using the Sequenase Version 2.0 DNA Sequencing Kit (USB).RNA was synthesized using the same protocol as for the in vitro transcription assays, with the exception that UTP was unlabeled. After phenol extraction and ethanol precipitation, 5 μg RNA was hybridized with a Phylogenetic group assignments of the prophage-carrying strains listed in  In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Gisela Storz as the Senior Editor. The reviewers have opted to remain anonymous.The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Thank you for submitting your article "Phage integration alters the respiratory strategy of its host" for consideration by torS and torT leads to significant changes in the respiratory strategy of E. coli. In prior work the authors had found that the mean response of cells /- O2 was unchanged but the variance changed. Now, they find that with HK022 integrated, cells exhibit a clear change in mean response with the TorS/R pathway on in anaerobic conditions and off in aerobic conditions. Follow up studies demonstrate that this difference likely stems from transcription of torS from a phage promoter. Although the authors do not specifically demonstrate how this metabolic change enhances host or phage fitness, it seems likely that there is some advantage to this prophage insertion under some conditions. Given all the possible places that a phage could integrate, the fact that HK022 lands in such a sensitive position and has a promoter to compensate for loss of the torS promoter indicates that there is an adaptive advantage to this phenomenon. Overall, the reviewers were enthusiastic about the paper and agreed that it warrants publication in eLife. There were, however, a few suggestions, collated below, for improvement that should be addressed in a revision.This paper presents evidence that integration of the temperate phage HK022 into its primary integration site between 2 condition there is higher variability in acetyl-phosphate, leading to the variability and higher response relative to the phage, O2 condition? E.g. maybe presence of the phage somehow affects central metabolism to reduce acetyl-phosphate levels, leading to a tighter OFF state in the phage, O2 condition? I think the experiments presented indicate that higher TorS levels are a more likely explanation for what's going on, but maybe there's a contribution from a change in other phosphodonors?1) I wondered about the role of non-specific phosphodonors for TorR, e.g. acetyl-phosphate, which can sometimes drive some RR phosphorylation, especially when a cognate kinase is not present or highly expressed. Is it possible that in the no phage, OtorCAD system. For example, what is TMAO and why is it important in the absence of oxygen. In the Discussion, this increased background might allow the authors to give a somewhat more detailed idea of what advantage the HK022 prophage could provide. For example, what kind of conditions produce high TMAO concentrations.2) In the Introduction, the authors should provide a small amount more background into the importance of the att. These phages have a completely different type of tail. Do these prophages all have the same integrase? Do any of them have different att sites but still integrate in the same region. This would strongly support the adaptive benefit of integrating in the torS region.3) In discussing the observation that different phages integrate at the same site as HK022, the authors should emphasize that these phages are definitely distinct phages from HK022. For example, some of the strains listed in Table 1 have P22-like phages integrated at the HK022 B. subtilis. This and some other examples can be found in recent reviews . Some of these examples could be mentioned to put the current work into context.4) While I could not find examples in the literature of the type of prophage-induced transcriptional re-wiring described here, there are examples of the insertion position of a prophage playing key roles in bacterial physiology. One example is the skn prophage in 5) No mention of spontaneous induction of the HK022 prophage is made. I submit that bet hedging might be going on at a lower level if there is a reasonable level of spontaneous excision going on. It would be easy to estimate the frequency of excision events using Q-PCR and properly selected primer pairs.E. coli strains that have the prophages. They (laboriously) tested 1 wt prophage strain and found the same abrogation of the stochastic regulation going on. Surely we should see whether the promoter is conserved or whether other promoters are there?6) The biggest omission is some sort of tabulation of the prophage promoters that should be found in the multiple wild type 7) One experimental concern is that the authors do not provide in vivo evidence for the activity of the prophage promoter. There are enough weird things that bacteria do to prophage sequences that I would like to see a Northern or some such from in vivo situations. 2 condition there is higher variability in acetyl-phosphate, leading to the variability and higher response relative to the phage, O2 condition? E.g. maybe presence of the phage somehow affects central metabolism to reduce acetyl-phosphate levels, leading to a tighter OFF state in the phage, O2 condition? I think the experiments presented indicate that higher TorS levels are a more likely explanation for what's going on, but maybe there's a contribution from a change in other phosphodonors?1) I wondered about the role of non-specific phosphodonors for TorR, e.g. acetyl-phosphate, which can sometimes drive some RR phosphorylation, especially when a cognate kinase is not present or highly expressed. Is it possible that in the no phage, OtorS strains show no expression of TorR-dependent genes . Based on this evidence, which was acquired under the same culture conditions as in the present work, nonspecific phosphodonors such as acetyl phosphate do not appear to play a significant role in phosphorylating TorR and activating torCAD transcription.Available data suggests phosphorylation of TorR is exclusively dependent on TorS kinase activity, as 2) In the Introduction, the authors should provide a small amount more background into the importance of the torCAD system. For example, what is TMAO and why is it important in the absence of oxygen. In the Discussion, this increased background might allow the authors to give a somewhat more detailed idea of what advantage the HK022 prophage could provide. For example, what kind of conditions produce high TMAO concentrations.torCAD and TMAO has been added to the Introduction, and some further speculation on the environments in which lysogeny could be advantageous has been added to the Discussion.Additional background on 3) In discussing the observation that different phages integrate at the same site as HK022, the authors should emphasize that these phages are definitely distinct phages from HK022. For example, some of the strains listed in Table 1 have P22-like phages integrated at the HK022 att. These phages have a completely different type of tail. Do these prophages all have the same integrase? Do any of them have different att sites but still integrate in the same region. This would strongly support the adaptive benefit of integrating in the torS region.E. coli genome sequences available from NCBI since the initial submission. In the 45 genomes containing a prophage upstream of torS, all have the prophage integrated at a site identical to attBHK022 . Additionally, the integrase of each of these prophages shares the highest conservation with HK022 integrase . We found the reviewer’s comment about the different tail types of some of the phages interesting and have accordingly added a column to the table listing the most similar known phage (again as identified by PHASTER) for the benefit of the intent reader.We have extended this table (now Supplementary file 1) to include new 4) While I could not find examples in the literature of the type of prophage-induced transcriptional re-wiring described here, there are examples of the insertion position of a prophage playing key roles in bacterial physiology. One example is the skn prophage in B. subtilis. This and some other examples can be found in recent reviews . Some of these examples could be mentioned to put the current work into context.Thank you for directing us to this interesting reference. In this revision we have included additional examples of integration site-dependent gene regulation by prophages and citation of the Feiner et al. article.5) No mention of spontaneous induction of the HK022 prophage is made. I submit that bet hedging might be going on at a lower level if there is a reasonable level of spontaneous excision going on. It would be easy to estimate the frequency of excision events using Q-PCR and properly selected primer pairs.attB site to assay for prophage excision events. We did find that HK022 excision occurs, but by PCR we do not know how to differentiate between excision events leading toward virion production/cell lysis and excision events not leading to cell death . However, if spontaneous HK022 excision were providing another layer of bet hedging regulation, we would expect to see a subpopulation of cells in the aerobic HK022 lysogen culture with high torCAD expression, and we do not see any such cells . This suggests that such excision events, if they occur, are rare.We grew an HK022 lysogen under culture conditions identical to those used in the bet-hedging experiments and performed PCR across the HK022 6) The biggest omission is some sort of tabulation of the prophage promoters that should be found in the multiple wild type E. coli strains that have the prophages. They (laboriously) tested 1 wt prophage strain and found the same abrogation of the stochastic regulation going on. Surely we should see whether the promoter is conserved or whether other promoters are there?HK022(torS)—the transcription start site that we identified—shows that this sequence is highly conserved across the 45 prophage-containing genomes. We have provided this alignment in the new Supplementary file 2.Unfortunately, we do not know the precise location of the prophage-encoded promoter, only the associated transcription start site. However, an alignment of the region upstream of TSS7) One experimental concern is that the authors do not provide in vivo evidence for the activity of the prophage promoter. There are enough weird things that bacteria do to prophage sequences that I would like to see a Northern or some such from in vivo situations.torS in vivo . However, we feel that our measurements of torS transcription with and without a synthetic terminator integrated between the prophage and torS coding sequence  provide strong evidence that the prophage promoter is active in vivo.We were unable to detect transcripts expressed from the prophage promoter in vivo in attempts to perform TSS mapping by primer extension using endogenous mRNA. Although disappointing, this was unsurprising considering the exceptionally low transcription rate of wild-type


