EhPgp5 gene in Entamoeba histolytica is induced by emetine stress. EhPgp5 overexpression alters the chloride-dependent currents that cause trophozoite swelling, diminishing induced programmed cell death (PCD) susceptibility. In contrast, antisense inhibition of P-glycoprotein (PGP) expression produces synchronous death of trophozoites and the enhancement of the biochemical and morphological characteristics of PCD induced by G418. Transcriptional gene regulation analysis identified a 59 bp region at position −170 to −111 bp promoter as putative emetine response elements (EREs). However, insights into transcription factors controlling EhPgp5 gene transcription are missing; to fill this knowledge gap, we used deletion studies and transient CAT activity assays. Our findings suggested an activating motif (−151 to −136 bp) that corresponds to a heat shock element (HSE). Gel-shift assays, UV-crosslinking, binding protein purification, and western blotting assays revealed proteins of 94, 66, 62, and 51 kDa binding to the EhPgp5 HSE that could be heat shock-like transcription factors that regulate the transcriptional activation of the EhPgp5 gene in the presence of emetine drug.Transcriptional regulation of the multidrug resistance  The multidrug resistance phenotype (MDR) is a phenomenon in which the cell is able to survive in the presence of a drug and shows cross-resistance to a variety of structurally unrelated drugs across membranes in a wide range of organisms Moons, . The MDRMDR genes (MDR1 and MDR3)  and drug-resistant (clone C2) trophozoites  an ATP-dependent membrane-bound transporter is the major protein involved in the MDR phenotype  that corresponds to a heat shock element (HSE). Gel-shift assays, UV-crosslinking, binding protein purification, and western blotting assays revealed proteins binding to the EhPgp5 HSE that could be heat shock-like transcription factors, which regulate transcriptional activation of the EhPgp5 gene in the presence of emetine drug.To investigate the relevance of ERE to Trophozoites of clones A and C2 (strain HM1: IMSS)  was used to identify potential transcription factor binding sites in the EhPgp5 gene core promoter fragments into multiple cloning sites of the pBSCAT-ACT plasmid . Electroporated trophozoites were transferred into plastic flasks  containing 50 ml of TYI-S-33 medium and were incubated for 48 h at 37°C. Two hours after electroporation, we added 8 μM emetine to clone A or 20 μM emetine to clone C2. CAT activities were measured by two-phase diffusion assay using 5 μg of trophozoite extracts and 200 μl of chloramphenicol (1.25 mM), which were incubated with [14C]-butyril-CoA  for 2 h. Protein concentrations were determined using Bradford's methods  and C2(20)] or absence (clones A and C2) of emetine, following Schreiber et al.'s protocol  were synthesized and labeled using T4 polynucleotide kinase  in the presence of [γ-32 P]-ATP. Specific activity was determined by scintillation counting. Gel-shift assays were performed in triplicate as described previously  and NE from trophozoites of both clones A[8] and C2[20]. Concatemeric polynucleotides were prepared by PCR amplification of the HSE probe. The oligomer was coupled to magnetic particles coated with streptavidin, as described by the manufacturer. Seventy-five micrograms of NE from trophozoites were mixed with the magnetic particles in protein buffer, poly [d (I-C)] and poly-L-lysine. Then, NE was incubated at 4°C for 60 min. After extensive washing, proteins bound to the particles were eluted with elution buffer containing 2 M KCl. All of the fractions were analyzed by 10% SDS-PAGE  and YY1 (−163 to −153 bp) transcription factors and, interestingly, a HSE at −151 to −136 bp  of the p Figure .EhPgp5 gene expression in E. histolytica drug-sensitive [A(8)] and drug-resistant [C2(20)] clones grown in the presence of 8 and 20 μM emetine, respectively, we performed a series of mutations and deletions within the 59 bp region. Different constructs were generated, as described in the Materials and Methods section   displayed significant CAT activity reductions of 80 and 78% in trophozoites from clones A[8] and C2[20], respectively.Transfection assays of all constructs were performed in trophozoites from clones A and C2. In transfected trophozoites of both clones grown without emetine, no CAT activities were detected with any constructs, compared to the CAT activity developed by pA5′A3′CAT plasmid ; pmHSE-TTCPgp5 had three base changes in the inverted repeated TTC and its flanking bases (ATAGAAATTTTTCATA → ATAGAAATTGATGAAA), and in p-sinHSEPgp5, the HSE sequence was completely replaced (GACTGCTCGACGTGAC). We transfected these constructs into trophozoites from clones A and C2 and then cultured them in the absence or presence of 8 or 20 μM emetine, respectively. The results showed that all of the constructs presented CAT activities in trophozoites from both clone cultures without emetine  and C2(20)], suggesting that the changes introduced into the GAA sequence from the HSE did not affect the transcriptional activation. Interestingly, the plasmid pmHSE-TTCPgp5 presented a strong CAT activity reduction in trophozoites from both clones, suggesting that the second repeat of the HSE was indispensable to EhPgp5 gene expression. Moreover, when the HSE was eliminated, CAT reporter gene activity was not detected, either in trophozoites of clone A[8] or in trophozoites of clone C2[20].To demonstrate that the putative HSE (−151 to −136 bp) is responsible for activating EhPgp5 gene, we analyzed DNA–protein interactions using gel shift and competition assays. Gel electrophoretic mobility shift assays were performed to obtain evidence of nuclear protein binding to the putative HSE. Double-stranded synthetic oligonucleotides containing the EhPgp5 HSE were incubated with NE from clones A, A[8], C2, and C2[20]. Additionally, oligonucleotides containing the wild-type consensus HSE and the HSEm mutated at the repeated GAA and the region of 34 bp of the EhPgp5 promoter from −170 to −136 bp, which contains the CdxA, YY1, and HSE sequences, were used as specific competitors. As demonstrated in Figures E. histolytica clones grown in the presence or absence of emetine, forming one DNA–protein complex that had identical electrophoretic mobility using NE from both clones. Interestingly, the intensity of the complex was enhanced when the DNA probe was incubated with NE from the trophozoites from clones A[8] and C2[20]  were found in both clones incubated with or without emetine  by western blot assays of the UV-crosslinked complexes from trophozoites of clones A, A[8], C2, and C2[20] using the human heterologous antibody anti-HSF1. As shown in Figure [20], trophozoites , NE from trophozoites of clones A, Figure , lane 3,] Figure , lane 3. Figures . These r[8] and C2[20]. The complexes were highly specific due to being completely inhibited by a 150-fold molar excess of the same unlabeled wild-type HSE and S34 oligonucleotides (lanes 3 and 5), and 350-fold molar excess of poly [d(I-C)] did not affect complex formation (lane 6). Interestingly, the HSEm did not affect complex formation  have functional relevance in different mechanisms of E. histolytica parasites. On the one hand, the antisense inhibition of gene expression enhances the PCD of trophozoites in the presence of G418; on the other hand, gene overexpression alters the chloride-dependent currents and confers drug resistance to emetine   is head-tail (GAA and TTC), while that of the Ehhsp100 is head-head (AAGGAACTTGAAGAA), containing a gap of four and three bases between them, respectively  in other organisms have demonstrated that the GAA and TTC motifs contain indispensable bases for DNA–protein interactions and consequently for promoter activation  to activate gene expression  translational modifications; (ii) formation of homodimers or homotrimers; or (iii) interactions with other proteins, for example, molecular weight changes in human HSFs by different posttranslational modifications, mainly phosphorylation  to regulate the transcriptional activation of the multidrug-resistant AN: Data acquisition and analysis or for the work. DP: interpretation of data and revision of intellectual content. EO: Revision of intellectual content. VS: Draft the work and revision of intellectual content. CG: Design of the work and revision of intellectual content.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling Editor declared a shared affiliation, though no other collaboration, with all the authors.


