Bacillus cereus is a Gram-positive spore-forming bacterium causing food poisoning and serious opportunistic infections. These infections are characterized by bacterial accumulation despite the recruitment of phagocytic cells. We have previously shown that B. cereus Haemolysin II (HlyII) induces macrophage cell death by apoptosis. In this work, we investigated the regulation of the hlyII gene. We show that HlyIIR, the negative regulator of hlyII expression in B. cereus, is especially active during the early bacterial growth phase. We demonstrate that glucose 6P directly binds to HlyIIR and enhances its activity at a post-transcriptional level. Glucose 6P activates HlyIIR, increasing its capacity to bind to its DNA-box located upstream of the hlyII gene, inhibiting its expression. Thus, hlyII expression is modulated by the availability of glucose. As HlyII induces haemocyte and macrophage death, two cell types that play a role in the sequestration of nutrients upon infection, HlyII may induce host cell death to allow the bacteria to gain access to carbon sources that are essential components for bacterial growth. Bacillus cereus group is composed of highly related pathogenic species, including B. thuringiensis, an insect pathogen, B. anthracis, the etiological agent of anthrax and B. cereus. B. cereus is an emerging human food-borne pathogen classified as the 3rd most important cause of collective food-borne infections in Europe, after Salmonella and StaphylococcusB. cereus infection generally causes mild disease characterised by gastroenteritis, but bloody diarrhea and emetic poisoning leading to some fatal cases have been reported B. cereus is also associated with severe local and systemic human infections, posing a public health problem B. cereusThe B. cereus produces several extracellular compounds  that might contribute to virulence B. cereus are not completely elucidated. The non-gastrointestinal infections are characterized by bacteremia despite the accumulation of inflammatory cells at the site of infection B. cereus is able to circumvent the host immune response. B. cereus spores survive, germinate and multiply in contact with macrophages Staphylococcus aureus, the ß-toxin of Clostridium perfringens and the B. cereus Cytotoxin K (CytK) in vivoB. subtilis, HlyII induces haemolysis and virulence in a crustacean infection model B. cereus virulence in insects and mice hlyII gene is present in several clinical isolates of B. cereusIn the early stationary phase, hlyII expression has so far been exclusively studied in the heterologous systems E. coli and B. subtilis. In those systems, it has been shown that hlyII expression is negatively regulated by the transcriptional regulator HlyIIR hlyII and hlyIIR genes are situated in the same chromosomal locus  but are not organized in an operon. In vitro analyses have shown that recombinant HlyIIR regulates hlyII expression by specific binding, as two dimers, to a 44 perfect inverted DNA repeat (22 bp x 2) centred 48 bp upstream of the hlyII transcription initiation point A precise knowledge of the conditions governing the expression of a gene improves our understanding of its role in the functioning of the bacterial cell, but also during pathogenesis. The regulation of hlyII expression and of its repressor hlyIIR. We highlight the major role of glucose 6P as a post-transcriptional activator of HlyIIR, and show that glucose 6P binds to and activates HlyIIR to inhibit hlyII expression.In this study, we investigated the regulatory process of B. thuringiensis 407 Cry_ strain was used as a model for B. cereus. This sequenced strain was originally described as a B. thuringiensis strain, but cured of its plasmid it is acrystalliferous and shows high phylogenic similarity with the B. cereus reference strain ATCC 14579 The hlyIIR gene was disrupted as follows. HindIII-XbaI (1024 bp) and SphI-BamHI (964 bp) DNA fragments corresponding to upstream and downstream regions of the hlyIIR gene were generated from the Bt407 chromosome by PCR using the primer pairs:The 5′-CCCAAGCTTGTACCAGCTTTAACAGGCTATGG -3′),hlyIIR-1 (5′- GCTCTAGACGTCTGCTCTCGAGATTTCCCC-3′), andhlyIIR-2 (5′-ACATGCATGCGGATGCCAGAAGACTTAGTGG -3′),hlyIIR-3 (5′-CGGGATCCCAGGCTCTAAGTTGGATAAGGGG -3′).hlyIIR-4 (R cassette carrying a tet gene was purified from pHTS1 XbaI and SphI) fragment. The amplified DNA fragments were digested with the appropriate enzymes and inserted between the HindIII and BamHI sites of pRN5101, with the selection marker cassette cloned in between the up- and downstream chromosomal fragments of the hlyIIR gene. The resulting plasmid was introduced into Bt407 by electroporation hlyIIR gene was deleted by a double crossover event as previously described 5′-GGGCCAAATGCCGATGCAAGTATCACAGGTAGC-3′) and downstream from hlyIIR-4: hlyIIR-6 (5′-CCAAGCGTTCAGCAAGTTGGTCTCATGATGTGG-3′), and in the TetR cassette (5′-CGGGTCGGTAATTGGGTTTG-3′), (5′-GCAGCTGCACCAGCCCCTTG-3′). The mutant strain was designated Bt407ΔhlyIIR.A TethlyII-lacZ and hlyIIR-lacZ fusions were constructed using DNA fragments corresponding to the promoter regions of hlyII and hlyIIR, generated by PCR using the primer pair PhlyII-1 (5′- AAAACTGCAGCCAGCTGTGTTGACAGAACTGGC-3′) and PhlyII-2 (5′-GCTCTAGACGGACGCTACCGCAACGCATTTAGC -3′), and hlyIIR-1 and hlyIIR-2, respectively. The PCR fragments were digested with the appropriate enzymes and inserted between the XbaI and PstI sites of pHT304-18Z hlyII’Z and pHT-PhlyIIR’Z were introduced into Bt407 and into the mutant strain Bt407ΔhlyIIR by electroporation. Transformants were named Bt407 [pHT-PhlyII’Z], Bt407 [pHT-PhlyIIR’Z] and Bt407ΔhlyIIR [pHT-PhlyII’Z].Transcriptional hlyIIR gene was amplified from the Bt407 chromosome by PCR using the primer pairs hlyIIR-GST-1 (5′-CGGGATCCATGGGGAAATCTCCAGAGCAGACGA-3′) and hlyIIR-GST-2 (5′-TCCCCCGGGCCCGTATGCAAATCGAAGAGCTTAT-3′). The corresponding DNA fragment was inserted between the BamHI and SmaI sites of plasmid pGEX6P1 , and the resulting plasmid was introduced into E. coli M15hellopREP4] (Qiagen). E. coli M15hellopREP4] strain harboring the plasmid pGEX6P1-GST-HlyIIR was grown in minimal M9 medium 600 0.8 was reached and protein expression was induced by addition of 1 mM IPTG. Growth was continued for 4 h after IPTG induction. Bacteria were then collected by centrifugation at 