The establishment of the embryonic and trophoblast lineages is a developmental decision underpinned by dramatic differences in the epigenetic landscape of the two compartments. However, it remains unknown how epigenetic information and transcription factor networks map to the 3D arrangement of the genome, which in turn may mediate transcriptional divergence between the two cell lineages. Here, we perform promoter capture Hi-C experiments in mouse trophoblast (TSC) and embryonic (ESC) stem cells to understand how chromatin conformation relates to cell-specific transcriptional programmes. We find that key TSC genes that are kept repressed in ESCs exhibit interactions between H3K27me3-marked regions in ESCs that depend on Polycomb repressive complex 1. Interactions that are prominent in TSCs are enriched for enhancer–gene contacts involving key TSC transcription factors, as well as TET1, which helps to maintain the expression of TSC-relevant genes. Our work shows that the first developmental cell fate decision results in distinct chromatin conformation patterns establishing lineage-specific contexts involving both repressive and active interactions. The role of the genome structure in the establishment of the embryonic and trophoblast lineages is still not well understood. Here the authors perform promoter capture Hi-C in mouse trophoblast and embryonic stem cells and find divergent networks of repressive and active chromatin interactions between the two lineages. Mammalian preimplantation development involves dramatic molecular changes that enable the consolidation of the information carried within the oocyte and sperm into a novel, and fast-evolving, gene expression programme that gives rise to the embryo.5.Importantly, during preimplantation development the embryo undergoes the first cell lineage differentiation process, with the inner cell mass  and trophectoderm (giving rise solely to extraembryonic tissues) being clearly delineated by the blastocyst stage. This morphological event constitutes a strict separation of cell fate, which is underpinned by the establishment of a strong epigenetic barrier set up by DNA methylation and histone modifications, such as H3K9me3, that prevent trans-differentiation from one cell type to the other6. Together with in vivo studies using mouse mutants, this has allowed for a dissection of the signalling events, transcription factors and epigenetic mechanisms that regulate the trophoblast lineage, maintaining its cellular identity and preventing trans-differentiation into embryonic stem cells (ESCs)7. Key transcription factors such as CDX2, EOMES, ELF5, TEAD4 and GATA3, among others, are essential for TSC self-renewal11. These networks are distinctly different from those operating in ESCs, which are prominently made up of OCT4, NANOG, KLF4, ZFP42 and others12. Intriguingly, some transcription factors, such as SOX2 and ESRRB, are seemingly pivotal in both cell lineages and stem cell types. However, despite this shared necessity, they operate in diverging protein complexes and bind to largely non-overlapping genomic loci in both stem cell types, which endows them with a cell type-specific function14.Our knowledge of the factors that drive the establishment and maintenance of the trophoblast lineage has greatly benefited from the ability to culture mouse trophoblast stem cells (TSCs) in a stable manner, which retain the full differentiative capacity of the early trophoblast15. Most prominently, the DNA in trophoblast cells is found in a globally hypomethylated state19. Interestingly, loss of DNA methylation in ESCs enhances trans-differentiation into TSCs, which is due in part to the activation of Elf5 expression1. TSCs are also characterised by overall low levels of the repressive H3K27me3 mark20, which in ESCs is often found co-localised with the active H3K4me3 mark at developmental genes, creating an epigenetic bivalency21. In TSCs, loss of H3K27me3 either enables the expression of lineage-determining genes such as Cdx2, or triggers the deposition of alternative repressive marks, namely H3K9me3 and DNA methylation23.These stem cell type-specific transcriptional networks are associated with dramatic differences in the epigenomic landscape between the trophoblast and the embryonic lineages25, have abundantly demonstrated that linear proximity in the genome cannot predict how localised epigenomic changes will affect gene expression. Namely, cis-acting enhancer elements can act on genes lying several hundreds of kilobases (or even megabases) away26. Interactions associated with repressive marks can also be detected at long distances within a chromosome or even across chromosomes, as was recently demonstrated for a large Polycomb-driven interaction network between Hox gene clusters27.Despite this large body of epigenomic data, it remains unknown how this information is integrated by the three-dimensional (3D) arrangement of the genome to establish a TSC-specific gene expression programme. Detailed studies in multiple cell types, including mouse and human ESCsHere we investigate the differences in chromosome conformation between the embryonic and extraembryonic lineages, using mouse ESCs and TSCs as models. We integrate these data with epigenomic information and transcription factor binding profiles to uncover key regulatory principles governing the regulation of the TSC-specific transcriptional programme. We find that TSC-specific genes are associated with: (1) repression in ESCs involving Polycomb-dependent interactions and (2) the action of key transcription factors and TET1 at cell-specific gene–enhancer contacts in TSCs.28 to gain an in-depth view of 3D contacts with gene promoters in mouse TSCs and ESCs. Two replicates of each cell type were performed, yielding 51–75 M mapped valid read pairs per replicate and an average of 1730 chimeric reads per captured promoter fragment . Sex chromosomes were excluded due to sex differences between the cell lines. This yielded a total of 690,074 and 672,062 interactions  between promoters and non-promoter regions ons Fig. . We alsoons Fig. . To gaug31. Notably, genes involved in TSC-specific interactions included a number of essential trophoblast-associated genes such as Tfap2c  1 in ESCs27. In line with reduced PRC activity in TSCs20, H3K27me3 levels at Hox gene clusters were low in TSCs and interactions within each cluster largely reduced in strength or even absent  that performs a direct comparison of the mapped data from both cell types, yielding a list of statistically significant differential interactions. These differential interactions also displayed strong differences in contact frequency in a genome-wide Hi-C dataset from an independently derived ESC line , hereafter called homotypic interactions. We found that a disproportionally large amount of H3K27me3 homotypic interactions were ESC-specific , we asked whether PRC1 is indeed necessary for all the ESC-specific homotypic interactions identified above. For this purpose, we analysed published PCHi-C data from a Ring1A knockout (KO) ESC line that also allows for tamoxifen-inducible deletion of Ring1B, thus generating Ring1A/B double knockout (DKO) cells27. We found that, compared to KO cells, DKO cells had substantially fewer of the H3K27me3 ESC-specific interactions that we identified  and PCR2 (EZH2 and SUZ12) proteins  are found in the active state in TSCs , poised (H3K4me1 + H3K27me3) and intermediate . Interactions with CTCF sites, as well as with intermediate and active enhancers, were enriched in TSC-specific interactions Fig. . This isTo investigate in more detail interactions with active TSC enhancers, we first defined a more stringent set of active enhancers by incorporating chromatin accessibility data (see Methods). As expected, genes interacting with at least one active TSC enhancer displayed higher expression than those with no active enhancer interactions  of peaks overlapping our stringent list of active enhancers .Differentiation between the embryonic and extraembryonic lineages involves the concerted effort of signalling cues, cell-specific activation of transcription factors, and remodelling of the epigenetic landscape. Here we have shown that the first cell lineage decision is also accompanied by pronounced reshaping of the 3D chromatin conformation, which reflects and possibly reinforces the reprogramming barrier imposed by other layers of gene regulation. While our study was limited to stem cell line models, ESC Hi-C data strongly resemble that from ICM cells and post-implantation epiblast31. Increased enhancer connectivity is also seen in foetal liver cells when compared with