Cga and Lhb. Guided by MNase mapping of the promoters of these genes, we reconstitute nucleosomes that mimic those located near the transcriptional start site and immediately downstream (+1), and measure the forces required to disrupt these nucleosomes, and their mobility along the DNA sequence. Our results indicate that these genes are basally regulated by two distinct strategies, making use of H2A.Z to modulate separate phases of transcription, and highlight how DNA sequence, alternative histone variants and remodelling machinery act synergistically to modulate gene expression.The structure and dynamics of promoter chromatin have a profound effect on the expression levels of genes. Yet, the contribution of DNA sequence, histone post-translational modifications, histone variant usage and other factors in shaping the architecture of chromatin, and the mechanisms by which this architecture modulates expression of specific genes are not yet completely understood. Here we use optical tweezers to study the roles that DNA sequence and the histone variant H2A.Z have in shaping the chromatin landscape at the promoters of two model genes,  Chromatin architecture at promoters can influence gene transcription. Here, the authors use a biophysical approach to elucidate how DNA sequence and histone variant usage modulate the properties of nucleosomes at the promoters of two target genes. Nucleosomes reduce the accessibility of the transcription machinery to DNA, acting as obstacles for the binding of transcription factors (TFs) and for the subsequent elongation of RNA polymerase II (Pol2). Hence, nucleosomal stability, position and dynamics act as a first and important layer in the complex modulation of gene expression levels. A general picture of how chromatin packaging is tailored at gene promoters to help regulate expression has emerged from genome-wide mapping of nucleosome positionsOf particular importance is the evolutionarily conserved and essential variant of histone H2A, H2A.Z, which has critical roles in development, differentiation, T-cell activation and moreClearly, the architecture of promoters is the result of the combined effect of a number of factors, including the sequence of DNA591112131416171819202122Cga and Lhb genes encode, respectively, the α and β subunits of luteinizing hormone (LH), a glycoprotein secreted by the anterior pituitary gonadotropes, which plays a pivotal role in regulating development and function of the gonads. LH is positively regulated by the hypothalamic gonadotropin-releasing hormone that activates transcription of both genes, involving a number of modifications to the chromatin262728Cga and Lhb are both expressed in the gonadotropes under similar hormonal control, the α subunit comprises also a part of the follicle-stimulating hormone, as well as part of the thyroid-stimulating hormone expressed in the thyrotropes; thus, their basal levels of expression differ, with the Cga gene being more widely expressed and at higher levels.The Cga and Lhb. Our aim was to elucidate how the structure and dynamics of chromatin at the promoters of these genes are shaped to achieve their relative expression levels. Our strategy was to use the results of bulk measurements on gonadotropes to inform single-molecule in vitro experiments. Hence, guided by MNase mapping of nucleosome positioning at the promoters of Cga and Lhb, we reconstituted nucleosomes that mimic those located near the transcription start site (TSS) and immediately downstream of the TSS (+1). Then, we measured the forces required to disrupt these nucleosomes, and their position along the DNA sequence. Our results shed light on two alternative strategies for gene regulation.In our work we have used single-molecule chromatin ‘unzipping' with high-resolution optical tweezers, in combination with micrococcal nuclease (MNase) mapping of nucleosomal occupancy in cultured cells, to study the roles that DNA sequence and histone variant H2A.Z play in shaping the chromatin landscape at the promoters of Cga and Lhb, we performed quantitative PCR (qPCR) analysis of MNase-digested DNA (MNase-qPCR). We measured occupancy in mature gonadotrope cells (LβT2) that express both genes , which skip over the most probable position as indicated by the high-coverage map. In addition, the +1 nucleosome was positioned immediately downstream of the TSS, between +29 and +179. These positions are consistent, and in good agreement with the DNA-based prediction and the positions in MEF cells. The high-coverage analysis for the Cga gene in gonadotropes revealed that the +1 nucleosome is positioned between +39 and +189, with a NDR immediately upstream of it, a result consistent with the previously observed pattern of +1 nucleosome positioning for active promotersTo obtain a more precise mapping of the nucleosomes found on ome maps  revealedCga +1 nucleosome is positioned further downstream than the +1 nucleosome on Lhb. Such a difference could be a consequence of high Pol2 occupancy at the promoter, as proposed in genome-wide studiesCga as compared with Lhb  to a luciferase reporter and transfected LβT2 cells. The luciferase assay allowed us to look at the activity of these promoters out of their native genomic context and devoid of their native packaging into chromatin. The measured luminescence shows only an ∼1.5-fold higher activity level for the Cga promoter over that of the Lhb  exists between the precise position of individual nucleosomes and the measured H3/H4 breaking force, which was not observed for the 601 sequence or the Lhb TSS. One could speculate that such a correlation may play a regulatory role, for example, allowing remodellers to affect the nucleosome's stability by modifying its position.Interestingly, although an effect for the sequence on the breaking force is not measured when averaged over the ensemble of measured nucleosomes, by looking at individual nucleosomes  it is clCga TSS nucleosome evicted in gonadotropes? Second, what mechanism is used to modulate the expression of Lhb, without affecting the overall positioning of this nucleosome?Overall, the fact that all four measured nucleosomes are relatively stable, and share comparable breaking forces, raises two questions about the regulation of these genes: first, how is the Cga TSS, and that this nucleosome exhibits a similar disassembly pattern as the other nucleosomes measured suggests, as previously shownin vivo is not the result of a DNA sequence that prevents the formation of a nucleosome, but is formed by the action of remodelling machinery. One such remodeller is the chromodomain-helicase-DNA-binding protein 1 (Chd1), whose activity is required to maintain nucleosome occupancy within promoter regions, as seen in genome-wide studies in MEFsCga promoter in gonadotropes, dependent on the presence of a distal enhancer non-coding RNA (eRNA) that is essential for active chromatin at this promoterCga is dependent on Chd1 recruitment, we performed MNase-qPCR in gonadotrope cells stably transfected with shRNA against the eRNA. This manipulation allowed a gene-specific perturbation of the recruitment of Chd1 to Cga, instead of knocking down Chd1 directly, which, due to the widespread roleCga  and 2 . This suggests a universal mechanism of action for H2A.Z, in which it weakens the interactions between both the H2A.Z/H2B dimers and the H3/H4 tetramer with the DNA. Reconstitution with H2A.Z also resulted in a small shift of ∼10 bp upstream, in the mean position of the Lhb TSS nucleosome , although there was no significant change in the mean position of nucleosomes on the Cga +1, or 601 sequence , which utilizes the same mechanism for transcription through a nucleosome in vitro as eukaryotic Pol II  and one binding site of paired-like homeodomain 1 (Pitx1), both of which are required for gonadotrope differentiation and Lhb expressionLhb. Interestingly, since higher mobility can also reduce the time-averaged exposure of other TF-binding sites, our results help shed light on the controversy between the repressive and activating roles of H2A.Z  to fulfil two distinct roles in regulating these genes.The incorporation of H2A.Z into the +1 nucleosome, as observed for cleosome . InteresLhb imposes well-localized nucleosomes, that is, an ordered nucleosome array, so both the TSS and +1 nucleosome positions are dominated by the sequence. This structure blocks TF binding and imposes severe obstacles to transcription so, in gonadotropes, the TSS nucleosome is remodelled with H2A.Z to lower these obstacles. Hence, the efficiency of transcription is dictated by a competition between the repressive characteristics of the sequence and the facilitating properties of H2A.Z  whose properties then control the rate and fate of Pol2 transcription. Hence, to tailor the expression levels of different genes, while also optimizing the use of cellular resources, the cell can utilize combinations of factors and the properties dictated by them. Our results, although specific for the two genes studied, highlight how DNA sequence, the usage of an alternative histone variant and remodelling machinery act synergistically to control the expression of genes via the properties of their promoter chromatin.−1 glucose (LβT2 cells), to which 10% fetal calf serum, 10 mM HEPES, 100 U ml−1 penicillin and 100 μg ml−1 streptomycin were added . The gonadotrope-derived αT3-1 and LβT2 cell lines have been authenticated in our lab to express the cell-specific gonadotropin genes: Cga abundantly in both cell lines; and Lhb at very much higher levels in the latter. This pattern of gene expression is constantly monitored in our lab by qPCR. All cell lines are regularly tested for mycoplasma contamination.Murine gonadotrope-derived αT3-1 and LβT2 cells , sieRNA αT3-1 cells6–1 × 107 cells were washed twice and then collected in cold PBS. After centrifugation , cells were permeabilized with 0.03% Triton X-100 and 10% fetal calf serum in PBS and incubated for 10 min at 37 °C. After centrifugation , cells were resuspended in reaction buffer , 50 mM NaCl and 2 mM CaCl2). The chromatin was digested with MNase  for 15 min and quenched on ice with EGTA to a final concentration of 25 mM, resulting in predominantly ∼150 bp fragments corresponding to mono-nucleosomes . For low-coverage nucleosome position maps, MNase-digested DNA was subjected to real-time PCR with primers designed according to the predicted nucleosome positions, amplifying ∼100 bp non-overlapping fragments of DNA regions between −550 and +250 bp of Cga and Lhb genes  was added for 5 min to quench the crosslinking. The cells were washed twice and then were collected in cold PBS. After centrifugation , cells were lysed with 750 μl lysis buffer (50 mM HEPES·KOH (pH 7.5), 140 mM NaCl, 1 mM EDTA 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS and protease inhibitors). To obtain average DNA fragments of <200 bp, cells then were sonicated 60 times  using a Sonics Vibra-Cell sonicator, while submerged in ice water. The cell debris was pelleted , and 12.5% of supernatant was removed to serve as the input. The remainder was immunoprecipitated by adding lysis buffer (750 μl), primary antibody (3 μg) and protein G magnetic beads , with overnight incubation on a roller at 4 °C. After three washes, the samples were eluted (1% SDS and 100 mM NaHCO3). The crosslinking in the immunoprecipitated and input samples was reversed by incubation at 65 °C overnight with the addition of 2 μl RNase A (0.5 mg ml−1), before purification using a PCR purification kit . Real-time qPCR with gene-specific primers  with reporter gene luciferase constructs (200 ng) of the mouse Cga (−507 to +46) or the mouse Lhb (−755 to +6) promoter sequences in the pGL2 plasmid, and a simian virus 40 (SV40)-Renilla luciferase reporter (2 ng). After 48 h, luciferase assays were carried out using the Dual-Luciferase reporter assay kit (Promega) according to the manufacturer's instructions, and levels of firefly luciferase