7700 g for 10 min. Pellets were lyzed using 1% triton X-100 and sonication in PBS. After centrifugation, the tagged HlyIIR contained in the supernatant was added on a Bulk GST Purification module . The HlyIIR protein was purified by cleavage of the GST tag using the Precission protease according to the manufacturer’s instructions. Protein concentration was calculated by Bradford staining and purity was assessed on SDS-Page gel.The plasmid pGEX6P1-GST-HlyIIR was constructed as follows. The hlyIIR were cultivated aerobically in LB at 37°C with agitation.Bt407 and Bt407Δ260 nm/OD280 nm >1,95 and RIN >9) and free of DNA traces (data not shown).1 ml samples were taken every hour between t0−2 h (t−2) and t0+4 h (t4), t0 h (t0) corresponding to the transition state between the exponential and the stationary phases of the bacterial culture. The bacterial cells were collected by a short centrifugation and immediately placed at −80°C. Total RNA of these cell pellets was extracted as described in ssu gene, encoding 16S RNA, as endogenous reference, as its expression level in Bt407 was stable between t-2 and t4 (data not shown). RT-qPCR samples contained 1 ng of RNA and 500 nM or 1 µM of each primer in a final volume of 25 µL and were run in a LightCycler 480 (Roche). The following primer pairs were used: F-hlyII-q (5′-CTGGAAAAACCATCAAGTTACTC-3′); R-hlyII-dq (5′-TCACCATTTACAAAGATACC-3′) 5′-GGTAGTCCACGCCGTAAACG-3′); LC-16S-R (5′-GACAACCATGCACCACCTG-3′) Target and reference gene mRNA abundance was measured by one-step RT-qPCR with the QuantiFast SYBR green RT-PCR kit, following the manufacturer’s instructions (Qiagen). We used the lacZ fusions were grown in LB medium at 37°C under shaking. Samples were taken every hour from t−2 to t4. Determination of ß-galactosidase activity was achieved as previously described Strains harboring plasmid transcriptional B. cereus HlyIIR was retrieved from the Protein Data Bank (PDB entry 2fx0) and hydrogen atoms added with the Biopolymer module of the SYBYL-X 1.3 package . The starting three-dimensional structure of glucose-6-Phosphate (glucose 6P) was built from the SYBYL fragment library. Docking of the ligand to the HlyIIR cavity was realized using two independent docking tools .The crystal structure of PLANTS GOLD v5.1 5′-GGAAATAATGTATCTGAATATAGGCTG-3′ and 5′-CACATGTTTCACTAATCTTCCTCTCAC-3′, and containing the HlyIIR binding region upstream of the hlyII gene Isothermal titration calorimetry (ITC) measures heat generated or absorbed upon the binding of two molecules The data were integrated to generate curves in which the areas under the injection peaks were plotted against the ratio of injected sample to cell content. Analysis of the data was performed using MicroCal Origin provided by the manufacturer according to the one-binding-site model. Changes in the free energy and entropy upon binding were calculated from determined equilibrium parameters using the equation:hlyII is independent of the pleiotropic regulator PlcR hlyII gene transcription. However, no study is so far describing hlyII expression regulation by HlyIIR from the B. cereus group. We therefore investigated the expression of hlyII in a Bt 407 hlyIIR deficient mutant. Quantitative RT-PCR assays showed that hlyII expression was significantly higher in the Bt407ΔhlyIIR mutant than in the wild type (wt) strain  strain . This dihlyII expression was 2 fold higher in the ΔhlyIIR mutant than in the wild type strain.The results obtained by qRT PCR were confirmed by ß–galactosidase assays . The tenhlyII expression during exponential growth phase was due to an increased expression of the regulator itself during this period, we assessed hlyIIR expression using a β–galactosidase assay .HlyIIR negatively regulates se assay . ExpresshlyII expression acted through the repressor HlyIIR, expression of hlyII was assessed in the presence or absence of glucose in the ΔhlyIIR mutant  to confirm and quantitatively characterize the interaction of glucose 6P with HlyIIR . The HlyThe N-terminal part of HlyIIR presents sequence similarity to regulators of the TetR repressor family which share common features such as a highly conserved helix-turn-helix motif implicated in DNA binding, dependence of cofactors for activity regulation, involvement in the adaptation to a changing environment and acting as homodimers. In view of the 3-D structure of HlyIIR and analogy with its structural homologues TetR, it has been proposed that binding of the appropriate ligand to HlyIIR could change the orientation of the DNA-binding domains leading to a different affinity of the ligand-bound form towards the specific DNA site We used ITC experiments to characterize whether the DNA binding affinity of HlyIIR is influenced by the presence or absence of glucose 6P binding. Calorimetric titrations were performed and revealed that the HlyIIR- glucose 6P complex binds to the specific DNA target with a Kd of about 28.7 nM . In the hlyII expression through its activation by binding glucose 6P.So far, the HlyIIR-binding ligand was unknown. Thus, to our knowledge, we present the first evidence that HlyIIR trigger its repression activity on hlyII expression in B. cereus. Glucose has been shown to be one of the preferred carbon sources during the initial growth phase of B. subtilisB. anthracis, glucose is a signalling molecule linking environmental sensing to virulence factor production by the way of the important transcription regulator of Gram-positive bacteria, CcpA nhe and hbl operons, two major enterotoxins of B. cereus, but not in hlyII expression In this study, we show that Glucose 6P binds and activates HlyIIR, the negative transcriptional regulator of hlyII gene expression through its post-transcriptional activation by glucose 6P. It has also been shown that hlyII expression depends on iron via activation of the global regulator Fur hlyII gene hlyII promoter, thus interfering with hlyII expression in vitrohlyII expression starts  and one specific (HlyIIR) regulator. Several hypotheses seem possible: i) the low production of HlyII may be sufficient to promote nutrient access and virulence. We have indeed shown that very low doses of protein are able to induce macrophage lysis and mice mortality Taken together, we have identified several mechanisms triggering Additionally, it has recently been shown that HlyII accumulates in high oxidation-reduction potential (ORP) conditions in contrast to low ORP conditions, suggesting a redox-dependent regulation of HlyII secretion hlyII, which plays an important role during the B. cereus virulence process. We demonstrate a new role of glucose 6P as a direct activator of a transcriptional regulator. As iron and carbon sources are essential and universal bacterial growth components, we believe that our data provide an important contribution to the understanding of bacterial infection strategies.Taken together, our data provide new insights into the regulatory pathways of