ESCs24. These different 3D chromatin arrangements probably reflect a switch from a transcriptionally poised state of developmental genes in pluripotent ESCs to the activation of specific subsets of these genes in more developmentally restricted lineages. However, unlike the neuronal lineage, the trophoblast lineage is not derived from the pluripotent state represented by ESCs, which does not have the potential to form extraembryonic tissues. An obvious question is therefore how the genome is spatially arranged in the lead up to the separation of these two lineages in the blastocyst. Recent work in preimplantation embryos has suggested that the 3D chromatin architecture undergoes a progressive maturation during early development, starting out with weak topologically associating domains and a global depletion of long-range interactions after fertilisation that become progressively stronger or more prominent as development progresses37. However, it remains unclear how finer scale gene–gene and gene–enhancer contacts develop during this period and into the separation of the embryonic and extraembryonic lineages. Future work on this question could explore the use of single-cell Hi-C technology39 to profile individual blastomeres as they acquire expression of key TSC markers such as CDX2.The chromatin folding features that differentiate ESCs and TSCs are: (1) an enrichment for repressive interactions in ESCs, mainly between gene promoters, but also involving H3K27me3-marked enhancers and (2) an enrichment for active enhancer–gene interactions in TSCs. Similar observations have recently been made during neuronal differentiation of ESCs, wherein PRC1-associated interactions are disrupted and enhancer–gene contacts become progressively more abundant20) and extraembryonic tissues are far less affected than embryonic tissues in mutants of the PRC2 catalytic subunit Ezh2 (ref. 40). H3K27me3 deposition increases throughout preimplantation development, with the majority of bivalent domains being established during the morula-to-blastocyst transition38, but it remains to be seen how this is integrated by the 3D arrangement of the genome to enable control of trophoblast-specific genes throughout this period. Notably, long-range interactions between PcG-bound regions similar to the ones we detected here in primed (serum-grown) ESCs are not present in ground-state (2i-grown) pluripotent ESCs41. Thus, PcG-dependent chromosomal interactions appear to be established in post-implantation pluripotent stem cells, and are then disrupted during cell fate specification during development31, most likely facilitating the activation of key germ layer genes.We have found that homotypic interactions between H3K27me3-marked loci in ESCs are associated with the repression of TSC-expressed genes. Interestingly, although virtually all of the genes involved in these interactions are devoid of H3K27me3 in TSCs Fig. , only di42. Therefore, similar to ESRRB14, TET1 is a factor shared by both ESCs and TSCs that gains context-specific functions, and our data suggest that the tissue-specific 3D conformation of the genome helps to enact such functions. Alternatively, it is the action of TET1 that modulates gene–enhancer contacts in TSCs. Interestingly, in gliomas with gain of function IDH mutations, interfering with TET function leads to aberrant gene expression, at least in part due to an increase in DNA methylation that impairs the binding of the methylation-sensitive chromosomal insulator protein CTCF43. DNA methylation of CTCF binding sites has also been shown to control enhancer–promoter interactions at the imprinted H19 locus45. Furthermore, in concert with other epigenetic mechanisms, DNA methylation has been shown to facilitate46 or weaken47 enhancer–promoter contacts at specific loci. It is therefore likely that proteins involved in DNA (de)methylation direct enhancer–promoter communication not only by altering the methylation status at enhancers and promoters themselves, but also through the establishment or disruption of spatial chromosomal domains that dictate the access of regulatory sequences to target genes. The precise role of DNA methylation in shaping ESC genome topology and promoter–enhancer interactions to maintain stem cell identity and to protect against trans-differentiation into TSCs1 remains a subject for future studies.We also found that TSCs bear a higher proportion of cell-specific interactions between genes and active enhancers when compared to ESCs. Our data suggest that the key TSC transcription factors ELF5 and ESRRB may play particularly important roles in the enhancer-driven regulation of TSC gene expression. Additionally, the DNA methylation oxidising enzyme TET1 also seems to play a key role, similar to what has been observed at other tissue-specific enhancersTaken together, we describe here the distinctly different 3D chromatin organisation in TSCs compared to ESCs and identify a highly TSC-specific set of TET1-bound enhancers that drive expression of key TSC genes.6 were cultured under routine conditions . J1 ESCs 48 were expanded on irradiated primary embryonic fibroblasts under standard pluripotent conditions  on tissue culture plates coated with 0.1% gelatin. To harvest the cells and remove contaminating feeder cells, ESCs were trypsinized and feeders allowed to re-attach for 30 min before collecting non-attached ESCs—this procedure was performed twice.Chimera-competent TS-EGFP cells 49, with minor modifications. Briefly, crosslinked chromatin was digested with HindIII (NEB), and restriction fragment ends were labelled using biotin-14-dATP (Life Technologies) and DNA Polymerase I . After ligation in nuclei50 , the crosslinks were reversed by Proteinase K digest (65 °C overnight), and the DNA was purified using phenol/chloroform (Sigma-Aldrich) extraction. After removal of biotin from unligated DNA ends, the DNA was sonicated (Covaris E220) to an average size of around 400 base pairs, and end-repaired using DNA Polymerase I (Large Klenow Fragment), T4 DNA polymerase, and T4 DNA polynucleotide kinase . dATP was added to the 3′ ends of the DNA (using Klenow Fragment (3′ → 5′ exo−); NEB), and the DNA was subjected to double-sided SPRI bead size selection . Biotin-marked ligation products were isolated using MyOne Streptavidin C1 Dynabeads (Life Technologies), and after adapter ligation  the bead-bound Hi-C DNA was amplified with seven PCR amplification cycles using PE PCR 1.0 and PE PCR 2.0 primers (Illumina).PCHi-C experiments were performed in duplicate for each cell line. Thirty to 40 million ESCs or TSCs were fixed in 2% formaldehyde (Agar Scientific) for 10 min at room temperature, and Hi-C libraries were prepared as described24, using a custom-made RNA capture bait system (Agilent Technologies) consisting of 39,021 individual biotinylated RNAs targeting the ends of 22,225 promoter-containing mouse HindIII restriction fragments. The Hi-C library DNA (500–750 ng) was mixed with hybridisation blockers (Agilent Technologies) and denatured for 5 min at 95 °C, then incubated with hybridisation buffer and the RNA capture bait system at 65 °C for 24 h (in a MJ Research PTC-200 PCR machine). After the hybridisation incubation, DNA/biotin-RNA was isolated using MyOne Streptavidin T1 Dynabeads (Life Technologies), following the manufacturer’s instructions . After the final wash in wash buffer 2 (Agilent Technologies), the beads were resuspended in 300 µl NEBuffer 2 (NEB), isolated on a DynaMag-2 magnet (Life Technologies), and resuspended in a final volume of 30 µl NEBuffer 2. After a post-capture PCR (four amplification cycles using Illumina PE PCR 1.0 and PE PCR 2.0 primers), the Promoter CHi-C libraries were purified with AMPure XP beads (Beckman Coulter) and paired-end sequenced  at the Babraham Institute Sequencing Facility.Promoter Capture Hi-C was performed as described51. For read yields and quality control metrics see Supplementary Fig. Raw sequencing reads were processed using the HiCUP pipeline (v0.5.8), which maps the ditags against the mouse genome mm10), filters experimental artefacts, such as circularised reads and religations, and removes duplicate reads0, filterq-value thresholds that ensured only visibly strong contacts with a given promoter were kept—based on empirical assessment of 4C-like tracks. To formally define this threshold, for each promoter we first plotted the cumulative distribution of the significance levels  of its interactions. We then used the inflection point of the first derivative of this cumulative significance curve to define a promoter-specific significance threshold. To take into account differences in biases affecting promoter–promoter interactions, these were called with GOTHiC using a modified logit background distribution as described in ref. 24.Significant promoter–genome interactions were called using the GOTHiC BioConductor package (v1.14.0). To exclude low frequency contacts, which are likely to be non-functional or have a role only in a very small proportion of cells, we established stringent Significant differential interactions were identified by a modified version of the GOTHiC pipeline, where two conditions are directly compared and the expected values are the number of reads observed in the reference condition. For scaling, the number of total captured reads per condition is used. To enable logarithmic transformation of data, for those interactions where no reads were detected for one of the cell types, we added 1 in the cell type with the missing value. For multiple testing correction we use Independent Hypothesis Weighting (IHW v.1.6.0) and a false discovery rate of 5%.30 , followed by filtering of uniquely mapped reads. ChIP-seq peak detection was performed using MACS2 v2.1.1 (ref. 55) with −q 0.05; for histone marks the option --broad was used. DNase-seq peak detection was performed using F-seq