activity normalized to those of the Renilla.For luciferase assays, cells were grown in 96-well plates and transfected using PolyJet Total RNA was extracted using TRIzol (Ambion), treated with DNase and reverse transcribed with random hexamers (Applied Biosystems) for qPCR using gene-specific primers , AbsolutCga and Lhb nucleosome reconstitution were amplified by PCR from mouse genomic DNA. Sequences for 601 nucleosomesDNA sequences used for E. coli BL-21-codonplus (DE3), grown for 5 h at 37 °C in LB and induced with 0.8 mM isopropyl-β-D-thiogalactoside at 18 °C overnight. A volume of 1 l of cells for each histone pair was collected  and washed twice with cold TBS+phenylmethyl sulfonyl fluoride. The pellet was snap-frozen in liquid nitrogen, thawed, resuspended with 15 ml of tris-buffered-saline (TBS)+phenylmethyl sulfonyl fluoride and 1 mg ml−1 lysozyme, (Sigma) and sonicated 10 times  using a Sonics Vibra-Cell sonicator, while submerged in ice water. The samples were cleared at 20,000g for 10 min and the supernatant was collected and treated with 0.1 M H2SO4 for 1 h on ice. An equal volume of Tris·Cl (pH 8) was added, the samples were cleared again at 20,000g for 10 min and the supernatant was collected. Subsequently, 0.2 M NaCl, 2 mM EDTA and 1 mM dithiothreitol (DTT) were added to final volume of ∼30 ml and the sample was loaded onto a SulfoPropyl cation exchange column (Sigma). The histones eluted ) at the first two fractions and were concentrated using Amicon Ultra (Merck).Histones were purified under acidic conditions as reported for mammalian cells2:1(H3/H4)4 molar ratio, 5 mM DTT was added and histones were snap-frozen with liquid nitrogen and kept at −80 °C. Histone purity and stoichiometry were verified using 12.5% SDS–PAGE stained with coomassie blue (Histone dimers were mixed at a 2(H2A/H2B)sie blue .3 and various amounts of NaCl as follows: 2 M NaCl for1 h; 1.5 M NaCl for 1.5 h; 1 M NaCl for 3 h; 0.75 M NaCl for 2 h; and 0 M NaCl—overnight.Nucleosomes were reconstituted under conditions reportedThe reactions were analysed using 1% agarose gels in 0.2 × TBE, post stained with ethidium bromide (EtBr) , and fra825202123At least three different nucleosome reconstitutions were performed for each nucleosome type. No significant difference was observed between the reconstitutions.3). Analysis of the data was performed using the nanoQ software (Cordouan Technologies).DLS measurements of the reconstituted nucleosomes (0.3 mg DNA per ml) were performed at room temperature with a Vasco DLS system (Cordouan Technologies), in reaction buffer (10 mM HEPES·KOH (pH 7.4), 1 mM EDTA and 0.02% NaNHWe generated two 600 bp DNA handles each incorporating a specific tag (digoxygenin and biotin) using commercially purchased 5′ modified primers in a standard PCR reaction . The oth2, 1 mM DTT, 3% v/v glycerol and 0.01% BSA). Tether formation was performed in situ  by trapping an anti-digoxigenin bead (bound by nucleosomes) in one trap, trapping a 0.9 μm streptavidin-coated polystyrene beads in the second trap and bringing the two beads into close proximity to allow binding of the biotin tag in the nucleosomal DNA to the streptavidin in the bead.Reconstituted nucleosomes were ligated to DNA handles using a rapid ligase system (Promega) in 3:1 molar ratio, 30 min at room temperature. The full construct  was incubated for 15 min on ice with 0.8 μm polystyrene beads (Spherotech), coated with anti-digoxigenin. The binding reaction efficiency was verified in a pull-down assay using 1% agarose gel in 0.2 × TBE post stained with EtBr  The reacet al.w0=4 mm, was split by a polarizing beamsplitter into two orthogonal polarizations, each directed into a mirror and combined again with a second polarizing beamsplitter. One of the mirrors is mounted on a nanometre-scale mirror mount . A X2 telescope expands the beam, and also images the plane of the mirrors into the back focal plane of the focusing microscope objective , ensuring that steering will not result in shifting from the objective aperture. Two optical traps are formed at the objective's focal plane, each by a different polarization and with a typical stiffness of 0.3–0.5 pN nm−1. The light is collected by a second, identical objective, the two polarizations separated by a polarizing beamsplitter and imaged onto two Position Sensitive Detectors (First Sensor). The position of the beads relative to the centre of the trap is determined by back focal plane interferometryExperiments were performed in a custom-made dual-trap optical tweezers apparatus, similar to the set-up used by Moffitt Stretching of naked DNA  tethers was used to find the polymer-model parameters under our experimental conditions. Stretching to 15 pN was used to fit an extensible worm-like-chain model (XWLC) for the stretching of the dsDNA handles, and the data at forces above the unzipping of DNA were used to fit a worm-like-chain model for the released ssDNA.Data were digitized at a sampling rate fs=2,500 Hz, and saved to a disk. All further processing of the data was done with Matlab (Mathworks). From the measured and filtered tether extension and force, the stretching of the dsDNA handles at each time point was subtracted from the measured extension. Then, the extension was divided by the extension of two ssDNA bases  to result in the number of unzipped base pairs.To improve the accuracy of the experiments, a 250 bp naked DNA segment was ligated to the reconstituted chromatin samples, and unzipped before the nucleosomeP value was no larger than 0.05.Differences in breaking forces and nucleosome positioning were checked using a two-sample Kolmogorov–Smirnov test, a non-parametric test that does not require assuming normality of the data. Differences in the variance of the position were checked using the non-parametric two-sample Ansari–Bradley test. Differences were considered statistically significant if the calculated Cga +1 reconstituted nucleosomes), was incubated with 0.2 U of E. Coli RNAP (New England Biolabs). Transcription was initiated and stalled by adding 250 μM ApU (Ribomed) and 50 μM rATP, rGTP and rCTP in transcription buffer  glycerol and 0.02% (v/v) BSA) for 20 min at 37 °C. To prevent multiple rounds of transcription, the reaction was diluted 10-fold and competitor DNA (50 bp DNA containing the T7A1 promoter) was added to 45 nM. Transcription was resumed at room temperature by addition of 1 mM of all four rNTPs for 1 min. The reactions were quenched by addition of 10 mM EDTA. RNA was purified using RNA clean and concentrator-5 kit (Zymo research) with subsequent on-column DNase I (Zymo research) treatment according to the manufacturer's instructions. After purification, RNase inhibitor (New England Biolabs) was added and samples were treated again with 1 U of DNase I (Invitrogen) to eliminate DNA template residuals. The RNA was then reverse transcribed using qScript (Quanta) and quantified using qPCR with primers specific for the full-length RNA product, Perfecta Sybr-Green ROX (Quanta) and the TProfessional Basic Thermocycler Real-time PCR (Biometra). To rule out the possibility of different transcription initiation efficiency between the samples, we exploited the restriction enzyme site of NcoI, located at +25 on the transcription template, relative to the TSS. RNAP should protect DNA from NcoI digestion if it is stalled at +26. To quantify the degree of protection, DNA was purified and subjected to qPCR with specific primers spanning the transcription construct.Transcription experiments were performed under conditions reportedThe data that support these findings are contained within the article or How to cite this article: Rudnizky, S. et al. H2A.Z controls the stability and mobility of nucleosomes to regulate expression of the LH genes. Nat. Commun.7, 12958 doi: 10.1038/ncomms12958 (2016).Supplementary Figures 1-7 and Supplementary Tables 1-7


Drosophila embryogenesis using characterized developmental enhancers. The authors demonstrate that while the timing of enhancer transcription is correlated with enhancer activity, the levels and directionality of transcription are highly varied among active enhancers and conclude that this is likely an inherent sequence property of the elements themselves.Here, Mikhaylichenko et al. investigate the transcriptional properties of enhancers during  Drosophila embryogenesis using characterized developmental enhancers. We show that while the timing of enhancer transcription is generally correlated with enhancer activity, the levels and directionality of transcription are highly varied among active enhancers. To assess how this impacts function, we developed a dual transgenic assay to simultaneously measure enhancer and promoter activities from a single element in the same embryo. Extensive transgenic analysis revealed a relationship between the direction of endogenous transcription and the ability to function as an enhancer or promoter in vivo, although enhancer RNA (eRNA) production and activity are not always strictly coupled. Some enhancers  can act as weak promoters, producing overlapping spatio–temporal expression. Conversely, bidirectional promoters often act as strong enhancers, while unidirectional promoters generally cannot. The balance between enhancer and promoter activity is generally reflected in the levels and directionality of eRNA transcription and is likely an inherent sequence property of the elements themselves.Gene expression is regulated by promoters, which initiate transcription, and enhancers, which control their temporal and spatial activity. However, the discovery that mammalian enhancers also initiate transcription questions the inherent differences between enhancers and promoters. Here, we investigate the transcriptional properties of enhancers during  Transcription in higher eukaryotes is regulated by the interplay of regulatory information encoded in promoters (where gene transcription initiates) and more distal enhancers. Despite sharing some features such as transcription factor (TF)-binding sites, promoters and enhancers have historically been considered as two distinct classes of regulatory elements. The term “promoter” implies the ability to recruit RNA polymerase II (Pol II) and initiate gene expression either at focused transcriptional start sites  or through more dispersed regions (broad promoters) . In contCaenorhabditis elegans, putative enhancers (intergenic TF-bound regions) have bidirectional eRNA transcription but form longer transcripts in the direction of the closest downstream gene , have been extensively studied in mouse and human cells, where several common properties emerged. Mammalian eRNAs are generally nonpolyadenlyated, low in abundance, unspliced, and retained within the nucleus ,b. They eam gene . This reeam gene . This sueam gene . In keepeam gene  and C. e elegans .The functional role of eRNAs remains unclear  and CAGE  data. Our results reveal that active developmental enhancers possess a range of eRNA levels and directionality. We explored the relationship between these properties and enhancer and promoter function using a new in vivo assay, simultaneously assessing both activities for the same element within the same embryo. This revealed that the ability of enhancers to function as promoters in vivo is highly correlated to both their level and directionality of eRNA transcription. Intergenic enhancers transcribed predominantly bidirectionally can generally function as weak promoters in both orientations. However, this was not the case for the majority of elements (both enhancers and promoters) with unidirectional transcription: The direction of endogenous transcription was generally correlated to the orientation in which the element can function as a promoter in vivo. Testing gene promoters for enhancer activity revealed that when bidirectionally transcribed, promoters can function as enhancers in vivo, while unidirectional promoters generally cannot. These proximal bidirectional