CpG islands are observed in mammals and other vertebrates, generally escape DNA methylation, and tend to occur in the promoters of widely expressed genes. Another class of promoter has lower G+C and CpG contents, and is thought to be involved in the spatiotemporal regulation of gene expression. Non-vertebrate deuterostomes are reported to have a single class of promoter with high-frequency CpG dinucleotides, suggesting that this is the original type of promoter. However, the limited annotation of these genes has impeded the large-scale analysis of their promoters.Ciona intestinalis, an invertebrate that is evolutionarily close to the vertebrates, and identified its transcription start sites genome-wide using a next-generation sequencer. We indeed observed a high CpG content around the transcription start sites, but their levels in the promoters and background sequences differed much less than in mammals. The CpG-rich stretches were also fairly restricted, so they appeared more similar to mammalian CpG-poor promoters.To determine the origins of the two classes of vertebrate promoters, we chose From these data, we infer that CpG islands are not sufficiently ancient to be found in invertebrates. They probably appeared early in vertebrate evolution via some active mechanism and have since been maintained as part of vertebrate promoters. Oikopleura dioica .More than 200 μg of total RNA was isolated from whole mid-tailbud-stage ascidian embryos (12-hour-old embryos), using ISOGEN (Nippon Gene) according to the manufacturer’s protocol. The RNA was subjected to oligo-capping method . In shortrans-spliced sequences were replaced with splice acceptor sequence “ag” for the subsequent mapping. The sequences were aligned to the KH assembly [cis-splicing. Because of the highly polymorphic genic features of this organism [CpG * N / C / G with C, G, CpG, and N observed numbers of C, G, and CpG and the fixed window size, respectively. The CpG content defined in the present study was CpG / (N - 1). The assembly used for the human genome was UCSC hg18. To select human CpG-poor (CpG score < 0.5) and CpG-rich promoters (CpG score > 0.6), we used DBTSS 6.0 (http://dbtss.hgc.jp/) and calculated CpG scores in 200-bp regions around representative TSSs [Illumina Pipeline (GAPipeline 1.0) was used to extract the sequenced reads from the image data. The spliced leaders (SLs) in the assembly  using Seassembly  for the assembly  for the organism , we usedive TSSs . The anaTSS: transcription start site; SL: spliced leaderThe authors declare that they have no competing interests.Ciona embryos and extracted RNA; Y.S. performed sequencing; and K.N. participated in the coordination of the study.K.O. conceived of the study, designed the study, and drafted the manuscript; R.Y. helped to analyze data; N.T, K.N., and T.G.K. prepared Ciona intestinalis embryos at mid-tailbud stage  A photo of two C. intestinalis embryos at mid-tailbud stage, taken 12 hours after fertilization.Click here for fileSummary of the TSS-seq experiments (Tables S1 and S2) Quantitative data for the 6312 non-trans-spliced and 8753 trans-spliced transcripts which were annotated by the nearest genes.Click here for fileChanges in dinucleotide scores in the vicinity of TSSs  The score changes are shown for ascidian promoters (A), human CpG-poor promoters (B), and human CpG-rich promoters (C).Click here for file


Saccharomyces cerevisiae to demonstrate that toxaphene affects yeast mutants defective in (1) processes associated with transcription elongation and (2) nutrient utilization. Synergistic growth defects are observed upon exposure to both toxaphene and the known transcription elongation inhibitor mycophenolic acid (MPA). However, unlike MPA, toxaphene does not deplete nucleotides and additionally has no detectable effect on transcription elongation. Many of the yeast genes identified in this study have human homologs, warranting further investigations into the potentially conserved mechanisms of toxaphene toxicity. Exposure to toxaphene, an environmentally persistent mixture of chlorinated terpenes previously utilized as an insecticide, has been associated with various cancers and diseases such as amyotrophic lateral sclerosis. Nevertheless, the cellular and molecular mechanisms responsible for these toxic effects have not been established. In this study, we used a functional approach in the model eukaryote  Toxaphene's most persistent congeners and degradation products have been detected in water, air, and sediment, and are known to bioaccumulate in wildlife and humans [Toxaphene is a complex mixture of polychlorinated camphenes and bornanes primarily used to control insects on cotton during the 1960-80s  [1]. Aftd humans . Epidemid humans , non-Hodd humans , melanomd humans . HoweverSaccharomyces cerevisiae is a valuable model in which to conduct toxicological studies. First, basic cellular processes, along with metabolic and signaling pathways, are conserved to higher organisms. Second, a close human homolog has been identified for a significant portion of yeast genes, with several hundred of the conserved genes linked to disease in humans [The eukaryotic yeast n humans . Finallyn humans , function humans ,11, withn humans -14.Here we present the results of a functional screen devised to identify yeast genes necessary for growth in the presence of toxaphene. It is the first known genome-wide study in any organism to examine the genetic requirements for toxaphene tolerance. Our results demonstrate that similar to the known transcription elongation inhibitors mycophenolic acid (MPA) and 6-azauracil (6-AU), mutants defective in processes linked to