v1.84 (ref. 56) with options −f 0 −t 6.Fastq files from publicly available datasets  between ESC-specific interactions and shared interactions.Enhancers were first defined based on the H3K4me1 peaks in each cell type: (1) H3K4me1 peaks overlapping H3K27ac but not H3K27me3 were classified as active enhancers, (2) those overlapping H3K27me3 as poised and (3) those overlapping neither mark as intermediate. Peaks within 1 kb of RefSeq transcriptional start sites were excluded. A second list of active enhancers in TSCs was defined by finding the intersection between DNase I, H3K4me1 and H3K27ac peaks, merging enhancers within 300 bp of each other, and filtering out particularly small (<100 bp) or large (>3 kb) enhancers.57) using a transcriptome index from Illumina’s iGenomes. For Ring1A KO and Ring1A/B DKO RNA-seq, mapped bam files were directly downloaded from the respective data repository. Transcript FPKM (fragments per kilobase of transcript per million mapped reads) values were extracted using Seqmonk.Fastq files from publicly available datasets . K-means clustering of expression data was performed using this minimum expression threshold, with the number of clusters (n = 7) decided empirically.To exclude low-expressing, high-variance transcripts when evaluating differences in expression, a minimum expression threshold was employed based on the distribution of log2 FPKM values (−3 for http://bioconductor.org).Custom scripts used to analyse the data in this study are available from the authors upon request. The differential interaction calling algorithm will be included in the next release of GOTHiC, available from Bioconductor (Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1


Here, we show that the ETS transcription factor, Etv6, positively regulates vegfa expression during Xenopus blood stem cell development through multiple transcriptional inputs. In agreement with its established repressive functions, Etv6 directly inhibits expression of the repressor foxo3, to prevent Foxo3 from binding to and repressing the vegfa promoter. Etv6 also directly activates expression of the activator klf4; reflecting a genome-wide paucity in ETS-binding motifs in Etv6 genomic targets, Klf4 then recruits Etv6 to the vegfa promoter to activate its expression. These two mechanisms (double negative gate and feed-forward loop) are classic features of gene regulatory networks specifying cell fates. Thus, Etv6’s dual function, as a transcriptional repressor and activator, controls a major signaling pathway involved in endothelial and blood development in vivo.VEGFA signaling controls physiological and pathological angiogenesis and hematopoiesis. Although many context-dependent signaling pathways downstream of VEGFA have been uncovered,  vegfa expression  to control blood formation in Xenopus.How vegfa expression is transcriptionally regulated in vivo is unclear. Here, the authors demonstrate that the ETS transcription factor Etv6 acts as a repressor and an activator of two direct regulators of  In angiogenesis, VEGFA is required for endothelial cell proliferation, migration and organization in three dimensions to form new vessels during vascular remodelling3. Finally, in cancer, VEGFA signaling plays essential roles in the regulation of tumour angiogenesis and metastasis4, and has emerged as a key anti-angiogenic target in a wide variety of cancer therapies5. However, inhibitors of VEGFA-mediated signaling often trigger limited responses in both human tumours and mouse models of cancer, due to the development of evasive and intrinsic resistance mechanisms5.Vascular endothelial growth factor A (VEGFA) signaling is critical for both physiological and pathological processes in the adult, including hematopoiesis, angiogenesis and solid tumour progression. In hematopoiesis, VEGFA regulates hematopoietic stem cell (HSC) survival and function in the bone marrow through both cell intrinsic and extrinsic mechanismsVegfA or VegfA receptor  genes in mice results in early lethality owing to severe defects in vascular development6. Mice deficient in Vegfr2  show an absence of yolk sac blood islands and reduction of CD34+ hematopoietic progenitors7. In vitro, VEGFA is required for the specification of FLK1+ mesoderm into hematopoietic and cardiovascular lineages in mouse embryonic stem cell differentiation models9. In embryos, vegfa is essential for HSCs emergence and has been shown to be required at several stages of their programming12. In Xenopus, acting in a paracrine manner from the somites, Vegfa is required for the establishment of definitive hemangioblasts  and HSCs) in the dorsal lateral plate (DLP) mesoderm12. Later, Vegfa secreted by the hypochord guides the migration of DA precursors from the DLP to the midline12.VEGFA signaling is also essential during embryogenesis where it plays pivotal roles in the development of the endothelial and hematopoietic systems. Knockout of vegfa during embryogenesis. Because developmental and tumour biology and, in particular, angiogenic and metastatic processes share signaling and transcriptional pathways13, a better understanding of the regulatory networks lying upstream of VEGFA during specification of the hematovascular lineage may help identify new druggable targets relevant to VEGFA-dependent diseases.Although critical to understand the development of the vascular and hematopoietic systems, very little is known about the regulation of the complex spatio-temporal expression pattern of 16, is a key regulator of hematopoietic, angiogenic and tumorigenic processes, and has been linked to vegfa regulation. It is essential for bone marrow hematopoiesis18 and is involved in many chromosomal rearrangements which lead to childhood leukaemias19. Additionally, germline and somatic mutations in ETV6 have recently been shown to be involved in other malignancies such as skin, colon, mammary and salivary gland cancers22. In mouse embryos, Etv6 homozygous deletion leads to early embryonic death because of defective yolk sac angiogenesis23.ETV6, a transcriptional repressor belonging to the ETS family of transcription factors (TFs)Xenopus embryos through positive regulation of vegfa expression in the somites at stage 22 of development12. Because Etv6 is considered a transcriptional repressor24, this suggested an indirect regulatory mechanism of action whereby Etv6 directly represses expression of a vegfa transcriptional repressor. To test this hypothesis, we set out to investigate the mechanism by which Etv6 activates vegfa expression in the somites. Using genome-wide and functional analyses, we identify Etv6 direct target genes and show that Etv6 employs multiple regulatory mechanisms. To allow vegfa expression, Etv6 directly represses foxo3, a known transcriptional repressor of VegfA in cancer cells26, thus participating in a double negative gate. Unexpectedly, Etv6 also directly activates expression of klf4, a known activator of VegfA in endothelial cells and a regulator of metastatic processes28. Consistent with a feed-forward loop mechanism, Etv6 also binds the vegfa promoter through a Klf4-dependent mechanism. Thus, our work demonstrates that vegfa expression in the somites is regulated by a complex gene regulatory network (GRN) with Etv6 acting both as a repressor and, unexpectedly, an activator. These findings further our understanding of the in vivo regulation of vegfa and provide a platform for the study of the processes underlying VEGFA-dependent tumorigenesis, such as those involving FOXO3 and KLF4, and for the establishment of future therapeutic strategies.Previously, we showed that Etv6 specifies DHs and HSCs in developing vegfa expression in the somites, we first set out to identify Etv6 genomic targets through ChIP-seq . Tal1, a hematopoietic gene not expressed in the somites at stage 2211, is an example of a gene devoid of Etv6 peaks at its TSS region.A total of 9128 Etv6 consistent peaks across the three biological replicates were identified using DiffBind30. Etv6 peaks showed significant enrichment of consensus binding sequences corresponding to known TFs 12 12 Fig. . Spearmates Fig. . Interestes Fig. , confirmmeox1 and meox2 regulate somitogenesis and myogenesis31. In zebrafish, meox1 also represses the expansion of somite-derived endothelial cells thereby limiting HSC development in the DA32. The single Xenopus meox gene is repressed by Etv6 in somites (WT/etv6 MO log2FC = −0.89 for meox2.L and −0.53 for meox2.S) and WISH confirmed higher expression in Etv6-deficient somites . WISH analysis confirmed downregulation of crim1 expression in the somites of Etv6-deficient embryos  Fig. . During tes Fig. . Crim1, yos Fig. . Sox18, yos Fig. , thus pr19, can also act as an activator of gene expression in the somites at stage 22. De novo motif analysis on the sequences underlying the peaks linked to genes normally activated by Etv6 identified Klf/Sp, Nfy and Creb motifs. Notably, only 9.94% of these peaks contained ETS motifs  to the list of DEGs 5186 genes). This revealed 540 putative direct target genes with 545 peaks in their TSS regions , a transcriptional repressor of vegfa in cancer cells26, and klf4 (Krüppel-like factor 4), a transcriptional activator of vegfa in endothelial cells and a regulator of metastatic processes28, are known to exert their functions through direct binding to the VegfA promoter27. In Xenopus somites, their expression is respectively repressed and activated by Etv6  are represented by two distinct homologous copies which can be regulated differently40. ChIP-seq tracks in Fig. foxo3.L and foxo3.S and Etv6 binding in these regions was confirmed by ChIP-qPCR , both sequences had the capacity to induce luciferase activity in wild type embryos, with the foxo3.S sequence exhibiting a stronger transcriptional activity 42 in the promoter region of Xenopus vegfa distantly localized upstream the Etv6 ChIP