elements appear to function as both an enhancer and a promoter for the same gene, thereby regulating both their spatial pattern and levels of expression. Taken together, our results indicate that there is a continuum of cis-regulatory activity, with some elements acting strictly as either an enhancer or a promoter, while others function predominantly as an enhancer with weak promoter activity, and yet others  have both strong promoter and enhancer activity. This spectrum of activities is highly correlated with the directionality of their eRNA transcription, which likely reflects the underlying sequence properties of the element.Although much of our understanding of how developmental enhancers function has come from model organisms, including nhancers  and putanhancers  availablDrosophila genome (with an annotated TSS every ∼6.5 kb), many Drosophila enhancers reside within genes (either protein coding or noncoding) and in proximity to the 3′ ends of genes. Drosophila enhancers therefore are often transcribed as a consequence of the activity of RNA Pol II running through the enhancer due to gene transcription; e.g., lncRNAs or transcriptional read-through at the 3′ ends of genes . To specifically measure transcription initiating at enhancers (eRNA) we used very deeply sequenced CAGE data, providing quantitative information on TSS usage at base-pair resolution , and 6–8 h AEL, mainly stages 10–11, when the major lineages within the mesoderm and neuronal primordia are specified (∼52 million mapped reads) . This was complemented by our previously published CAGE data .Due to the compact nature of the solution , and PROsolution , measurisolution . We perfAGE data , performAGE data , we combDrosophila, we first estimated the relative abundance of eRNA using measurements of nascent transcription in both species: The levels of PRO-cap signal in Drosophila embryos (this study) was compared with published GRO-cap  signal in human K562 cells  and estimated transcript abundance at intergenic DNase 1 hypersensitivity sites (DHSs)  . This cod humans A.Drosophila eRNA. In contrast to Drosophila promoters, which are generally directionally transcribed, transcription at putative enhancers in S2 cells appears more bidirectional . The OI represents the fraction of total reads transcribed on the strand with the highest PRO-cap signal  are transcribed with no particular directionality preference, similar to what is observed in mammals , while these motifs have the expected distribution at promoters .R motifs C. We exaR motifs C. This rR motifs C. IntereR motifs C. Taken R motifs  at putatDrosophila and vertebrates, strongly suggesting that they arise from a similar mechanism. Given that eRNAs have also been observed in worms . In addition, we included 63 regions that do not function as enhancers (at least within the tested context) at any stage of embryogenesis .eRNA transcription is highly correlated with enhancer activity and induced gene expression in vertebrates, as seen in a number of studies in both mice and humans . Given t embryos . Both PR embryos  during eogenesis , referreSupplemental Fig. S5A,B). Developmental enhancer transcription in Drosophila therefore is highly correlated with the timing of the enhancer's activity. To confirm that eRNA expression also matches the specific tissue in which the enhancer is active, we dissociated 6- to 8-h embryos and sorted mesodermal cells using FACS to >95% purity and performed CAGE on the isolated RNA . The level of mesoderm-specific eRNA transcription  is significantly higher at enhancers that are active in the mesoderm at 6–8 h compared with enhancers that are inactive in the mesoderm but active in other tissues at these embryonic stages  have significantly higher levels of PRO-cap and CAGE signal compared with inactive enhancers (enhancers that are inactive at 6–8 h of embryogenesis but active in transgenic embryos at earlier or later time points) A,B. Simic stages C. eRNA le models .Supplemental Table S5) and then visualized where active enhancers (126), inactive enhancers (42), and nonenhancers (63) are located along this quantitative spectrum  by their level of PRO-cap signal . We selected eight elements: four with high (arbitrary cutoff >240 PRO-cap reads) and four with low  levels of eRNA. Each element was inspected manually to confirm that no other source of transcription was located in their proximity, which could confound the eRNA signal . Five of these regions were shown previously to function as an enhancer in vivo  and inserted in an orientation where the maximum transcription occurred in the direction of the reporter gene. The derived 16 constructs were stably integrated into the same genomic location (51C landing site of the J27 line) using the φC31 integrase system  and one that has low levels  of endogenous eRNA. This suggests a relationship between the level of eRNA and an enhancer's inherent capacity to act as a promoter in vivo, although we note that the number of tested enhancers is low. In all four cases, the relative intensity of promoter signal was much weaker than that of the enhancer-driven expression, a feature also observed in mammalian cells  . Therefore, when the element is functioning as both an enhancer and a promoter, the TFs that bind to the enhancer are still able to direct Pol II activity in a specific spatial pattern. For the remaining enhancer (CRM3316) : While CRM3316 drives enhancer activity in the mesoderm, it regulates promoter activity in the central nervous system  B, the sps system B. This slacZ, a long (4309 bp), stable, polyadenylated mRNA. Therefore, what we are classifying based on RNA sequencing (RNA-seq) methods as “high” and “low” eRNA likely reflects both eRNA stability and inherent promoter strength. Enhancers that can act as promoters in vivo may have stronger inherent promoter sequences  and/or other properties that contribute to transcript stability.In summary, these results demonstrate that intergenic enhancers can act as weak promoters in vivo. While, in three cases, the element acted as both an enhancer and promoter, driving activity in overlapping patterns, in the fourth case, the enhancer possibly functioned as an ectopic promoter for an external enhancer. We note that not all transcribed enhancers (∼50%) have detectable promoter activity, and this seems to correlate with their levels of eRNA production. In the transgenic assay, promoter activity is measured by in situ hybridization against Drosophila pH Pelican and pH Stinger vectors . However, with this arrangement, it is impossible to discriminate between an enhancer's inherent promoter or enhancer activity, as transcription may originate from the minimal promoter or the candidate region inserted directly upstream of it. To address this, we generated a new vector that can simultaneously assess both enhancer and promoter activity from the same element in the same embryo. The dual enhancer–promoter activity vector contains a multiple cloning site in between an upstream GFP reporter  and a downstream LacZ   does not affect its enhancer activity, we first directly compared the activity of a characterized enhancer (BN5-lf-p) . As expected, placing the enhancer further away from the target gene's promoter (GFP) led to a reduction in the level of gene expression . Importantly, by placing the enhancer downstream from the reporter gene, the enhancer–promoter dual vector can unequivocally distinguish between enhancer and promoter activities.The standard enhancer reporter constructs used for transgenic studies in both flies and mice place the tested enhancer upstream of a minimal promoter. This includes the widely used  vectors  and the romoter) B. If theN5-lf-p)  cloned ipression . HoweverDrosophila , we focused on enhancers with relatively high levels of eRNA (>22 CAGE counts in at least one orientation) but differences in the directionality in which the eRNA is transcribed. We selected 20 DHS regions overlapping in vivo validated or putative enhancers from TF occupancy  was cloned in both the sense and antisense orientation, leading to 41 transgenic lines, including an empty control line. While enhancer orientation (relative to the reporter gene's promoter) is expected to have no effect on enhancer activity, to our knowledge, the effect of orientation on promoter activity (LacZ expression) has not been assessed in vivo.Of the 20 tested regions, 10 were intergenic DHSs (including one enhancer that we tested in the standard upstream assay) , which hGFP expression in specific tissues and stages . In all 10 cases, the enhancer functioned in both orientations, regulating qualitatively similar spatio–temporal activity . Notably, seven out of the 10 intergenic enhancers (including one previously tested in the single activity assay [BN5-lf-p])  can also function as promoters in vivo, regulating weak but spatially restricted LacZ expression. This suggests that at least a fraction of Drosophila enhancers that initiate transcription can function as weak promoters in vivo. We note that as the dynamic range of transcription measured by deep RNA-seq is much higher than that detected by in situ hybridization, we may have missed low levels of promoter activity in our in vivo reporter assay and thereby underestimated this function. In five out of seven cases, the promoter activity at least partially recapitulates the enhancer activity: CRM5130 , CRM1149, and rpr4s4 . In the remaining two cases (CRM1594 and CRM3105) , two somatic and visceral muscle enhancers drive weak promoter expression in the brain, mirroring the result observed for CRM3316 inserted in the same location , suggesting that their promoter activities might be controlled by an external enhancer. Interestingly, both CRM1594 and CRM3105 produced this promoter pattern in an orientation-dependent manner . Why only three of the 27 tested elements  were susceptible to this extrinsic enhancer's activity in the promoter assay is unclear. It may reflect potential enhancer core promoter specificity, as shown in other cases . The majority of tested elements has ∼10-fold higher endogenous transcription in one direction (the direction of the gene's expression) and is therefore highly unidirectional. We used these elements and the 10 intergenic elements, which have a range of bidirectional and unidirectional transcription, to determine whether the orientation of the endogenous eRNA's transcription is predictive of promoter activity. Assessing promoter activity of each element when inserted in both orientations revealed a strong association between the direction of its endogenous transcription and the element's ability to function as a promoter in the in vivo assay. Of the 15 elements that function as promoters, eight had detectable promoter activity in both orientations and corresponded to elements that were endogenously bidirectional yet often asymmetrically transcribed . In the case of BN5-lf-p, the promoter activity appears stronger in the orientation of endogenous transcription , although we note that the in situ hybridizations are not strictly quantitative.Eight of the 10 tested promoters  can function as promoters in vivo, as expected . Five of these overlap gene promoters that are strongly unidirectionally transcribed, either main or alternative, where the direction of the endogenous transcription is clearly linked to the orientation in which it can function as a promoter.In contrast, seven elements could function only as a promoter in one orientation, and, in all cases, this corresponds to the direction in which the bulk of their endogenous eRNA is transcribed  or bidirectional  transcription that can only function as a weak promoter in one orientation (CRM1594) .An overview of all of the elements’ activity and directionality (OI) is shown in CRM4566 D and BN5VT2049 and VT1617) were reported to have no enhancer activity  are alternative TSSs; in all four cases, the elements functioned as both a promoter and an