transcription elongation are sensitive to toxaphene. While toxaphene does display synergism with MPA, it apparently exhibits a mechanism of action distinct from that of MPA/6-AU and additionally does not appear to directly affect transcription elongation. Many yeast genes required for toxaphene resistance have human homologs that may play a role in human toxicity.MATa/MATα his3Δ1/his3Δ1 leu2Δ0/leu2Δ0 lys2Δ0/LYS2 MET15/met15Δ0 ura3Δ0/ura3Δ0, Life Technologies). Yeast growth was performed in liquid rich media  at 30°C with shaking at 200 revolutions per minute (rpm). For the elongation assays, the GAL1-YLR454 strain [The diploid non-essential yeast deletion strains used for functional profiling and confirmation analyses were of the BY4743 background (Dose-finding and growth curves were performed as in . Toxaphen = 4607) were cultured for 15 generations in YPD at various toxaphene concentrations. Following extraction of genomic DNA with the YDER kit (Pierce Biotechnology), DNA sequences unique to each strain were amplified by PCR and subsequently hybridized to TAG4 arrays (Affymetrix). Arrays were incubated overnight, stained, and scanned at 560 nm with a GeneChip Scanner (Affymetrix). Data files are available at the Gene Expression Omnibus database (http://www.ncbi.nlm.nih.gov/geo/).The functional screen and differential strain sensitivity analysis (DSSA) were performed as previously described . Brieflyp value cutoff of 0.002 and Bonferroni correction. Gene interaction networks were generated in Cytoscape [Saccharomyces cerevisiae functional interaction network. The jActiveModules Cytoscape plugin identified sub-networks enriched with fitness data, within which the BiNGO plugin discovered overrepresented GO categories. The Functional Specification (FunSpec) resource  identifiytoscape  by mappit-test. Analyses of relative strain growth were performed as described . BrieflyGene Length Accumulation of mRNA (GLAM) assays were performed as described . Chromatn = 4607) were grown for 15 generations at the toxaphene IC20 (640μM), 50% IC20 (320μM), and 25% IC20 (160μM) to identify genes important for growth in toxaphene. DSSA deemed 130 strains sensitive and 542 strains resistant to at least one dose of toxaphene  confirmed as sensitive to toxaphene (mft1Δ and thp2Δ) lacked components of THO, a complex required for efficient transcription elongation [hpr1Δ and tho2Δ), finding growth defects (spt20Δ), the Paf1p complex (rtf1Δ), and the TREX-2 transcriptional export machinery (sac3Δ and thp1Δ) were sensitive to toxaphene (RPB9), along with genes implicated in RNA polymerase II activation  or reactivation through deubiquitylation (BRE5 and UBP3) were required for toxaphene tolerance , which reduces ribonucleotide levels and increases dependence on transcription elongation factors for transcriptional efficiency [IMD2, the only yeast IMPDH homologue that provides resistance to MPA [imd2Δ strain did not display altered growth in toxaphene (data not shown). Finally, we found toxaphene did not affect strains deleted for URA2 or URA4, enzymes involved in the de novo biosynthesis of pyrimidine ribonucleotides [IMPDH family of enzymes.Similarities between the mutant sensitivity profiles for toxaphene and the transcription elongation inhibitors MPA and 6-AU  promptedficiency ,27. As aficiency , and tunficiency . Both MPficiency ,26. A fle to MPA , for toxleotides  (data noPHO5-lacZ or PHO5-LAC4) versus short transcription unit (PHO5) for BY4743 wild-type cells in the presence or absence of toxaphene, but did not find altered ratios upon toxaphene exposure  was a false positive (data not shown) and (2) most are petite and/or slow growing, which causes inherent competitive fitness defects during pool screens and may thus misinform further analyses. Future studies may elucidate the effects, if any, of toxaphene on aerobic respiration in yeast. The most sensitive strain identified by DSSA (and confirmed by growth curve assays) was pdr5Δ, which, not unexpectedly, lacks a drug resistance transporter involved in detoxification  of toxicity remain ambiguous and findings directly related to transcription elongation have not been reported. Perhaps most relevant to this study is a report indicating that toxaphene altered expression of transcription termination genes in largemouth bass . Woo and Yum  performeSAC3, SPT4, THP1, and those encoding components of the THO complex, are associated with other processes tightly coupled to transcription elongation, such as mRNA processing, mRNA nuclear export, or transcription-coupled nucleotide excision repair [in situ hybridization, however, our findings were inconclusive (data not shown). Alternatively, toxaphene sensitivity of transcription elongation mutants may be attributed to defective expression of yet to be defined toxaphene tolerance protein(s). This type of indirect effect occurs in elongation mutants exhibiting sensitivity to the nucleotide depleting compound 6-AU; these deletions cannot sufficiently induce Imd2p, the protein that combats 6-AU toxicity by restoring nucleotide levels [Toxaphene-sensitive mutants also experience growth defects in MPA and 6-AU  was calculated for strains treated with 640μM or 960μM toxaphene and expressed as a percentage of the AUC for the untreated strain. All bars represent the mean and SE for three independent cultures. (A) Mutants lacking drug resistance genes are sensitive to toxaphene. (B) Various mutants were confirmed to display sensitivity to toxaphene.(TIF)Click here for additional data file.Table S1Primers utilized in transcription elongation assays. Primer sequences are listed.(PDF)Click here for additional data file.Table S2Mutants displaying altered growth in toxaphene. DSSA identified strains sensitive or resistant to various doses of toxaphene. A negative log2 value signifies sensitivity at the corresponding dose, while a positive log2 value signifies resistance.(XLS)Click here for additional data file.Table S3Mutants displaying sensitivity to both toxaphene and MPA, 6-AU, or 4-NQO. A literature search was conducted to identify overlapping mutant sensitivities between toxaphene, the GMP synthesis inhibitors mycophenolic acid (MPA) and 6-azauracil (6-AU), and the model carcinogen 4-nitroquinoline-N-oxide (4-NQO).(PDF)Click here for additional data file.