peak 28 to activate VegfA expression through direct binding to its promoter. We, therefore, hypothesized that Etv6 could activate vegfa expression in the somites through Klf4.The tes Fig. . InteresXenopus klf4 genes, klf4.L and klf4.S . Finally, to further confirm that the transcriptional activity of the klf4 promoter is regulated by Etv6 in the somites, the DNA fragment corresponding to the Etv6 peak in the klf4 promoter was functionally tested in in vivo luciferase assays in both wild-type and Etv6-deficient backgrounds. We observed a significant decrease of the transcriptional activity of this sequence in Etv6-deficient embryos  as well as TALENs (transcription activator-like effector nucleases) which efficiently generate null mutations in the second exon of klf4 . Analysis of the genomic structure of vegfa.S indicated that it is a gene undergoing degeneration and it is not transcribed during embryogenesis . Thus, only one vegfa gene, vegfa.L, is functional in X. laevis (referred to as vegfa hereafter) and bound by Etv6 at its TSS. To test whether vegfa could be directly bound by Etv6, we performed Etv6 ChIP-qPCR on the vegfa promoter and confirmed the enrichment of Etv6 in this region , these are Ets2, shown to down-regulate VegfA expression in HUVECs treated with arsenic trioxide (ATO)39, the tumour suppressor Rbl2 , that inhibits tumour formation in mice by inhibiting VegfA-mediated angiogenesis37, the transcriptional repressor Mecp2, that represses VegfA expression by binding to methylated CpG islands in the VegfA promoter35, and the transcriptional regulator Creg1, that regulates human endothelial homoeostasis in vivo and promotes vasculogenesis in mouse ES cells through the activation of VegfA36. These findings validate our experimental design and the robustness of the list of Etv6’s direct target genes.To establish how Etv6 controls 24. Surprisingly, in Xenopus somites, around two thirds  of Etv6 direct target genes are normally transcriptionally activated. Therefore, during the early stages of specification of the HSC lineage, Etv6 acts both as a transcriptional activator and a repressor. As detailed below, molecular and functional examination of Etv6 target genes revealed that Etv6’s dual function is required for the regulation of vegfa expression.ETV6 and its Drosophila orthologue, Yan, are well-established transcriptional repressors44. As all ETS TFs bind to the same motif, additional mechanisms regulating the selection of specific transcriptional targets within biological contexts are required. It has been suggested that high-affinity ETS motifs found in the promoter of housekeeping genes can be bound by any ETS TF whereas lower-affinity ETS-binding sites found in tissue-specific promoters and only bound by a subset of ETS TFs are flanked by binding sites of other TFs46. Cooperative binding with other TFs in sequences with composite binding sites results in a higher affinity and stable binding to DNA, and in synergistic repression or activation of specific target genes48.Members of the ETS family of TFs are defined by a highly conserved ETS domain that recognizes a core sequence 5’-GGA(A/T)-3’ motif within the context of a 9- to 10-bp DNA sequenceHere, de novo motif analysis of the 545 peaks associated with the promoters of genes normally activated or repressed by Etv6 showed (i) high prevalence of Klf/Sp binding sequences in both sets of targets; (ii) absence of ETS-binding motif enrichment in the peaks associated with genes repressed by Etv6; and (iii) ETS-binding motif enrichment in the peaks associated with a small fraction of genes activated by Etv6 (9.94%). This suggested that members of the Klf/Sp family are required for both the activating and repressive transcriptional activities of Etv6 and that Etv6 could be recruited to DNA through its interaction with other DNA-binding TFs. What determines Etv6 to act either as a transcriptional repressor or an activator remains to be fully explored.klf4 was one of the target genes activated by Etv6 in somites. KLF4 is a multifunctional TF which is critical for pluripotency, tissue homoeostasis, stemness, maintenance of cancer stem cells and normal hematopoiesis49, and has previously been implicated in the expression of VegfA in human and mouse cells both positively and negatively. KLF4 binds the VegfA promoter and represses Vegfa expression during EMT in mouse mammary gland cells50, whereas, in HRMECs and HUVECs, it promotes angiogenesis by transcriptionally activating VegfA28. Here, we show that Klf4 not only binds to conserved Klf/Sp motifs but also recruits Etv6 to the vegfa promoter region to activate vegfa expression. Our in vivo and in vitro co-immunoprecipitation (co-IP) assays indicate that these two proteins form part of the same transcriptional complex controlling gene expression in the Xenopus somites but are likely to interact indirectly. In support of this, it has been shown, in overexpression experiments with tagged proteins, that the ETS protein ERG co-immunoprecipitates with KLF251. Similarly, in situ proximity ligation assays have demonstrated that KLF4 and the phosphorylated form of the ETS TF, ELK1, physically interact in human coronary artery cells52. Moreover, analysis of EHF  ChIP-seq peaks indicates that a significant number of peaks contained no ETS-binding motifs and many of them harboured Klf4/5 binding motifs only53. Interestingly, EHF is involved in the maintenance of corneal transparency through the repression of angiogenic factors such as VegfA53. In conclusion, the mechanistic relationship between Etv6 and Klf4 in regulating vegfa expression is compatible with a coherent feed-forward loop, a regulatory circuit where two pathways downstream of a TF control expression of the same gene: Etv6 activates klf4, Klf4 binds and activates vegfa and Etv6 binds vegfa55 42 in the Xenopus vegfa promoter region, and negatively regulates its expression in the somites as (i) overexpression of this TF results in robust vegfa promoter occupancy and repression of vegfa expression, and (ii) downregulation of foxo3 in Etv6-deficient embryos rescues vegfa expression. We propose that this double negative gate57 (Etv6 repressing Foxo3 that represses vegfa), together with the feed-forward loop described above, unlocks the endothelial cell fate in the lateral plate mesoderm through tight control of vegfa expression  and linker domains. These domains can repress transcription independently through different mechanisms. The linker domain represses transcription by complexing with co-repressors which recruit histone deacetylases (HDACs)vegfa expression in the somites unveils the foundations of a complex Etv6 GRN. It indicates that Etv6 has the capacity to regulate vegfa expression through the deployment of pathways involving both positive and negative regulators of gene expression and begins to unveil the complexity of vegfa regulation during hematovascular development. This work also further strengthens the parallel between oncogenic and developmental processes13. We propose that this Etv6-vegfa GRN is conserved through vertebrate evolution, from Xenopus to human, and that Etv6 uses, depending on tissue and context, particular components and/or branches of this network to ensure the correct levels of vegfa expression. Further investigation will establish whether Klf4, or additional members of the Klf/Sp family of TFs, are required for the regulation of some of the other Etv6 targets identified in our study and known to regulate Vegfa in the tumorigenic processes. If so, studying these molecular mechanisms could lead to new strategies for the treatment of pathological processes driven by the Etv6-vegfa GRN.In conclusion, our investigation of the functions of Foxo3 and Klf4 in Xenopus laevis embryos were injected in 0.35 × MMR  supplemented with 3% Ficoll. After 6 h, embryos were washed and cultured in 0.1×MMR at 17–19 °C59 until they were collected for analysis. Embryos were staged according to Nieuwkoop and Faber60. The somites of stage 22 embryos were manually dissected in 0.35 × MMR using forceps, these explants also contained the hypochord, neural tube, notochord, and some dorsal endoderm.Animal experiments were performed according to UK Home Office regulations under the appropriate project licence and approval of the University of Oxford Animal Welfare and Ethical Review Body. Wild type (WT) 59. Briefly, embryos were fixed overnight in MEMFA  and then dehydrated to 100% methanol and stored at −20 °C. Embryos were rehydrated through a methanol gradient in PBST  and then incubated in a 5% formamide, 0.5% SSC, and 10% H2O2 solution to remove embryo pigmentation. Bleaching solution was washed with PBST (3 × 5 min). At this point, stage 39 embryos were treated with 8 µg/ml Proteinase K for 1 min and then washed in glycine  to inactive Proteinase K (younger embryos do not require Proteinase K treatment). Embryos were then washed in PBST (3 × 5 min) and incubated in 0.1 M Triethanolamine-HCl  before treating with acetic anhydride . Embryos were rinsed with PBST (3 × 5 min) and then pre-hybridized in hybridization buffer  for 6 h at 67 °C before incubating in 0.5 ml hybridization buffer containing DIG-labelled probes (1 µg/ml each) for 16 h at 67 °C. After hybridization, embryos were sequentially washed in: (i) 50% formamide/5xSSC, 67 °C, 10 min; (ii) 25% formamide/2xSSC, 65 °C, 10 min; (iii) 12.5% formamide/2xSSC, 63 °C, 10 min; (iv) 2×SSC, 0.1% Tween, 61.5 °C, 10 min; (v) 0.2×SSC, 0.1% Tween, 60 °C, 30 min. After washing in PBST (3 × 5 min) and MAB buffer , embryos were blocked for 6 h in MAB blocking solution (MAB buffer + 2% Boehringer Blocking Reagent). After blocking, embryos were incubated overnight at 4 °C in MAB blocking solution containing anti-DIG-AP antibody . Next, embryos were washed in MAB buffer (6 × 1 h) while rocking gently, followed by 3 × 5 min washes in AP