enhancer, giving largely overlapping spatial patterns of activity . In three cases, both promoter and enhancer activities occurred in both orientations, in keeping with the bidirectional transcription of these elements  . In contrast, the fourth region overlapping an alternative TSS, VT1617, has orientation-dependent activity and unidirectional transcription. This element does not have enhancer activity when tested in one orientation , as observed previously . This region has very strong unidirectional transcription (>4500-fold higher on the antisense strand) , in keeping with its directional ability to function as a promoter. However, this orientation dependence for the enhancer activity is surprising. The enhancer and promoter spatial patterns driven by this element are very similar, although, intriguingly, the two activities were observed in the opposite orientations.In five out of 10 cases, the tested promoter region can function as a developmental enhancer in vivo. Interestingly, four of these five regions . The tested region contains a single DHS that spans the gene's main promoter and a characterized enhancer—the Twist promoter-proximal element (Twist_440 bp_promoter)—that is located in very close proximity to the main twist TSS . This orientation-dependent enhancer activity may be due to transcriptional interference from antisense gfp transcription, although we note that there was no detectable antisense gfp transcription by in situ hybridization (data not shown).Interestingly, none of the main TSSs possesses enhancer activity, with the exception of the wist TSS . This elwist TSS B, plus: Taken together, these results indicate that some elements, either TSS-overlapping or TSS-proximal, can function as both a promoter and an enhancer  with that of developmental enhancer activity (using hundreds of in vivo characterized embryonic enhancers), we assessed the general properties of C. elegans enhancers do not overlap transcription initiation clusters (TICs)  and place (tissue) of enhancer activity. This is consistent with similar global comparisons in cell culture models and suggests a mechanistic link to TF occupancy or some other property of enhancer function. However, we also observed that active enhancers have a wide range of eRNA levels, with many active enhancers having very low or undetectable eRNA at the stages when the enhancer is active. Similarly low levels of eRNA may also occur in other species; 35% of putative s (TICs) , while 6s (TICs) . While ts (TICs) . In the The presence of Pol II and the basal transcriptional machinery at enhancers  and theiDrosophila, supported by the presence of potential “pairs” of INR motifs within bidirectional enhancers at the two points of maximal divergent transcription  from different genomic locations and with different transcriptional properties for both enhancer and promoter activity. Our results indicate that highly transcribed developmental enhancers can function as weak promoters in vivo. The spatial pattern of promoter activity was generally a subset of the tissues in which the enhancer was active, indicating that both activities can occur in the same cells from the same element. This promoter function depended largely on the orientation in which the element was inserted, matching the direction of enhancer transcription in its endogenous location: Bidirectional elements (both enhancers and promoters) can generally function as promoters in both orientations, while unidirectional elements have orientation-dependent activity C. This icription C. Intragcription . In the Recent high-throughput studies indicate that the same sequences can function as both promoters and enhancers in vitro, although gene promoters generated more promoter activity compared with distal elements . While otwist gene and is transcribed in a unidirectional manner. We show that this element can function as both a promoter and an enhancer but, interestingly, can perform both functions in only one orientation and is inactive in the other (cis-regulatory elements (either enhancers or promoters) can often function as both enhancers and promoters  in an orientation-independent manner. In contrast, unidirectional elements generally function only in the orientation in which they are transcribed.However, we also found interesting intermediate cases between the two, which suggests a relationship between the directionality of eRNA transcription and the ability to function as an enhancer or promoter in vivo. When bidirectionally transcribed, alternative gene promoters can function as both strong promoters and enhancers in vivo in both orientations A,C. In che other B. These cis-regulatory elements  were extracted, and PRO-cap was performed according to ist gene , and CAGSupplemental Table S9. All constructs were integrated at chromosomal position ZH-51C using the J27 landing site  or lacZ and gfp . All images were taken with a Zeiss LSM 780 confocal microscope.For transgenic analysis, genomic regions tested in either the single readout vector or the dual enhancer–promoter assay are listed in ing site . Homozyghttp://furlonglab.embl.de/data.All raw sequencing data have been submitted to the European Molecular Biology Laboratory-European Bioinformatics Institute (EMBL-EBI) ArrayExpress database under accession numbers E-MTAB-6154  and E-MTAB-6159 . Processed files for visualization are available at


S. cerevisiae terminators contain a hexamer sequence, the efficiency element, that strongly resembles the eukaryotic TATA box i.e. the promoter sequence recognized and bound by RNA polymerase II.In bacteria, transcription units can be insulated by placing a terminator in front of a promoter. In this way promoter leakage due to the read-through from an upstream gene or RNA polymerase unspecific binding to the DNA is, in principle, removed. Differently from bacterial terminators, yeast By placing different yeast terminators  in front of the CYC1 yeast constitutive promoter stripped of every upstream activating sequences and TATA boxes, we verified that the efficiency element is able to bind RNA polymerase II, hence working as a TATA box. Moreover, terminators put in front of strong and medium-strength constitutive yeast promoters cause a non-negligible decrease in the promoter transcriptional activity.S. cerevisiae.Our data suggests that RNA polymerase II molecules upon binding the insulator efficiency element interfere with protein expression by competing either with activator proteins at the promoter enhancers or other RNA polymerase II molecules targeting the TATA box. Hence, it seems preferable to avoid the insulation of non-weak promoters when building synthetic gene circuit in yeast The online version of this article (doi:10.1186/s13036-016-0040-5) contains supplementary material, which is available to authorized users. A commonly accepted way to characterize biological parts has not been established yet, although some have been proposed  and othent (KH) . In thisklk is the mRNA production rate constant due to promoter leakage, ktr is the transcription initiation rate, T represents a transcription factor that binds p and regulates m synthesis, kd is m decay rate, and c is a coefficient equal to 1 if T is an activator or 0 when T is a repressor. In principle, the leakage term can be omitted if promoter p is repressed since the Hill function equals zero only for an infinite amount of repressor T, whereas it has to be present in case of transcription activation. In this scenario, the leakage is entirely due to RNA polymerase II binding to a promoter in an inactive configuration i.e. either already occupied by repressor proteins or in absence of any activator that can help RNA polymerase II recruitment. However, leakage effects can be due also to RNA polymerase II moving along the DNA after a read-through from an adjacent terminator or an unspecific binding upstream a promoter TATA box–the entry point for RNA Polymerase II along a promoter sequence [insulate the promoter, perhaps with the insertion of a terminator upstream its sequence [where sequence . Therefosequence .S. cerevisiae and mammalian cells. However, the TATATA hexamer, i.e. the strongest terminator efficiency element, is also reported to be a strong TATA box [Yeast terminators are characterized by three specific small sequences: the efficiency element, the positioning element, and the poly(A) site . The effTATA box . TherefoIn this work we show that the TATATA and other efficiency elements can indeed work as TATA boxes since they are able to recruit RNA polymerase II to the DNA and lead the transcription of a green fluorescence protein. This was proved with the construction of new synthetic promoters where a native or a synthetic terminator was placed in front of the yeast CYC1 promoter stripped of its two upstream activating sequences (UAS) and its three TATA boxes , 13. As In order to check if a terminator containing a TATA-box-like efficiency element can recruit RNA polymerase II molecules and initiate mRNA transcription, we constructed four synthetic promoters made of a terminator placed in front of the the yeast CYC1 promoter stripped of its two upstream activating sequences (UAS) and its three TATA boxes , 13. We Among the terminators we chose, two contains the TATATA efficiency elements. They are the yeast DEG1  (DEG1t) S. cerevisiae promoters is able to activate TSSes that lie from 40 up to 120 nucleotides downstream [We characterized the strength of our promoters via fluorescence measurements , is clearly above both the background (4.3 AU) and the shortADH1t-pCYC1noTATA (5.9 AU) fluorescence level. If we regard shortADH1t-pCYC1noTATA as an insulated pCYC1noTATA, we can conclude that leakage effects due to unspecific binding of RNA polymerase II upstream the pCYC1noTATA sequence account only for few (about 12) arbitrary units of fluorescence (with the machine setup we chose for our measurements). Therefore, they are negligible for strong promoters such as pGPD, whose average fluorescence level is about 612 AU.In order to further ensure the role of the terminator efficiency element as an RNA polymerase II binding site, we mutated the TATATA sequence inside DEG1t-pCYC1noTATA and Tsynth8-pCYC1noTATA into GAGATA. As shown in Fig. U−A bond between mRNA and DNA in the active site once the T-rich motif has been transcribed, RNA polymerase can escape from the DNA.Transcription units into bacterial synthetic gene circuits can be insulated by placing a terminator in front of the promoter. Bacterial promoters contain, around position −10, a TATA-like hexamer recognized by RNA polymerase. However, bacterial terminators are very different from the eukaryotic ones: they have a palindromic region rich in guanines and cytosines followed by at least six thymines. Upon transcription, the G-C-rich sequence folds into an hairpin that slows down the motion of RNA polymerase. Taking advantage of the weak As shown above, the efficiency element of yeast terminators is able to recruit RNA polymerase II due to its resemblance to a TATA box. Once placed in front of a constitutive yeast promoter, the efficiency element of a terminator is too far away from any TSS to increase gene transcription. However, it might interfere somehow either with promoter activation, by reducing the binding of activator proteins to the corresponding enhancers, or directly with the transcription initiation process. The latter circumstance can arise, for instance, if the terminator efficiency element gets spatially closer to the promoter TATA box because of the DNA bending provoked by an activator or the general transcription factor see Fig. . As a reS. cerevisiae [To test this hypothesis, we placed each of the four terminators described in Fig. revisiae  and measrevisiae  . As it is shown in Fig. So far, we have shown that the efficiency element of yeast terminators is able to recruit RNA polymerase II molecules due to its similarity to a TATA box. This prevent