Methylophilaceae, Methylotenera mobilis, Methylotenera versatilis and Methylovorus glucosotrophus, in order to determine which metabolic pathways are actively transcribed in cultures grown in laboratory on C1 substrates and how metabolism changes under semi-in situ conditions. Comparative analyses of the transcriptomes were used to probe the metabolic strategies utilized by each of the organisms in the environment. Our analysis of transcript abundance data focused on changes in expression of methylotrophy metabolic modules, as well as on identifying any functional modules with pronounced response to in situ conditions compared to a limited set of laboratory conditions, highlighting their potential role in environmental adaptation. We demonstrate that transcriptional responses to environmental conditions involved both methylotrophy and non-methylotrophy metabolic modules as well as modules responsible for functions not directly connected to central metabolism. Our results further highlight the importance of XoxF enzymes that were previously demonstrated to be highly expressed in situ and proposed to be involved in metabolism of methanol by Methylophilaceae. At the same time, it appears that different species employ different homologous Xox systems as major metabolic modules. This study also reinforces prior observations of the apparent importance of the methylcitric acid cycle in the Methylotenera species and its role in environmental adaptation. High transcription from the respective gene clusters and pronounced response to in situ conditions, along with the reverse expression pattern for the ribulose monophosphate pathway that is the major pathway for carbon assimilation in laboratory conditions suggest that a switch in central metabolism of Methylotenera takes place in response to in situ conditions. The nature of the metabolite(s) processed via this pathway still remains unknown. Of the functions not related to central metabolism, flagellum and fimbria synthesis functions appeared to be of significance for environmental adaptation, based on their high abundance and differential expression. Our data demonstrate that, besides shared strategies, the organisms employed in this study also utilize strategies unique to each species, suggesting that the genomic divergence plays a role in environmental adaptation.We carried out whole transcriptome analysis of three species of  Methylophilaceae are ubiquitous in natural environments, with the exception of extreme environments, and are found in fresh and saline waters, soils, air, industrial waste-water treatment reactors etc., pointing to the environmental importance of this group  have served for decades as models for studying the biology of methylotrophs utilizing the ribulose monophosphate pathway (RuMP) for formaldehyde assimilation , typically methanol and methylamine, resulting in high biomass yields  with 13C-labeled C1 compounds, Methylotenera sequences were enriched not only in communities accumulating heavy (13C) carbon originating from methylamine, but also in communities accumulating heavy carbon from methanol or methane  or methanol (100 mM) as substrates, as previously described (escribed . M. versid media . Solidifid media . M. glucescribed .M. mobilis JLW8 and M. glucosotrophus SIP3-4 cells were grown on an appropriate substrate in liquid MM2 medium as described above to an OD600 of approximately 0.45 ± 0.05. Stop solution (5% buffer-equilibrated [pH 7.4] phenol in ethanol) was added, and cells were harvested by centrifugation at 5000 × g for 15 min at 4°C and immediately used for RNA extraction. M. versatilis 301 cells were grown on plates with solidified diluted MM2 medium supplemented with methylamine as described above. For methanol-induced samples, cells were grown on methylamine as described above, collected, centrifuged, washed with fresh diluted MM2 medium, and resuspended in MM2 plus methanol . After 2 h of incubation at 30°C, with shaking, cultures were pelleted as described above and immediately used for RNA extraction.The schematic of experimental design is depicted in in situ incubations, cells of M. mobilis JLW8 and M. versatilis 301 were grown on methylamine, and cells of M. glucosotrophus SIP3-4 were grown on methanol. Slide-A-Lyzer 3.5 K MWCO Dialysis Cassettes  were used for sediment incubations, and these were prepared and cells were inoculated exactly as previously described . The RNA samples were submitted to a sequencing facility , where cDNA libraries were generated using a platform-specific (Illumina HiSeq 2000) chemistry. Sequencing was carried out on an Illumina HiSeq 2000 instrument using platform-specific protocols and producing reads of 36 bp in length.RNA extraction was performed as described before . The intReads corresponding to each sample were aligned to the respective reference genome  using thin situ conditions, i.e., after incubation in semi-penetrable dialysis cassettes placed into freshly-sampled lake sediment cores, assuming that metabolic processes that naturally occur in this environmental niche continued over the short duration of time (48 h) in the laboratory. Note that control samples, i.e., samples growing under laboratory conditions, were prepared differently for each strain  should provide good clues as to which transcriptional responses are specific to in situ conditions. A total of 18 samples were compared in this study  was expressed at a very low level in each condition, and no homolog of mmol_1769 was present near m301_1208 or elsewhere in the genome, suggesting no role for this gene in either methylotrophy or in environmental niche adaptation for this organism (mmol_1770 in M. glucosotrophus SIP3-4 (msip34_0016) was significantly (100-fold) over expressed in in situ conditions compared to laboratory conditions, but the expression level was relatively low (msip34_0015) predicted to encode a cytochrome (not homologous to either mmol_1769 or mxaG) revealed a similar expression pattern, suggesting a role in electron transfer from Msip34_0016.We have previously noted that multiple homologs of organism . Thus thcultures . Along worganism –S4. A neorganism –S7. The vely low –S10. ThexoxF homolog in M. mobilis JLW8, mmol_2048 was also slightly over-expressed in in situ conditions. The corresponding genes in M. versatilis 301 and M. glucosotrophus SIP3-4 were also most highly expressed in situ, compared to the methylamine- and methanol-grown or induced cultures. xoxJ and xoxG, along with an oxidoreductase of unknown function as these genes form tight and highly conserved clusters in all known Methylophilaceae genomes (The second  genomes .xoxF homolog in M. versatilis 301 (m301_1659) and two additional homologs in M. glucosotrophus SIP3-4 (msip34_1813 and msip34_2549) were expressed at low levels. Of the three organisms, only M. glucosotrophus SIP3-4 encoded the well-characterized (MxaFI) methanol dehydrogenase (msip34_0734-0737) were highly expressed on all substrates as has been previously shown for other methylotrophs , is well characterized  showed different transcription patterns, as follows. mmol_1253 that is part of a sensing/chemotaxis gene cluster , in linking formaldehyde to tetrahydromethanopterin  was not greatly influenced by the