buffer  and then stained in BM purple solution (diluted 1:1 with AP buffer) in the dark. After staining, embryos were fixed in MEMFA for 1 h, dehydrated in 100% methanol and stored at -20 °C. Before photography, embryos were cleared in benzylbenzoate:benzyl alcohol (2:1). All procedures were performed at room temperature unless otherwise indicated.For probe details see Supplementary Table Whole-mount in situ hybridization (WISH) was performed using a standard protocolEtv6 MO targeting both etv6.L and etv6.S was previously published12. A klf4 MO targeting both klf4.L and klf4.S was designed  and the other targeting foxo3.S (foxo3.S MO), were co-injected in a 1:1 ratio in order to generate Foxo3-deficient embryos  were obtained from GeneTools LLC . ned Fig. . As sing61. TALEN sequences were designed using the online design tool, Mojo Hand . TALEN mRNA for injection was generated by linearizing the plasmids with NotI and transcribing with SP6 RNA polymerase using the mMESSAGE mMACHINE Kit (Ambion). TALEN mRNAs were injected at the 1-cell stage of development. To determine the mutagenesis caused by the TALENs, DNA was extracted from the somites of stage 22 TALEN-injected embryos and the genomic DNA fragment containing the TALEN binding sites was amplified and Sanger sequenced  was used to construct the TALEN plasmids containing the homodimer-type FokI nuclease domainced Fig. .X. laevis etv6 and foxo3, including the sequences targeted by the MOs . Etv6-HA, foxo3-HA and klf4-HA mRNAs were synthesized using mMESSAGE mMACHINE T3 Kit (Ambion). mRNAs were injected at the 2-cell stage of development.To make mRNA for injection, the full-length coding sequence for X. laevis Etv6 antibodies were generated in collaboration with NovoPro Bioscience Inc. . In brief, the amino acid sequence of X. laevis Etv6 (GeneBank number EU760352) was analyzed using a proprietary algorithm, NovoFocusTM antigen design, to identify epitopes with good hydrophilicity, surface probability and high antigenic index. Based on this information, three epitopes with little or no conservation with other proteins, including ETS TFs, were selected  at a 1:1000 dilution. HA-tagged protein was detected with anti-HA antibody -G) at a 1:500 dilution. Histone H3 protein was detected using anti-Histone H3  antibody at a 1:100,000 dilution. GFP protein was detected using anti-GFP antibody  at a 1:5000 dilution. Lmo2 antibody  was used in the immunoprecipitation assays. Myc-tagged protein was detected with anti-Myc antibody  at a 1:1000 dilution. Foxo3 antibody  was used for ChIP-qPCR. Clean-blot IP detection kit  was used as a secondary antibody in the Western blot for the immunoprecipitation experiments and was used at a 1:200 dilution. Protein extracts were prepared by lysing 10 somites from stage 22 embryos in 50 µl of oocyte lysis buffer , and centrifuging at 13,000 rpm at 4 °C for 10 min. An equal volume of 2xRSB  was added to the supernatants and boiled at 100 °C for 5 min and stored at −20 °C. 20 µl of protein extract  was ran in SDS-PAGE gels and transferred to 0.2 µm nitrocellulose. Membranes were blocked with 1% milk in PBST, and probed with appropriate primary antibodies and horseradish peroxidase-conjugated secondary antibodies. Uncropped blots for Figs. Abcam, ab06629 at 63 with some modifications. Briefly, the somites of 40 stage 22 embryos were dissected, homogenized, using a pestle, in 1 ml ice-cold PBS containing protease inhibitors and 20 mM sodium butyrate, and centrifuged at 1300 rpm for 5 min at 4 °C. Pellet was re-suspended and cross-linked in 2 mM EGS (ethylene glycol bis(succinimidyl succinate)) for 20 min at RT, followed by fixation in 1% formaldehyde for 20 min at RT and gentle rocking before treating with glycine  for 5 min. Samples were centrifuged  and pellets rinsed twice with cold PBS, lysed and chromatin sonicated to an average size of ~200 bp using Covaris S220 . Samples were centrifuged for 10 min at 14,000 × g and supernatant recovered. After saving 1% of each sample as input, Triton X-100  was added to the supernatant and incubated overnight at 4 °C with slow shaking in the presence of anti-Etv6 antibody already bound to Dynabeads protein A. Beads were washed in RIPA buffer  followed by 2 washes (5 min) in TEN buffer . Complexes were eluted in 1% SDS elution buffer  by shaking the Dynabeads for 15 min at 65 °C in a thermomixer set to 1000 rpm. Eluted samples were centrifuged at full speed for 1 min at RT. The supernatants and the reserved input samples were de-crosslinked by adding 1/20 volume of 5 M NaCl and incubating at 65 °C overnight. After treating with RNase A  and Proteinase K , DNA was extracted using phenol:chloroform:isoamyl alcohol purification followed by ethanol precipitation. Immunoprecipitated DNA and input DNA were quantified using Qubit Fluorometer and sequencing libraries were constructed with 1 ng of DNA using NEBNext Ultra DNA Library Prep Kit (NEB#E7370) for Illumina sequencing. DNA was sequenced  using NextSeq 500. Sequencing was performed on three independent biological replicates with corresponding inputs as control.Etv6 ChIP-seq was performed using a standard procotolTrimmomatic (Version 0.32)64, and then mapped to the X. laevis V9.140 with BWA version 0.7.12. Peaks were analyzed using MACS2 peak calling software65 at default thresholds, with the input samples as control for each replicate. The package DiffBind (Bioconductor package)29 was employed to identify consistent peaks between replicates using the function dba.peakset with the parameter minOverlap = 3. The consensus peak output of DiffBind was generated by adding and merging the three replicate peak sets. Peaks located in TSS regions were identified using Homer software and used to perform TFs binding site analysis with Homer software (findMotifsGenome.pl)30. Examples of the Etv6 peaks for individual replicates are shown in Supplementary Figure vegfa promoter region (−2 kb to +1 bp) and the Etv6 peak region (−552 to −244 bp upstream of vegfa.L TSS), DNA sequences were analyzed using Jaspar41. ChIP-seq data were visualized on the Integrative Genome Viewer (IGV).Raw sequence reads were checked for base quality, trimmed and filtered to exclude adapters using Total RNA was extracted from somites dissected from 20 WT or Etv6-deficient embryos at stage 22. Triple biological replicates were generated. Indexed libraries were constructed with 1 µg of total RNA using the KAPA Stranded RNA-seq Kit with RoboErase (KK8483) and NEBNext Mutiplex Oligos (NEB#E7500S) for Illumina sequencing. DNA was sequenced  using NextSeq 500.Trimmomatic (Version 0.32)64 and then mapped to the X. laevis V9.140 using STAR66 with default parameters. Aligned read features were counted using Subread tool: featureCounts method (version 1.4.5-p1). Differential gene expression analysis was carried out using EdgR (Bioconductor Package)67. For this analysis, samples were CPM (counts per million)-normalized in order to filter out genes with low counts and, then, TPM (transcript per kilobase million)-normalized for differential expression analysis. CPM normalization accounts for library size differences between samples and produces normalized values that can be compared on an absolute scale.Sequenced reads were checked for base qualities, trimmed and filtered to exclude adapters using 68 was performed between WT and MO samples, using the generalized linear model69 followed by Estimates Dispersion, fitted with negative binomial model and estimates Generalized linear model likelihood ratio. Genes with a false discovery rate (FDR) above 0.05 were filtered out. The functional annotation  of Etv6-regulated transcriptome was performed using DAVID Bioinformatics Resources70.An ANOVA-like test analysisodc1 gene. Details of primer sequences for RT-qPCR and ChIP-qPCR are indicated in Supplementary Tables qPCR (quantitative PCR) was performed using Fast SYBR Green Master Mix  and StepOne Real-Time PCR system. For RT-qPCR, cDNA was made from 1 µg of total RNA and relative expression levels of each gene were calculated and then normalized to vegfa promoter region containing a conserved Foxo3 binding site as well as the klf4 and foxo3 promoter regions containing Etv6 peaks were amplified from X. laevis genomic DNA by PCR using primers described in Supplementary Table vegfa promoter fragment. All the produced sequences were subcloned into the pGL4.10 vector (Promega). 100 pg reporter plasmid DNA together with 5 pg Renilla luciferase construct pRL1-TK (Promega) and etv6 MO or foxo3 mRNA, were co-injected into 2-cell stage embryos. Somites were dissected at stage 22, and assayed for luciferase activity using the Dual Luciferase Reporter Assay Kit (Promega).The Xenopus Etv6-HA, Klf4-HA, Ldb1-Myc, mouse Lmo2, and GFP proteins were synthesized from mRNAs . After sonication for 20 min at 4 °C using Bioruptor Plus (30 s on and 30 s off for each round of sonication), the extracts were centrifuged at 13,000 rpm at 4 °C for 10 min. After saving 1% of each sample as input, the supernatants were incubated overnight at 4 °C with slow shaking in the presence of anti-Etv6 or IgG antibody already bound to Dynabeads protein A. After washing three times in lysis buffer containing protease inhibitors, protein complexes were eluted in 1% SDS elution buffer by shaking the Dynabeads for 15 min at 65 °C in a thermomixer set to 1000 rpm. Eluted proteins were separated by SDS-PAGE and transferred onto a 0.2 μm nitrocellulose membrane for western blotting. For in vitro co-IP, synthesized proteins were first incubated in lysis buffer for 1 h at 4 °C. After saving 10% of each sample as input, the lysates were used for immunoprecipitation as described above.Embryos were injected with t-test was performed .In RT-qPCR and ChIP-qPCR experiments, error bars represent Standard Error of Mean (±SEM). The data shown summarize the results of three biological replicates. Two-tailed Student’s WISH images and numbers shown in Figures are from one experiment and are representative of three biological replicates.No statistical methods were used to predetermine the sample size.No data or animal have been excluded from this study.Xenopus laevis embryos were allocated to experimental groups on the basis of different treatments and randomized within the given group. Investigators were not blinded to the group allocation during experiments and outcome assessment.Further information on experimental design is available in the Supplementary InformationSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Supplementary Data 6Peer Review FileReporting SummaryDescription of Additional Supplementary Files