terminators–particularly DEG1t–from working as proper insulators when placed in front of the strong yeast GPD promoter since they disturb its transcriptional activity. We tried, then, to assess if DEG1t has a similar impact on the strength of other yeast promoters such as: TEF2 promoter  pTEF2, . pADH1 aS. cerevisiae is chosen as a chassis for synthetic gene circuits is the relative easiness of genome manipulation (genomic integration). One common technique exploits homologous recombination and auxotrophic selection. However, yeast strains are usually mutated only in a handful of auxotrophic markers. Therefore, the construction of complex circuits made of several interacting genes would require to place two or more transcription units inside a unique vector such that they are later on integrated into the same locus. Our previous analysis on single transcription units indicates that an insulator is likely to reduce the transcription efficiency of strong and medium-strength promoters considerably. What happens if we place an insulator between two transcription units? To answer this question we built a simple system where two adjacent transcription units express diverse fluorescent proteins (yomKate2–red fluorescence [One of the reasons why rescence –and the rescence ). We insUsed as an insulator, a terminator should stop RNA polymerase II that bound to an unspecific site upstream the promoter sequence from reaching the promoter TATA box and start mRNA transcription. This leakage effect should be removed to characterize promoter strength properly and, therefore, improve synthetic gene circuit design. However, yeast terminators contain a hexamer, the efficiency element, that is in general very similar to a TATA box. In particular, the strongest efficiency element, TATATA, is identical to one of the strongest eukaryotic TATA boxes.By fusing four different terminators in front of what we called pCYC1noTATA  we proved that the efficiency elements are able to recruit RNA polymerase II molecules from the cell nucleus and lead to fluorescent protein expression. Moreover, we could estimate that the contribution of leakage effects to constitutive promoter transcriptional activity is very low.When we placed the same terminators in front of the strongest yeast constitutive promoter, pGPD, fluorescence production dropped remarkably. This reduction could not be explained by the sole leakage removal. Hence, we concluded that the terminator efficiency elements interferes with transcription initiation by recruiting RNA polymerase II molecules.Among the four terminators we used in this work, DEG1t proved to be the one with higher affinity towards RNA polymerase II. We insulated five yeast constitutive promoters of different strength with the DEG1 terminator. Only the weakest among these promoters (pACT1) turned out to be insensitive to the insulator presence, the other four underwent a non negligible reduction in transcription efficiency. In particular, ADH1 promoter fluorescence level dropped to the 7% of its original value. This effect was due to the short distance between the DEG1t efficiency element and pADH1 UAS (43 nucleotides only). RNA polymerase II binding the insulator efficiency element is, however, able to interfere with the promoter transcriptional activity also when a high distance separates the efficiency element from the promoter UAS. This was apparent from our experiments on a re-engineered GPD promoter where the strong bipartite UAS was almost completely mutated such that transcription was activated by a weaker UAS downstream. Once insulated with DEG1t, the fluorescence level of the mutated pGPD dropped by about one half, despite the fact that the distance between the efficiency element and the weak UAS was higher than 450 nucleotides.S. cerevisiae genome, the effect of DNA bending is somehow lowered. This would explain why DEG1t, once inserted between two transcription units, caused only a marginal reduction in the expression of the downstream protein.Probably, DNA bending is a factor that enhances the interaction between RNA polymerase II binding the insulator efficiency element and either the activators targeting the enhancers or other RNA polymerase II binding the promoter TATA box. We argue that, by integrating two adjacent transcription units into the same locus of the S. cerevisiae. Insulation, indeed, would provoke a reduction in protein expression that might have high repercussions on the performance of a whole synthetic gene circuit.Overall, our results point out clearly that a single transcription unit containing a strong or medium-strength promoter should not be insulated with a terminator when integrated into the genome of yeast S. cerevisiae genome  following the procedure in [The yeast integrative shuttle-vector plasmid pRSII406   was usededure in . pCYC1 sedure in   or the yeast enhanced green fluorescent protein (yEGFP) obtained from pRS31-glag . Here, ttetR gene. The constructs genCYC1t-pCYC1noTATA, Tsynth8-s100-pCYC1noTATA, DEG1-pCYC1noTATA  was constructed by using, as a template, the MIT Registry part BBa_K801012. Insulated promoters were extended via PCR to be preceded by DEG1t (50 nt long), Tsynth8 (49 nt long) or their mutated version. The spacer s100 was taken from the bacterial Plasmids were constructed either via isothermal assembly  or MoCloμl BsaI 20 units /μl, NEB-R0535S; 2 μl Cutsmart buffer NEB; 1 μl T4 ligase 400 units /μl, NEB-M0202S; 2 μl10 mM ATP, Sigma-Aldrich-A7699) to a final 15 μl volume. The thermocycler program was set to: 3 cycles of 10 min at 40 °C and other 10 min at 16 °C. These cycles were followed by 10 min at 50 °C, 20 min at at 80 °C, and the final temperature was set to 16 °C. E. coli competent cells  transformed with our plasmids (30-s heatshock at 42 °C) were grown overnight at 37 °C either in LB broth or plates  supplied with the necessary antibiotic (ampicillin or spectinomycin). Plates were spread with 100μl100 mM IPTG (Merck) and 100μl20 mg/ml X-gal for blue/white screening. Plasmid extraction from bacterial cells was carried out with standard methods [Touchdown PCR was employed to select and amplify DNA sequences. DNA elution from agarose gel was carried out with the QIAGEN-28604 “DNA Elution kit”. Isothermal assembly required always one hour at 50 °C. In order to assemble 1-module plasmids the insert (0-modules) and our ypL1F-1_406 1-level acceptor vector were combined in 2:1 molar ratio and mixed with a master mix , Euroscarf . Genomic integration was carried out as described in [μg of plasmidic DNA were linearized either along the URA3 marker with the restriction enzyme StuI (NEB-R0187S) or along the LEU2 marker with the restriction enzyme BstXI (NEB-R0113S). Transformed cells were grown on plates containing synthetic selective medium  for about 36 h at 30 °C.Our new, synthetic plasmids were integrated into the genome of the yeast ribed in . About 5OD600 was always between 0.2 and 2.0 . Fluorescence measurements were performed with a BD FACScalibur machine . The FACS machine set-up was reproduced at each experiment by using fluorescent beads . We placed their peak  as close as possible to 400 AU. The measurement was repeated at the end of each experiment to assure that the machine conditions did not change considerably over the whole experiment. We considered as reliable only the measurements where the relative difference between the initial and the final value of the peaks of the beads was lower than 5%. Data were analyzed with the flowcore R-Bioconductor package [p-value <0.05). Fluorescence levels were estimated as the mean values of at least three independent experiments (i.e. carried out in different days–each time 30000 samples were recorded). Standard deviations were calculated on these mean values. The error on a relative fluorescence value (ratio) was finally computed via the error propagation formula. Box plots and histograms of representative experiments are provided in the Additional file Yeast cells were grown overnight in synthetic complete medium (SDC) at 30 °C. They were diluted, in the morning, approximately 1:100 and let them grow (in synthetic medium again) up to five more hours such that their  package . StatistAdditional file 1Supplementary Material. (PDF 494 kb)


SER3 gene in Saccharomyces cerevisiae positions nucleosomes over the upstream activating sequences, leading to strong repression of SER3 transcription. To explore the possibility of other regulatory roles for ncDNA transcription, we selected six candidate S. cerevisiae genes that express ncRNAs over their promoters and analyzed the regulation of one of these genes, ECM3, in detail. Because noncoding transcription can lead to changes in the local chromatin landscape that impinge on the expression of nearby coding genes, we surveyed the effects of various chromatin regulators on the expression of ECM3. These analyses identified roles for the Paf1 complex in positively regulating ECM3 transcription through methylation of histone H3 at lysine 4 (K4) and for Paf1 in controlling the pattern of intergenic transcription at this locus. By deleting a putative promoter for the noncoding transcription unit that lies upstream of ECM3, we provide evidence for a positive correlation between intergenic transcription and ECM3 expression. Our results are consistent with a model in which cotranscriptional methylation of histone H3 K4, mediated by the Paf1 complex and noncoding transcription, leads to activation of ECM3 transcription.Transcription of nonprotein-coding DNA is widespread in eukaryotes and plays important regulatory roles for many genes, including genes that are misregulated in cancer cells. Its pervasiveness presents the potential for a wealth of diverse regulatory roles for noncoding transcription. We previously showed that the act of transcribing noncoding DNA (ncDNA) across the promoter of the protein-coding  Xist and HOTAIR, two important developmental regulators, associate with and direct the Polycomb repressive complex, PRC2, to specific genetic loci. PRC2 is then able to alter the local chromatin state and lead to the regulation of gene expression  during transcription elongation. During transcription of SRG1, these histone chaperones place nucleosomes over the upstream regulatory sequences for the SER3 gene, creating a barrier that prevents the transcriptional machinery from accessing the SER3 promoter  molecule itself plays a regulatory role. The act of transcribing ncDNA can also alter the local chromatin environment and the regulation of neighboring genes. Transcription can alter the occupancy and positions of nucleosomes, posttranslational histone modifications, and potentially higher order chromatin structures. Our previous studies in  of SER3 . The mecSchizosaccharomyces pombe, the fbp1+ gene is activated by stepwise displacement of nucleosomes over the fbp1+ promoter, which is mediated by induction of a series of long noncoding transcripts across the promoter  and methylation of H3 K79 throughout the bodies of transcription units, and methylation of H3 K36 at the 3′ ends of transcription units  at 5′ ends of transcription units, monoubiquitylation of H2B  analyses.32P-dATP (EUC1 (−541 to −100), ECM3 (+545 to +976), ARO2-CUT (−494 to −49), ARO8-CUT (−429 to −28), CLN3-CUT (−671 to −321), FET4-CUT (−479 to −114), KNH1-CUT (−496 to −235), and SCR1 (−182 to +284). SCR1 RNA levels serve as an internal loading control. The oligonucleotides used to generate probe templates are listed in Table S2. Images were generated by phosphorimaging and quantified using ImageJ software. For each experiment, the data from at least three biological replicates were averaged.Northern blot analyses were performed using 20 μg total RNA samples resolved in gels containing 2% agarose, 6.5% formaldehyde, and 1 × MOPS as previously described . Double-32P-dATP . Probe t32P-ATP and T4 polynucleotide kinase using standard protocols , which are short transcripts that are terminated by