conditions, and the levels of expression were similar for the three organisms. All three organisms expressed genes for a NAD-linked formate dehydrogenase known as FDH1  possess genes encoding the methylcitric acid cycle (MCC), whose role remains unknown in these species , suggesting a role for this gene in switching the pathway on in response to environmental conditions. Transcription of the RuMP genes followed a reversed pattern in these organisms, being highly expressed in laboratory conditions (on both methylamine and methanol) and transcribed at lower levels in situ.Some  species . We founnditions , in genehilaceae . One of in situ conditions, genes with such a pattern mostly represented the following major functional categories: motility, adhesion, signaling and sensing, and transport. The gene cluster(s) encoding the flagellum functions, including regulatory functions, were some of the most highly transcribed genes in all three organisms  in in situ conditions  element (mmol_1456-1493) encoding transport/efflux functions in M. mobilis JLW8 (m301_2524-2533) in M. versatilis 301  nitric oxide dioxygenase, a sigma 24 factor and a transcriptional regulator (m301_0769-0776), a cytochrome complex and a two-component transcriptional regulation system (m301_0064-0069), a cytochrome oxidase along with sensing and regulation functions (m301_844-858), and a sensing/chemotaxis/regulation cluster  previously identified by observations of a dramatically different GC content  genes.One of the notable outcomes of this study is further evidence for the importance of XoxF enzymes in the metabolism of the fae also demonstrated different responses to in situ conditions, further suggesting that different homologs may respond to different signals, and, in turn, that different homologs may have different additional functions, as proposed earlier  processed via this pathway still remains unknown.This study reinforces the prior observations on the apparent importance of the methylcitric acid cycle (MCC) in the Methylophilaceae strains, the former being implicated in not only mobility but also attachment and adhesion (Of the functions not related to central metabolism, flagellum and fimbria synthesis functions appeared to be of significance for environmental function, based on their high abundance and differential expression. Obviously, solely from gene/protein homology it is impossible to predict the major functions of the flagella or of different pili in different adhesion , and theadhesion . Finding10.7717/peerj.115/supp-1Table S1Click here for additional data file.10.7717/peerj.115/supp-2Table S2Methylotenera mobilis JLW8.Comparative RNA-Seq statistics for core vs. induced with methanol in Click here for additional data file.10.7717/peerj.115/supp-3Table S3Methylotenera mobilis JLW8.Comparative RNA-Seq statistics for core vs. methylamine in Click here for additional data file.10.7717/peerj.115/supp-4Table S4Methylotenera mobilis JLW8.Comparative RNA-Seq statistics for induced with methanol vs. methylamine in Click here for additional data file.10.7717/peerj.115/supp-5Table S5Methylotenera versatilis 301.Comparative RNA-Seq statistics for core vs. induced with methanol in Click here for additional data file.10.7717/peerj.115/supp-6Table S6Methylotenera versatilis 301.Comparative RNA-Seq statistics for core vs. methylamine in Click here for additional data file.10.7717/peerj.115/supp-7Table S7Methylotenera versatilis 301.Comparative RNA-Seq statistics for induced with methanol vs. methylamine in Click here for additional data file.10.7717/peerj.115/supp-8Table S8Comparative RNA-Seq statistics for core vs. methanol in Methylovorus glucosotrophus SIP3-4.Click here for additional data file.10.7717/peerj.115/supp-9Table S9Comparative RNA-Seq statistics for core vs. methylamine in Methylovorus glucosotrophus SIP3-4.Click here for additional data file.10.7717/peerj.115/supp-10Table S10Comparative RNA-Seq statistics for methanol vs. methylamine in Methylovorus glucosotrophus SIP3-4.Click here for additional data file.10.7717/peerj.115/supp-11Table S11Gene designations, functions and protein identifiers for key enzyme systems known to be or potentially involved in methylotrophy.Click here for additional data file.


C. elegans TCF) also activates target genes through HMG and Helper site interactions. Helper sites enhanced the ability of a synthetic enhancer to detect Wnt/ß-catenin signaling in several tissues and revealed an unsuspected role for POP-1 in regulating the C. elegans defecation cycle. Searching for HMG-Helper site clusters allowed the identification of a new POP-1 target gene active in the head muscles and gut. While Helper sites and the C-clamp are essential for activation of worm and fly Wnt targets, they are dispensable for TCF-dependent repression of targets in the absence of Wnt signaling. These data suggest that a fundamental change in TCF-DNA binding contributes to the transcriptional switch that occurs upon Wnt stimulation.Regulation of gene expression by signaling pathways often occurs through a transcriptional switch, where the transcription factor responsible for signal-dependent gene activation represses the same targets in the absence of signaling. T-cell factors (TCFs) are transcription factors in the Wnt/ß-catenin pathway, which control numerous cell fate specification events in metazoans. The TCF transcriptional switch is mediated by many co-regulators that contribute to repression or activation of Wnt target genes. It is typically assumed that DNA recognition by TCFs is important for target gene location, but plays no role in the actual switch. TCF/Pangolin (the fly TCF) and some vertebrate TCF isoforms bind DNA through two distinct domains, a High Mobility Group (HMG) domain and a C-clamp, which recognize DNA motifs known as HMG and Helper sites, respectively. Here, we demonstrate that POP-1 (the  Drosophila. This work thus reveals an unexpected complexity in POP-1 DNA binding, which is likely to be relevant for its human counterparts, which play important roles in stem cell biology and cancer.The DNA of cells must be correctly “read” so that the proper genes are expressed. Transcription factors are the primary “DNA readers”, and these proteins bind to specific DNA sequences. Using nematodes as a model system, we investigated the rules of DNA binding for a particular transcription factor, called POP-1, which mediates Wnt signaling, an important cell-cell communication pathway. In addition to its known DNA binding site, we found that POP-1 recognizes additional sequences, termed Helper sites, which are essential for activation of Wnt targets. We used this knowledge to discover that Wnt signaling is active in pacemaker cells in the nematode intestine, which control defecation, a rhythmic behavior with parallels to the vertebrate heartbeat. POP-1 has a dual role in regulating Wnt targets, repressing target genes in the absence of signaling and activating them upon signal stimulation. Surprisingly, we found that Helper sites are only required for activation and not repression, and that this is also the case in the fruit fly  Transcriptional switches are common in the regulation of gene expression by cell-cell signaling pathways cis-regulatory modules that control