AGAGAGAGGA/motif RB and AGAAAAATTC/motif RV) and one K8 binding motif (i.e. AAAATGAAAA/motif KB), respectively. The binding of K-RTA/K8 proteins with these motifs and resulting transcriptional modulation of downstream genes was further confirmed by DNA electrophoretic gel mobility shift assay (EMSA), reporter promoter assay, Chromatin Immunoprecipitation (ChIP) assay and mRNA quantitation assay. Our data conclusively shows that K-RTA/K8 proteins specifically bind to these motifs on the host/viral genomes to modulate transcription of host/viral genes during KSHV lytic reactivation.The oncogenic Kaposi’s sarcoma-associated herpesvirus (KSHV) has two distinct life cycles with lifelong latent/non-productive and a sporadic lytic-reactivating/productive phases in the infected immune compromised human hosts. The virus reactivates from latency in response to various chemical or environmental stimuli, which triggers the lytic cascade and leads to the expression of immediate early gene, i.e. Replication and Transcription Activator (K-RTA). K-RTA, the latent-to-lytic switch protein, activates the expression of early (E) and late (L) lytic genes by transactivating multiple viral promoters. Expression of K-RTA is shown to be sufficient and essential to switch the latent virus to enter into the lytic phase of infection. Similarly, the virus-encoded bZIP family of protein, K8 also plays an important role in viral lytic DNA replication. Although, both K-RTA and K8 are found to be the ori-Lyt binding proteins and are required for lytic DNA replication, the detailed DNA-binding profile of these proteins in the KSHV and host genomes remains uncharacterized. In this study, using chromatin immunoprecipitation combined with high-throughput sequencing (ChIP-seq) assay, we performed a comprehensive analysis of K-RTA and K8 binding sites in the KSHV and human genomes in order to identify specific DNA binding sequences/motifs. We identified two novel K-RTA binding motifs, (i.e.  Infectious agents account for approximately 15% of all human cancers per year worldwide . Kaposi’KSHV lytic DNA replication initiates at the lytic origin of replication (ori-Lyt) and requires both viral as well as cellular trans-acting elements. Two ori-Lyts (i.e. ori-Lyt-L/R) have been identified in the KSHV genome and KSHV viral K-RTA and K8 proteins have been demonstrated as ori-Lyt-binding proteins. K-RTA, also referred to as the latent-to-lytic master switch protein, is an immediate-early protein that activates the early/E and late/L lytic genes by transactivating multiple viral promoters. Multiple studies have shown that K-RTA is a direct transactivator of specific viral promoters even in the absence of other viral lytic genes . AnotherIn this study, we performed a chromatin immunoprecipitation assay coupled with high throughput sequencing (ChIP-seq) to comprehensively characterize the binding sites of these two DNA binding proteins, K-RTA and K8 of KSHV onto the viral and host genomes. These binding sites were further confirmed by ChIP-quantitative real-time PCR analyses. The identified host and viral binding sites were analysed to discover the motifs specific for K-RTA or K8 bindings. Furthermore, the ability of K8 and K-RTA to bind to these motif sequences and modulate transcription of downstream ORFs was confirmed by reporter promoter assay and DNA electrophoretic mobility shift assays. Finally, the effect of K-RTA and K8 on transcription of set of six representative host genes containing the discovered motifs in their promoter was analysed. Overall, our findings revealed novel viral and cellular DNAs associated with K-RTA and K8 proteins during lytic DNA replication that could be further exploited to understand the mechanisms of KSHV reactivation and associated tumorigenesis.HEK293 cells were purchased from ATCC and maintained in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum , 2 mM L-glutamine, 25 U/mL penicillin, and 25 μg/mL streptomycin. The KSHV-positive/BCBL-1 and KSHV-negative/BJAB cells were maintained in RPMI medium supplemented with 10% FBS, 2 mM L-glutamine, 25 U/mL penicillin, and 25 μg/mL streptomycin. TRExBCBL-1/RTA cells were provided by Dr. Jae Jung  and were maintained in RPMI supplemented with 10% FBS, 2 mM L-glutamine, 25 U/mL penicillin, 25 μg/mL streptomycin, and 20 μg/mL hygromycin B. The iSLK RGB (containing KSHV BAC16) cells were provided by Dr. Jae Jung and were maintained in DMEM with 10% FBS, 2 mM L-glutamine, 25 U/mL penicillin, 25 μg/mL streptomycin, 600 μg/mL hygromycin B, 1 μg/mL puromycin and 250 μg/mL G418. The pLVX-GFP-RTA and pXi-K8-FLAG plasmids were described previously. The reporter vector, pGL4.23 luc2/minP for determining the activity of a minimal promoter was purchased from Promega (Promega Corporation). The sequences of K-RTA binding to the viral genome (RV) was cloned into pGL4.23 luc2/minP to obtain pGL4.23-RV. Similarly, K-RTA binding on cellular genome (RB) and K8 binding region (KB) was cloned into pGL4.23 luc2/minP to obtain pGL4.23-RB, and pGL4.23-KB. The integrity of the clones were confirmed by sequencing. K-RTA and K8 antibody were obtained from Dr. Izumiya  and Dr. Rossetto’s laboratory , respectively. The mouse anti-control IgG antibody (ChIP grade # C15400001-15) was purchased from Diagenode Inc.Approximately, 20 million TRExBCBL-1/RTA induced with doxycycline for 24 hours (h) were harvested and fixed using 1% formaldehyde for 10 mins at room temperature, followed by addition of glycine at a final concentration of 125 mM for 5 mins to block the cross-linking. The cells were rinsed three times with ice-cold PBS and lysed in cell lysis buffer  supplemented with protease inhibitors for 10 mins on ice. The nuclei were enriched by low speed centrifugation and the obtained pellets were resuspended in nuclear lysis buffer . Chromatin was sonicated to an average length of 500 to 800bp and centrifuged for 10 mins at 13,000 rpm to remove the cell debris. Samples were diluted 6-fold with ChIP dilution buffer  and pre-cleared with Protein A/G sepharose beads (GE Biotechnology) pre-treated with 1 mg/mL BSA and 1 mg/mL sheared salmon sperm DNA (GE Biotechnology) at 4°C for 30 mins. For each immunoprecipitation, the lysate was incubated with 10 μL of each antibody at 4°C overnight. No-antibody and antibody-isotype control immunoprecipitations were also performed. Protein A/G sepharose beads were blocked with 10 μg of sheared salmon sperm DNA and BSA at 4°C overnight and then washed with ChIP buffer. The blocked and washed protein A/G sepharose beads were incubated with the lysate at 4°C for 2 h. The beads were washed once with low-salt buffer , once with high-salt buffer , once with LiCl buffer , and twice with Tris-EDTA. Beads were then resuspended in TE, incubated with RNase A at 37°C for 30 mins, treated with proteinase K/10% SDS at 37°C for 4 h, followed by incubation at 65°C overnight. For input control samples, NaCl was added to the sonicated lysate to a final concentration of 0.3 M and incubated at 65°C overnight. The antibody, no-antibody, and isotype control immunoprecipitated samples were extracted with phenol-chloroform and precipitated using ethanol. The enriched DNA was quantified using a Qubit spectrophotometer and 10 ng of total DNA was used with the ChIP-seq DNA library preparation kit (Bioo Scientific), according to the manufacturer’s instructions. The ChIP-seq libraries were validated using a Bioanalyzer 2100 with a high-sensitivity DNA ChIP (Agilent) and quantified by qPCR (Kappa kit). ChIP-seq libraries were sequenced using a MiSeq (Illumina). The resulting sequencing data were analysed for determining the ChIP peaks using CLC Workbench (CLC Bio).32P-labeled probes containing specific K-RTA or K8 binding motifs sequences. The probes were synthesized as double-stranded DNA containing three copies of motif sequences in tandem . These probes were end-labeled with α-32P and purified on a GE Illustra ProbeQuant G-50 micro column . A 50 μl DNA-protein binding assay mix containing 12 mM HEPES (pH 7.9), 12% glycerol, 60 mM KCl, 5 mM MgCl2, 0.12 mM EDTA, 0.3 mM DTT, 0.1 μg poly (dI/dC), and 0.05 μg of denatured salmon sperm DNA as carrier was used for EMSA. For detecting the super shift, K-RTA or K8 nuclear extracts were incubated with anti K-RTA or anti-K8 antibody along with the labeled probe. The reaction mix was incubated on ice for 30 mins and the bound complexes were resolved on 5% non-denaturing polyacrylamide gel in 0.5X TBE buffer . The gel was run at 4°C at 100V for 10–12 h and then exposed overnight on a phosphoimager screen. The signals were detected by autoradiography .Nuclear extracts containing K-RTA and K8 from HEK293 cells were prepared by transiently transfecting K-RTA and K8 expression plasmids. Binding of these proteins to DNA were evaluated by EMSA using HEK293 cells were transiently co-transfected using appropriate pGL4.23, internal control pRL-TK Renilla luciferase-expressing plasmid constructs (Promega), and expression vectors for eukaryotic expression of K-RTA or K8. The 4 μg of DNA was used per sample and all the transfections for reporter assays were done using metafectene (Biontex Laboratories GmbH), according to the manufacturer’s protocol. Following 48 h post-transfection, cells were harvested, lysed in cell lysis buffer (Promega) and 50 μl of the cell lysate was used for the reporter assay using dual luciferase reporter assay kit . The relative luciferase activity was calculated by normalizing K-RTA and K8 luciferase activity to that of control Renilla luciferase activity to account for the transfection efficiencies. The results were plotted as a percentage of the activity to that of the empty pGL4.23 vector. A portion of the cell lysates was used for western blotting to detect K-RTA, K8 and GAPDH. All experiments were repeated multiple times, and the data shown are means of three independent experiments.CT method as described earlier [For quantitative Reverse-Transcriptase PCR assays, total mRNA from the cells was extracted using Illustra RNA spin Mini kit , according to the manufacturer’s instruction and cDNA was made using High-Capacity cDNA reverse-transcription kit . Each PCR reaction consisted of 10 μl of 2X SYBR GREEN Universal master mix , 5 μl of forward and reverse primers (0.5 μM), and 5 μl of the sterile water diluted cDNA. The qRT-PCR was performed to analyse the expression of RAB2A, NAP1L1, HDX, MBL2, SLITRK3, DSERG1, COL4A3BP, DMBT1, CDC7, MAGEC3, UBE3A and ROCK1P1 on an ABI Step One plus real-time PCR instrument  and relative gene copies or the transcripts were calculated by the ΔΔ earlier . Table 1Briefly, 20 million TRExBCBL-1 RTA and iSLK RGB cells were induced with doxycycline or doxycycline and sodium butyrate, respectively, for 24 h. The cells were subjected to cross-linking with 1% formaldehyde followed by re-suspending them in the chromatin shearing buffer after washing with PBS. Chromatin Immunoprecipitation was performed using the iDeal ChIP-qPCR kit for transcription factors (Diagenode Inc.). The ChIP enriched DNA was subjected to qRT-PCR, performed with 10 μl of 2X SYBR GREEN Universal master mix , 5 μl of forward and reverse primers (0.5 μM), and 5 μl of the sterile water diluted ChIP-enriched DNA. Specific primers corresponding to the region of RTA and K8 binding sites on viral and host genomes were used, which are listed in The next generation sequence data of ChIP-seq data for RTA and K8 are available at the NCBI GenBank under the accession numbers GSE82283.In order to identify the genome-wide K-RTA binding sites on the KSHV and human genomes, chromatin immunoprecipitation (ChIP) with K-RTA and next generation sequencing (ChIP-seq) of the DNA was performed on KSHV-infected tetracycline-inducible TRExBCBL-1/RTA stable cells. The lytic cycle was triggered by treating the cells with doxycycline (1μg/ml) for 24 h and performing ChIP using anti-K-RTA antibody. The purified ChIP DNA was used for making ChIP-seq libraries using ChIP-seq kit (Bioo scientific). The libraries were sequenced using MiSeq (Illumina) and the sequences were mapped to the reference KSHV genome using ChIP-seq analysis tool of the CLC Genomic Workbench (CLC Bio). The resulting sequencing reads were mapped to the reference KSHV genome (NC_009333) and reference human genome (hg19) using CLC Genomics Workbench software. The ChIP-seq analysis was performed using reads from the input samples as reference in the analysis. Isogenic control antibody, IgG was used in the ChIP assay and the reads were also mapped to the viral genome. Importantly, K-RTA ChIP showed specific peaks on the viral genome as compared to the control antibody (IgG) with some previously known sites including the ori-Lyt regions . MappingOur results indicated that K-RTA binds to KSHV genomic DNA upstream of K4.1, K4.2, T1.5, K6, PAN RNA, ORF59, K12, and K15 . To veriWe also performed ChIP-seq analysis to discover genome-wide binding sites of K8 on lytically active KSHV genome as well as on the host genome. The virus reactivation was triggered by treating the cells with doxycycline (1μg/ml) and cells were harvested after 24 h induction for the analysis. K8-associated chromatin was isolated by ChIP using K8-specific antibody and subjected to Illumina sequencing. The resulting sequencing data was then aligned with the KSHV genome (GenBank accession no (NC_009333) and the human genome hg19 using CLC software. The aligned sequences were then analysed for K8 enriched region on the viral and cellular genomes using input as reference. The comparison of ChIP peaks, K8 specific vs IgG control ChIP data revealed that K8 associates with multiple regions on the KSHV and host genomes . Our anaK8 enriched regions on the KSHV genome were upstream to ORF2, ORF4, ORF6, ORF7, ORF8, ORF10, ORF70, K6, K7, K7, ORF16, ORF17, ORF23, ORF25, ORF28, ORF33, ORF34, ORF39, ORF44, ORF46, ORF49, K8, K8.1, ORF52, ORF54, ORF55, ORF57, vIRF1, vIRF4, vIRF3, ORF59, ORF62, ORF64, K12, ORF73, ORF75, and K15 .AGAAAAATTC/motif RV with a strong central enrichment sequence among the K-RTA bound sequences identified on the viral genome. The analysis of motif RB using TOMTOM motif comparison tool revealed it to be a novel motif without a significant similarity with any previously reported motif in JASPAR CORE database  or RV (AGAAAAATTC) or KB (AAAATGAAAA) in tandem repeats were further established by an electrophoretic mobility shift assay (EMSA). For EMSA, radiolabeled double-stranded RB, RV and KB motifs oligos were used as probe. Protein from nuclear extracts from HEK293 cells transiently transfected with the corresponding transient expression plasmids were evaluated by EMSA for their DNA binding ability, with HEK293 cells transfected with empty vector serving as a control. The radiolabeled RB, RV and KB probe bound to K-RTA and K8 proteins, respectively, and showed a specific shift probe mobility  in a luciferase vector encoding a synthetic firefly luciferase gene luc2 . This vector pGL4.23 carries minimal promoter (minP) with a TATA-box promoter element immediately upstream of the luc2 gene and immediately downstream of the multiple cloning sites. These are referred to as pGL4.23-RB, pGL4.23-RV, and pGL4.23-KB. HEK293 cells were co-transfected with DRA-Luc reporter plasmid, together with increasing concentrations of either K-RTA or K8 expression vectors. Dual luciferase assay was performed according to the manufacturer’s instructions . Our data showed that increasing amount of both K-RTA and K8 proteins resulted in an upregulation (up to 10 fold) of downstream reporter gene expression. The expression of corresponding amount of K-RTA and K8 in equal amount of lysates was also confirmed by western blot for K-RTA, K8 along with GAPDH as a loading control . These rTo further confirm the binding sites of K-RTA and K8, identified using ChIP-Seq, and to investigate their functional significance in the regulation of viral and host gene expression, we performed ChIP-qPCR analyses on TRExBCBL-1 and iSLK RGB cells. We selected 6 host genes each with K-RTA binding motifs  and K8 binding motifs  identified from our ChIP-seq assay. We also chose three viral genes each with K-RTA binding motifs  and K8 binding motifs  in their promoter regions . Relativvia their binding to their specific binding sites, we performed qPCR assays to detect the mRNA levels of genes, which were in proximity to the binding sites identified in our ChIP-seq assay. We selected those 6 representative cellular genes, used in ChIP-qPCR assay  in doxycycline-induced KSHV-positive TRExBCBL-1/RTA stable cells. We identified 21 K-RTA binding sites and 38 K8 binding sites on host genome and 7 K-RTA binding sites and 32 K8 binding sites on the KSHV genome, respectively Tables . Our datAdditionally, K-RTA also binds to KSHV genomic DNA promoter regions upstream to K4.1, K4.2, T1.5, K6, PAN RNA, ORF59, K12, and K15 as well of K6, PAN RNA, ORF 59, K12, and K15 [Correspondingly, our K8 ChIP-seq analysis also showed 32 K8 binding sites upstream of 43 ORFs of KSHV  as well AGAGAGAGGA/motif RB that had a strong central enrichment among the DNA sequences on both host and viral genomes bound by K-RTA as well as AGAAAAATTC/motif RV that had a strong central enrichment among the DNA sequences on viral genome bound by K-RTA . EMSA results consequently confirmed the preferential enrichment of K-RTA and K8 at novel motif sequences identified in our ChIP-seq analysis. Furthermore, our EMSA results consistently showed that K-RTA can directly bind to oligos containing RB or RV motif sequences, whereas K8 can directly bind to KB motif containing oligos. This was also confirmed by the super-shift observed using specific anti K-RTA and K8 antibodies. These data clearly substantiates the ability of RB, RV and KB motifs to bind to KSHV viral proteins K-RTA and K8, respectively .In order to investigate whether enrichment of K-RTA or K8 at their binding motifs can modulate the transcription of downstream target genes, we performed a dual luciferase assay. Our data consistently showed that increasing amount of both K-RTA and K8 proteins led to an upregulation of luciferase reporter gene expression . The expTo confirm the bindings of these two proteins on viral and cellular genomes, we selected gene promoters with K-RTA and K8 binding sites, identified in our ChIP assay and tested their association with these proteins. Our data clearly showed that both, K-RTA and K8 can efficiently bind to the chromatin of those identified regions confirming the validity of our ChIP-seq. In order to understand the functional significance of these interactions onto the viral and cellular genome, in the regulation of genes expression, we performed a qPCR assay on KSHV-positive TRExBCBL-1 RTA and iSLK RGB cells and on KSHV-negative BJAB cells stably expressing K-RTA or K8 proteins. Interestingly, all the selected host genes with K-RTA binding motifs  and K8 binding motifs  in their promoter regions showed upregulation following expression of the indicated proteins . Taken t