the Nrd1-Nab3-Sen1 termination pathway, polyadenylated by the TRAMP complex, and degraded by the nuclear exosome , which were also detected by TIF-Seq analysis (ECM3 promoter as the ECM3 upstream CUT (EUC1). Upon deletion of the RRP6 gene, we did not detect a significant change in ECM3 expression, suggesting that ECM3 expression is not regulated by stability of the EUC1 transcript itself , although ECM3 mRNA levels remained low  . This analysis identified a conserved putative TATA sequence 344 nucleotides upstream of the ECM3 start codon. In ChIP-exo analyses, ECM3 start codon. Guided by this information, we generated promoter deletion mutations at the endogenous ECM3 locus using a two-step integration method, which replaced the putative EUC1 promoter sequence with an unrelated DNA sequence (177 bp encoding the 3XHA tag and linker DNA). Deletion 1 (pEUC1∆1) replaced bases −400–−350 and deletion 2 (pEUC1∆2) replaced bases −400–−300 relative to the ECM3 start codon. These deletions eliminate the detected site of PIC assembly and pEUC1∆2 also eliminates the putative TATA sequence  in the presence and absence of EUC1 transcription as well as in paf1∆ and set1∆ mutants, which served as controls for loss of the modification. As expected, H3 K4me3 levels were nearly undetectable in the paf1∆ and set1∆ mutants , which is enriched in paf1∆ strains . Neither the SC nor the LC isoform is detected with an antisense strand-specific northern probe. In addition, the LC transcript is not detected in paf1∆ strains carrying a wild-type RRP6 allele, indicating that it is also unstable . Because Paf1 is required for proper termination by the Nrd1-Nab3-Sen1 pathway  the entire ECM3 ORF  may remove a transcription factor binding site that could directly activate the ECM3 promoter, several pieces of data indicate that this sequence most likely contains key regulatory elements of the EUC1 promoter. First, our ChIP data show two distinct peaks of H3 K4me3 for the EUC1 and ECM3 transcripts, suggesting that these promoters are distinct. Second, preliminary data in environmental conditions where we have observed slight changes in ECM3 expression show a corresponding change in expression of the short EUC1 isoform. Although these effects are subtle, they suggest that the correlation between EUC1 and ECM3 expression is not limited to a context where the EUC1 promoter has been deleted. Third, we report multiple lines of evidence for the methylation of H3 K4 as a positive regulatory event for ECM3 expression. As this histone modification is coupled to transcriptional activity, it is likely that transcription of EUC1 plays a role in placing this mark across the ECM3 promoter. Though we are not aware of any transcription factors that both bind to the sequence deleted in pEUC1∆1 and upregulate ECM3 transcription in a manner not dependent on EUC1, we cannot rule out their existence. Our attempts to prevent EUC1 transcription through strategies other than generating the pEUC1∆1 and pEUC1∆2 mutations were unsuccessful. Introduction of an exogenous terminator sequence within EUC1 had nonspecific effects on ECM3 transcription start site selection, and targeted mutation of the predicted TATA element failed to eliminate EUC1 transcription. This is consistent with previous reports detecting PIC assembly further upstream of the putative TATA sequence and indicates that this TATA element is not likely to be the predominant site of PIC assembly for the EUC1 transcript. The overall AT-richness of this region may allow for transcription initiation at multiple sites that may not be abolished by targeted mutations.One explanation for our observations is that transcription of ECM3 promoter, EUC1 transcription and the Paf1 complex also have separable functions in facilitating ECM3 expression. A double mutant lacking both EUC1 transcription and PAF1 is more severely compromised for ECM3 expression than either single mutant strain. This indicates that the Paf1 complex stimulates ECM3 transcription through a mechanism in addition to its role in promoting H3 K4 methylation. This is further evidenced by the Leo1 subunit of the Paf1 complex having a positive role in ECM3 regulation, despite the fact that Leo1 is not required for H3 K4me3 (EUC1 transcript is altered in a paf1∆ strain, our results of Nrd1 depletion do not support a major role for alternative termination of EUC1 in regulating levels of ECM3 expression. However, it is possible that there are transient isoform-specific effects or that these isoforms have different roles under biologically relevant conditions. An intriguing feature of the locus is that the EUC1 and ECM3 transcripts overlap in a region of DNA occupied by a nucleosome (ECM3. This nucleosome could also be a determinant in which isoforms of EUC1 and ECM3 are expressed. It would be interesting to relate the position of this nucleosome with where termination of EUC1 and initiation of ECM3 occur.While both appear to be important for establishing the H3 K4 methylation pattern across the GAL1-10, DCI1, and DUR3 loci (Set3. Our data indicate that the functionally important readers of the H3 K4 methyl marks at ECM3 are the HAT complexes SAGA and NuA3, which would explain the opposite effect of H3 K4 methylation on ECM3 expression compared to GAL1-10, DCI1, and DUR3. Differential recruitment of readers could depend on whether the histones at the promoters of these genes display predominantly tri- or dimethylation of H3 K4 or other differences in the local chromatin environment. The observation that one histone modification associated with noncoding transcription can have opposite effects on the regulation of a neighboring gene adds to the increasing diversity of mechanisms by which noncoding transcription can regulate gene expression. This points to a potential plethora of regulatory mechanisms imparted by noncoding transcription that will be important to study at individual genes.Our finding of a positive role for H3 K4 methylation at a gene that lies downstream of a noncoding transcription unit is interesting, as others have shown this modification to be repressive to initiation of transcription at the


RUNX1 gene is frequently translocated or mutated in leukaemia. Runx1 is transcribed from two promoters, P1 and P2, to give rise to different protein isoforms. Although the expression of Runx1 must be tightly regulated for normal blood development, the mechanisms that regulate Runx1 isoform expression during haematopoiesis remain poorly understood. Gene regulatory elements located in non-coding DNA are likely to be important for Runx1 transcription. Here we use circular chromosome conformation capture sequencing to identify DNA interactions with the P1 and P2 promoters of Runx1, and the previously identified +24 enhancer, in the mouse multipotent haematopoietic progenitor cell line HPC-7. The active promoter, P1, interacts with nine non-coding regions that are occupied by transcription factors within a 1 Mb topologically associated domain. Eight of nine regions function as blood-specific enhancers in zebrafish, of which two were previously shown to harbour blood-specific enhancer activity in mice. Interestingly, the +24 enhancer interacted with multiple distant regions on chromosome 16, suggesting it may regulate the expression of additional genes. The Runx1 DNA contact map identifies connections with multiple novel and known haematopoietic enhancers that are likely to be involved in regulating Runx1 expression in haematopoietic progenitor cells.The transcription factor Runx1 is essential for definitive haematopoiesis, and the  Runx1 in mouse embryos is lethal in embryonic stage (E) 12.5 due to the complete absence of definitive blood cell progenitors accompanied by extensive haemorrhaging2. Runx1 is crucial for haematopoietic stem cell (HSC) emergence and maintenance during development3, since conditional ablation of Runx1 in adult mice results in HSC exhaustion4. In acute myeloid leukaemia (AML) and myelodysplastic syndrome, RUNX1 function is frequently altered through mutations or translocations5, resulting in dysregulation of its target genes. While mutations directly affecting the RUNX1 protein are common in leukaemia, mutations in regulatory elements that affect RUNX1 expression remain enigmatic. As yet unidentified mutations in regulatory elements, such as enhancers, could alter Runx1 expression, resulting in abnormal haematopoiesis.Runx1 is a key regulator of haematopoietic development. Deletion of Runx1 is transcribed from two promoters, P1 and P2, to give rise to different protein isoforms6. Expression of these isoforms is tightly controlled during haematopoiesis. At the onset of mouse haematopoiesis (E7.5), preceding the generation of HSCs, expression of the P2 isoform(s) is predominant8. P1 is expressed soon after P2, and its expression is synchronised with the generation of HSCs9. P1 expression is predominant in the mouse fetal liver, the main site of definitive haematopoietic stem/progenitor cell (HSPC) development from E12.5 onward9.10. One previously identified Runx1 enhancer is located 24 kb downstream of the P1 transcriptional start site12. The +24 enhancer  is active in HSCs that express Runx1 during mouse embryogenesis13. The human equivalent of the +24 enhancer (+32 kb downstream of P1) directly contacts the promoters of RUNX1 in leukaemia cell lines14. In addition to the +24 enhancer, putative regulatory elements for RUNX1 have been identified upstream of RUNX1-P1 and between P1 and P2; however, whether they directly contact the RUNX1 promoters has not been investigated16.Regulatory elements, such as enhancers, can control the expression of genes via long-range chromatin interactionsRunx1 P1 promoter, versus an inactive P2 promoter. While Hi-C provides genome-wide contact profiles, and Capture Hi-C enriches for interactions with preselected genomic features , 4C-seq can generate very high resolution contact profiles from ‘baits’ of particular interest17. Therefore, 4C-seq can yield richer information about a selected genomic region than Hi-C or Capture Hi-C. HPC-7 is a well characterised mouse HSPC line with genomic annotations, including transcription factor (TF) binding, histone modifications and chromatin accessibility20. 