Wnt target gene transcription, through direct binding to TCFs The T-cell factor (TCF) family of transcription factors (TFs) offers a prominent example of a transcriptional switch Drosophila and C. elegans each possess a single TCF gene, TCF/Pangolin (TCF/Pan) in flies and pop-1 in nematodes. Genetic evidence indicates that both these TCFs operate as transcriptional switches. For example, in C. elegans embryos, Wnt signaling activates transcription of end-1 and other endoderm-specific genes in the E blastomere pop-1 mutants, there is a reduction of end-1 expression in E cells end-1 and other endoderm markers end-1 WRE reporter results in a similar pattern of GFP expression as seen in POP-1 mutants TCF/Pan results in both loss of activation and expansion of Wnt targets The current model for the TCF regulatory switch is that ß-catenin promotes a dramatic change in transcriptional co-regulators associated with TCFs. Transducin-like enhancer of Split (TLE) co-repressors such as Groucho in flies C. elegans utilize an additional mechanism, often referred to as the “Wnt/ß-catenin asymmetry” (WßA) pathway. WβA signaling promotes nuclear influx of the ß-catenin homolog SYS-1, while also promoting nuclear efflux of POP-1 In addition to this classic switch, many Wnt dependent targets in Drosophila and mammalian cell culture All TCFs contain a HMG domain, which recognizes a 9 bp consensus of SCTTTGATS (S = G/C) with high affinity ceh-22, psa-3 and end-1 loci. We found that all three had Helper site motifs near the functional HMG sites, and these Helper sites were crucial for expression of WRE reporters in transgenic worms. The presence of Helper sites dramatically increased binding of POP-1 to HMG sites in vitro. In some cases, multiple Helper sites functioned with a single HMG site to enhance POP-1 binding and mediate expression in vivo. An in silico search of the C. elegans genome for similar Helper sites-HMG site clusters uncovered a new WRE upstream of the K08D12.3 gene, which is expressed in head muscles and the gut. In addition, a synthetic reporter containing concatamerized HMG and Helper sites displayed a novel POP-1 dependent pattern in the int9 cells of the larval intestine, leading us to discover that pop-1 regulates the C. elegans defecation cycle. As previously reported end-1 WRE reporter results in derepression in MS blastomere, but this was not observed upon mutation of two nearby Helper sites, although these motifs were required for expression of the reporter in the E blastomere. This differential requirement of Helper sites in the transcriptional switch was also observed in a fly WRE. Consistent with the cis-regulatory mutagenesis data, we also found that the C-clamp is not required for TCF-mediated repression in the developing fly wing, but is essential for activation. These data support a model where basal repression of WREs occurs through HMG-HMG site interactions, whereas conversion of TCF to a transcriptional activator requires HMG-HMG site and C-clamp-Helper site recognition of DNA. Our data indicate that the TCF binding site is not passive during the transcriptional switch, but rather plays a more active role than previously suspected.In this study, we extended our analysis of Helper sites to three WREs directly regulated by the WßA pathway, from the C. elegans WREs. Therefore, we examined three targets that have previously been shown to be directly regulated by the WßA pathway, ceh-22psa-3end-1Like TCF/Pan and vertebrate TCF1E and TCF4E isoforms, POP-1 contains a C-clamp downstream of its HMG domain ceh-22 encodes a homeodomain TF that is required for specification of the distal tip cells (DTCs) ceh-22 locus produces three isoforms, but the ceh-22b/c isoforms (termed ceh-22b) (ceh-22 mutants ceh-22b isoform (ceh-22b::VENUS) is expressed in the somatic gonadal precursors (SGPs) and their descendants Z1.a and Z4.p, the distal daughters of which become the DTCs sys-1 and pop-1, and two HMG sites in the ceh-22b::VENUS reporter ceh-22b)  are suffceh-22b::VENUS reporter revealed that they all contribute to expression in SGP descendants. Transgenic worms expressing stably integrated versions of the wild type (WT) or mutant ceh-22b::VENUS reporters were generated  with recombinant POP-1 and DNA probes containing Helper 1, HMG2 and Helper 2 from the ceh-22b reporter . Based on the organization of functional Helper and HMG sites in ceh-22b, psa-3 and end-1 (ceh-22b and end-1 WREs (list provided in K08D12.3) or the quality of the HMG and Helper sites (RO5G6.5 and Y66d12A.9).We next wanted to test whether a computational search for HMG and Helper site clusters could identify new POP-1 targets in the ed below . Becausend end-1 , we utilK08D12.3/ZNF9 gene had significant binding to POP-1 , posterior intestine and hindgut during larval stages till adulthood  mutants or animals depleted of POP-1 via RNAi feeding (data not shown). However, these conditions only partially reduce pop-1 gene activity To test whether the region upstream of reporter , and tesK08D12.3 reporter, late L4 hermaphrodites containing a wild-type, a HMG mutant or a triple Helper mutant reporter were scored based on VENUS expression in the hindgut and midgut, as well as the head and pharyngeal muscles, and characterized as having strong, intermediate or weak expression  contains seven copies of a high affinity HMG site, which displays a wide range of expression including several cells where Wnt/POP-1 signaling is known to occur POP-1 and HMG-Helper Optimal Promoter) and tested it for expression in C. elegans.Synthetic reporters containing concatemerized high-affinity HMG sites have been used as Wnt/ß-catenin signaling readouts in several systems pop-1(hu-9)  . In sum,C. elegansC. elegans.The expression pattern of POPHHOP in int9 cells was intriguing, given the central role that these cells are known to play in regulating the defecation cycle in pop-1 are early larval or embryonic lethal pop-1, both containing single amino acid substitutions in the ß-catenin binding domain, were used to examine the role of POP-1 in the defecation cycle. pop-1(hu9) homozygotes survive to adulthood pop-1(q645) make it to adulthood pop-1 mutants in early L2 larvae, well before any lethality was apparent in the pop-1(q645) animals.Strong alleles of pop-1(hu9) and pop-1(q645) alleles had a 11–18% increase in mean cycle time and greater arrhythmia than N2 controls (pop-1(q645) mutants, while such failures were never seen in control or pop-1(hu9) animals . RNAi depletion of pop-1 resulted in a significant increase (8.8% in the mean period) in the defecation cycle and greater variability compared with control fed animals  causes a reduction in expression in E cells, accompanied by a dramatic elevation of expression in MS cells end-1 by POP-1 provides an ideal system in which to test the role of Helper sites in a target where both sides of the transcriptional switch are robustly apparent.Although the TCF transcriptional switch is the prevailing model for Wnt/β-catenin gene regulation, for many WREs, there is little evidence for repression in the absence of signaling on end-1 . In earlend-1::GFP::H2B reporter fusions were generated , originally identified through a computational search for HMG-Helper site clusters