4C-seq in HPC-7 cells identified nine haematopoietic enhancers that interact with the P1 promoter and +24 enhancer, and that are occupied by haematopoietic TFs. Four of these were previously identified, three of which were functionally tested and two out of the three showed activity during mouse haematopoiesis15. Here we assessed the activity of all nine enhancers in zebrafish, and show that eight are active in zebrafish haematopoiesis. Further, the +24 enhancer was highly interactive both within a topologically associated domain (TAD) harbouring Runx1, as well as with loci outside the TAD. Collectively, our results point to the formation of a local ‘active chromatin hub’ controlling Runx1 expression in haematopoietic cells.Here we used circular chromosome conformation capture sequencing (4C-seq) to identify regulatory elements that interact with an active Runx1  were similar to their nearby corresponding baits at P1, +24 and P2 Fig. . We inveRunx1 domain 21 did not reveal obvious tissue-specific expression patterns. However, among the genes contacted by +24 are Erg, a haematopoietic TF, and Tiam1 (T-cell lymphoma invasion and metastasis 1), involved in cell adhesion and cell migration. These distant connections suggest that +24 may also regulate other haematopoietic genes in adjacent domains.Most of the significant interactions for P1, P2 and the +24 enhancer occur within the 1.1 Mb domain Fig. . There aain Fig. . Many ofRunx1 expression. To identify putative enhancers, we aligned significantly interacting sites with the occupancy of thirteen TFs involved in haematopoietic progenitor cell production; enhancer histone modifications and DNase I hypersensitivity sites20; and conserved non-coding elements (CNE), which were identified based on comparative genomics alignment and retroviral integration site mapping to indicate potential DNaseI hypersensitive sites11. We note that the +24 enhancer binds all thirteen haematopoietic progenitor TFs in HPC-7 cells , or fall between the P1 and P2 promoters (+24 and +110). Four out of nine of the identified putative enhancers  were identified previously based on the binding of at least three blood TFs and the presence of a H3K27ac peak (denoting active chromatin) from a subset of the same HPC-7 datasets that we have used here15.We selected putative enhancers based on the binding of at least six TFs involved in haematopoietic progenitor cell development Fig. , and the110 Fig. . An addi110 Fig. . The +24Runx1 promoters  form long-range interactions with the P1 promoter, the +24 enhancer, or both Fig. . The excers Fig. . In conters Fig. . A speci22, and cohesin is involved in transcription regulation at active genes23. We found that four of the nine enhancer loci (in addition to the +24 enhancer) coincide with Rad21 (cohesin) binding in the absence of CTCF .We compared the 4C interactions identified with recently published Capture Hi-C data in HPC-7 cellsRunx1 recruit haematopoietic TFs in HPC-7 cells, therefore we determined if these regions act as enhancers in vivo. Each of the putative enhancers was tested for the ability to drive tissue-specific GFP expression in zebrafish embryos. Eight out of nine drove GFP expression specifically in the intermediate cell mass and posterior blood island, which are sites of haematopoietic progenitor cell production at 20–24 hours post-fertilisation (hpf)24 24 Fig. . Two of hpf Figs , S4. Intin vivo function to multiple putative enhancers upstream of Runx1 that were previously identified in silico18. These regions not only drive haematopoietic expression, but also physically connect with the Runx1-P1 promoter, lending confidence to the concept that they are bona fide regulators of Runx1 transcription.In summary, we have assigned Runx1, and confirmed previously identified upstream connections from P1 to putative regulatory elements18. Runx1 appears to be contained within a ~1 Mb chromatin domain, consistent with Hi-C analyses in other cell types26. We observed multiple connections both up- and downstream from all 4C-seq baits . Significantly, there were many more upstream connections anchored by the active elements (Runx1-P1 and +24).4C analysis in HPC-7 cells generated a high-resolution connectivity map of the genomic region harbouring Runx1 TAD. These comprise up to one-third of all connections formed and include other haematopoietic genes, such as Tiam1 downstream, and Erg upstream. Erg and Tiam1 dysregulation is associated with several tumor types, including AML and B- and T-cell lymphomas28. Strikingly, these genes are located over 2 Mb away from Runx1. These findings raise the interesting possibility that the +24 enhancer acts as a scaffold to recruit multiple promoters, enhancers and TFs over long distances in cis. Connections to +24 identified by our 4C-seq may point to the identity of some of these genes.Surprisingly, the +24 enhancer forms many up- and downstream connections outside of the 20. Eight out of nine of these DNA regions are able to drive haematopoietic expression in zebrafish, strongly suggesting an in vivo function. Although previous studies had identified transcriptional activity for a subset of these enhancers15, or an interaction with the P1 promoter for an overlapping subset18, no single prior study has shown that enhancers both interact with Runx1 promoters (and +24), and have transcriptional activity. Table LacZ expression in mice15; these are equivalent to the −58 and +110 enhancers identified here. Our data show in addition that these regions are in contact with P1 and the +24 enhancer  supplemented with 3.024 g/L sodium bicarbonate, 10% fetal bovine serum , 10% stem cell factor conditioned media and 0.15 mM monothiolglycerol (Sigma-Aldrich) as previously described36. SCF-conditioned media was obtained from culturing BHK-MKL cells maintained in Dulbecco’s modified Eagle media (Sigma-Aldrich) supplemented with 3.5 g/L glucose, 3.7 g/L Sodium Bicarbonate and 10% FBS.HPC-7 cells were maintained at a density of 1–10 × 1037 with modifications. Three libraries were generated, two replicates (passage 8 and passage 10 cells) and one control (a 1:1 mix of both replicates for which the first ligation step was omitted). Cells were cross-linked in 2% formaldehyde, 5% FBS and 1x PBS for 10 minutes at room temperature while rotating. Formaldehyde was quenched with a final concentration of 125 mM glycine for 5 minutes on ice while inverting several times. Cell pellets were washed twice with ice-cold 1x PBS.4C library preparation was performed as previously describedDpnII restriction buffer (New England Biolabs) and 0.3% SDS and incubated for 1 hour at 37 °C while shaking. Triton X-100 was then added to a final concentration of 1.8% and the reaction was left at 37 °C while shaking for another hour. Chromatin was digested with 800 U of DpnII overnight at 37 °C while shaking. DpnII was inactivated by adding SDS to a final concentration of 1.3% and incubating at 65 °C for 20 minutes. Nuclei were diluted into a volume of 7 mL containing 1.01x T4 DNA ligase buffer (Life Technologies) and Triton-X100 at a final concentration of 1% and incubated at 37 °C for 1 hour. Ligations were carried out with 100 U of T4 ligase (Life Technologies) for 4.5 hours at 16 °C and 30 minutes at room temperature while shaking. For control libraries, ligase was omitted. Samples were proteinase K treated and reverse-crosslinked overnight at 65 °C. Samples were then treated with RNase A at 37 °C for 30 minutes. DNA was purified by phenol/chloroform extraction and ethanol precipitated.Nuclei were harvested by lysing the cell pellets in ice-cold lysis buffer  for 10 minutes on ice. Nuclei were then resuspended in 1.2x BfaI (New England Biolabs) for P1 and P2 baits or MseI (New England Biolabs) for +24 baits in restriction buffer overnight at 37 °C while shaking. Restriction was inactivated by adding SDS to a final concentration of 1.3% and incubating at 65 °C for 20 minutes. A second ligation was performed in the same way as the first ligation, except that ligations were incubated overnight. DNA was purified with two phenol/chloroform extractions and one chloroform extraction followed by ethanol precipitation. DNA concentrations were measured using a Qubit® 3.0 Fluorometer (Life Technologies) and Qubit® double-stranded DNA (dsDNA) High Sensitivity Assay kit (Life Technologies).A second digestion was performed with 25 U of TM DNA Library Prep Kit from IlluminaTM (Affymetrix) and TruSeq® adaptors (Illumina). Libraries were mixed equimolarly and sequenced as 125 bp paired-end reads on two IlluminaTM HiSeq 2500 lanes by New Zealand Genomics Limited.For each bait, a total of 1 μg of DNA was amplified by PCR using Q5® High-Fidelity DNA Polymerase (New England Biolabs). Bait primer sequences are listed in Supplementary Table 38, and visualised using the University of California, Santa Cruz (UCSC) genome browser (http://genome.ucsc.edu/) with mouse assemblies mm9 or mm10 or with the R package ggplot241. Baits were demultiplexed based on bait primer sequences up to and including the digestion site using a custom awk script, allowing 0 mismatches. Only read pairs that had the forward and reverse bait sequences in the correct orientation were selected. Adapter sequences, bait sequences up to but excluding the digestion site, and bases with a Phred quality score under 20 were then trimmed from the reads, using the fastq-multx, fastq-mcf and cleanadaptors v1.24 tools43. Quality of reads was assessed using FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/)44.4C-seq data was analysed in command-line and the R statistical environment45, allowing 0 mismatches. Mapped reads were assigned to DpnII digestion fragments using fourSig46. The following reads were removed from the files: 1) self-ligated reads, 2) uncut reads (fragments adjacent to baits), and 3) reads at fragments that have at least 1 read in the control (non-ligated) library. The running mean was calculated from the sum of read counts from nine successive fragments, which was obtained using fourSig46, and was read per million normalised.Reads with a minimum length of 30 bp were mapped to the mm10 reference genome using Bowtie146. Significant interactions were called for two regions: 1) the whole of chr16, and 2) from chr16:92,250,000-93,635,000 (within the domain). Significant interactions in both replicates were overlapped using the bedIntersect tool from UCSC47.Significant interaction calling was performed using the R package fourSig with the following settings: window size of 3, 1000 iterations, fdr of 0.01, fdr.prob of 0.05 (which selects the top fifth percentile of interactions with a FDR of <0.01), and only included mappable fragments46. For this study, only category 1 and 2 interactions that overlap between both replicates were included, as they are more likely to represent true interactions (because they span multiple fragments), and were previously shown to be more reproducible between replicates than single-fragment interactions46. Furthermore, we distinguished category 1 interactions that overlap between both replicates from other category 1 and 2 interactions by colouring them red and orange, respectively, to visualise the most significant interactions. For conversion from assembly mm10 to mm9, the liftOver tool from UCSC was used (http://genome.ucsc.edu/)47. Gene annotations used in Figures are UCSC reference genes.In the fourSig package, significant interactions can be categorised into three categories: 1) interactions that are significant after the reads from the fragment with the highest read count is removed, 2) interactions that are significant when the fragment with the highest read count is averaged to the read counts of the neighbouring fragments, and 3) interactions that are significant only when all fragment read counts are includedRunx1 regulatory regions were amplified from HPC-7 gDNA or from I-SceI-zhsp70 plasmid containing the −368, +24 and +110 sequences11 (primer sequences are in Supplementary Table 48. Plasmid purifications were performed with the NucleoSpin® Plasmid or NucleoBond® Xtra Midi prep kits (Machery-Nagel). Primers amplified the TF binding peak +/− 200 bp, except for −321 which (due to a repetitive region) did not have a 200-bp extension on the 3′ end. The −368, +24 and +110 fragments are 471, 529 and 579 bp, respectively11. A mixture of 30 pg vector DNA and 120 pg Tol2 transposase mRNA49 was injected into 1-cell zebrafish embryos. Embryos were imaged at 20–24 or ~48 hpf using a Leica M205FA stereomicroscope with a DFC490 camera and LAS software (Leica Microsystems), images were processed using Adobe Photoshop. Zebrafish were maintained as described previously50 and zebrafish handling and procedures were carried out in accordance with the Otago Zebrafish Facility Standard Operating Procedures. The University of Otago Animal Ethics Committee approved all zebrafish research under approval AEC 48/11.11.Mouse conserved non-coding elements (mCNEs) were identified as described previously20. Rad21, Smc3 and CTCF chromatin immunoprecipitation sequencing (ChIP-seq) data in MEL and CH12 cells were obtained from ENCODE51.Occupancy of the transcription factors Erg, Fli1, Scl, Runx1, Gata2, E2A, Ldb1, Lyl1, Lmo2, Gfi1b, Meis1, Myb, phospho-Stat1, Pu.1, Stat3, Eto2, Cebp-α, Cebp-β, Elf1, Nfe2, p53, cMyc, Egr1, E2f4, cFos, Mac and Jun; Rad21 and CTCF; H3K27ac and H3K4me3; DNase I hypersensitivity sites; and Capture Hi-C data in HPC-7 cells was obtained from previously published datahttps://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc = GSE86994).The 4C-seq dataset is accessible through GEO Series accession number GSE86994 (Supplementary Information