in the fly genome pxb-WRE reporter was also expressed at low levels in the anterior midgut , or LEF1 with the C-clamp of TCF/Pan  but strongly suppressed the formation of ectopic bristles  ; Table 2pxb WREs , supportC. elegans WREs expressed in a variety of tissues and developmental stages  was tested in a transgenic GFP reporter assay  working in cell-cell signaling pathways pop-1 dependent pop-1(q645)egl-20, cwn-1 and lin-44 are expressed close to or overlapping the int9 cells The defecation behavior is comprised of rhythmic contractions of the posterior body wall muscles, followed by the anterior body wall muscle and eventually the enteric muscles expelling the fecal matter. Under conditions of constant feeding, these steps are highly regular rhythmia . These dpop-1 is in a class of mutants that show defecation cycle length and expulsion defects  lead to a dramatic increase in the cycle length with mutants displaying severe arrhythmia and expulsion defects pop-1 mutants show a subtle phenotype compared to egl-8, which could be attributed to the hypomorphic alleles used in our study. Interestingly, egl-8 is expressed in the posterior intestine egl-8 that could contribute to this pattern  GCATGCCTGCAGCCGATTGCCGGAATGGCTTTGCGC3′5′TCGAA. The POPHHOP plasmid was generated by cloning 6×HMG-Helper (6× AAGATCAAAGGGGGTAGCCGCCAGTGG) pPD107.94  vector using the NheI sites. 6×Histagged full length POP-1 plasmid (pRSETA-POP-1) was kindly provided by Dr. David M. Eisenmann and the C-clamp mutant used in pxb-lacZ plasmid was generated by subcloning the pxb cluster3 WRE into the pH-Pelican vector as described previously pUAS LEF1V5 and pUAS LEF1C-clamp V5 were generated by PCR based cloning into pUAST vector. pUAS LEF1-V5 was generated by subcloning a human LEF1 fragment from an expression plasmid (kindly provided by Dr. Marian L. Waterman) into pActin5.1 to introduce a V5 tag. PCR based amplification of LEF1 was carried out using forward primer GGTACCATGCCCCAACTCTCCGGA3′5′CCCC and reverse primer GAATTCTGCGATGTAGGCAGCTGTCATTCTTGG3′5′CAGT and inserted into the KpnI and EcoRI sites of the pActin5.1 vector, with the stop codon mutated (underlined). Lef1V5 was digested using the KpnI and PmeI sites and inserted into the KpnI and XbaI sites of pUAST. Prior to insertion, pUAST vector was restricted with XbaI and sticky ends filled in by Klenow to create blunt ends, followed by a restriction with KpnI. pUAS-Lef1C-clamp V5 plasmid was generated by PCR based amplification of the C-clamp  from fly TCF/Pan and insertion into the EcoRI site of pUAS Lef1V5 by non-directional cloning.For C.elegans N2 Bristol strain and cultured using standard protocols. Transgenic strains with extrachromosomal arrays were generated by injecting WT or mutant versions of ceh-22b::VENUS (100 ng/µl), psa-3::GFP (50 ng/µl), end-1::GFP::H2B (100 ng/µl) or K08D12.3::VENUS (150 ng/µl) plasmid into N2 worms, along with coinjection marker myo-2::RFP plasmid (3 ng/µl) and pActin5.1 . Stable integrants were generated by UV irradiation using a Stratalinker (Stratagene) at power 325. POPHHOP plasmid (1 ng/µl) was injected along with a dpy-20(+) plasmid (50 ng/µl) and pBluescript (100 ng/µl) and stable integrants generated by gamma-irradiation. Animals with integrated transgenes were outcrossed at least three times. Transgenic POPTOP (7× HMG::mCherry) strain was kindly provided by Dr. Paul W. Sternberg POPHHOP was crossed into a pop1(hu9)psa-3::GFP transgenic analysis during which synchronized L1s were grown at 20°C for 9 hours only for the reporter analysis. pop-1(q645) strain (JK2944) was obtained from Caenorhabatitis Genetics Center (CGC).Worm strains were derived from the wild-type pxb-lacz, Lef1 and Lef1 C-clamp flies were generated by P-element transgenesis (performed by BestGene Inc.). w1118 was obtained from Bloomington Stock Center. C96::Gal4 was kindly provided by Dr. Rolf Bodmer TCF/Pan RNAi line (#25940) was obtained from Vienna Drosophila RNAi Center. All fly crosses were performed at 25°C.Transgenic C.elegans larvae and embryos by Nomarski optics have been described previously ceh-22b::VENUS, psa-3::GFP, end-1::GFP::H2B, K08D12.3::VENUS and myo-2::RFP reporter expression was analyzed by fluorescence on a Olympus BX61 motorized X-drive microscope. Images were taken using a Hamamatsu ORGA-ERCA-CCD camera, with a specific exposure time for each WRE reporter and multiple focal planes were merged to obtain the representative image. Deconvolution was performed using slidebook 5.0 software and the nearest neighbors method. POPHHOP and POPTOP reporter expression was analyzed using a Zeiss Axioscope microscope equipped with a Zeiss Axiocam digital camera. pxb-lacZ WRE images were obtained using a Leica triple channel confocal microscope DM6000B-CS and multiple focal planes were merged to obtain the representative image. All images were processed using Adobe Photoshop 8.0.Methods for mounting and viewing E.Coli and purified using nickel beads (Sigma). ESMA was performed as described previously Full-length 6×His-POP-1 was expressed in ceh-22b::VENUS fluorescence analysis, worm larvae were fixed in 4% paraformaldehyde (in M9) for 15 min, using a protocol adapted from the whole-mount freeze-cracking method pxb-lacZ WRE analysis, fly embryos were fixed and immunostained using anti-lacZ and anti-Wg as described previously For pop-1 mutants were maintained at 20°C, and scored for defecation at room temperature (∼23°C) using a Leica MZ16 F stereoscope at a 115× magnification. The protocol was adapted from a previous report pop-1(hu9) worms were maintained as a homozygotes pop-1(q645)  strain segregates as WT- green-fluorescing heterozygotes, and non-fluorescing homozygotes. pop-1(q645) hermaphrodites are sterile due to severe gonadal development defects pop-1(q645) larvae were scored for defecation cycles, transferred to individual plates and allowed to grow to adulthood to confirm that they were sterile and had a protruding vulva pop-1(hu9) (G to A)(E47 to K) pop-1(q645) (T to A)(D9 to E) 5′CTCCATGGCCTAACTTCCGCGGACC3′ and reverse primer 5′GTCGAAAGGCAATTGAGGTGGTCC 3′.N2 worms and RNAi feeding of N2 and OLB11 strains were performed as described Adult flies were stored in 70% ethanol and soaked in 100% ethanol for two hours before dissection in 100% ethanol. Dissected wings were transferred into Xylene (Fisher) and mounted on a slide with Permount (Fisher). Wing notch and bristle images were obtained using the Nikon Eclipse 800 microscope. All images were processed using Adobe Photoshop 8.0.Figure S1ceh-22b and psa-3 WRE probes. EMSA analysis of a 69 bp ceh-22b WRE probe (A) and 47 bp psa-3 WRE probe (B) both of which show a POP-1 dependent shift (lane 2). For ceh-22b, an excess of unlabeled wildtype (WT) oligonucleotide competes with the labeled probe for POP-1 binding (lanes 3 & 4). Much less competition is observed when oligonucleotides containing mutations in the Helper 1 (lanes 5 & 6) or Helper 2  sites are used. No competition is observed with DNA containing mutations in both Helper sites (lanes 9 & 10) or the HMG2 site (lanes 11 & 12). For the psa-3 probe, efficient competition is observed with excess of unlabeled WT DNA (lanes 3–5), which is greatly reduced with Helper mutant (lanes 6–8) or HMG mutant (lanes 9–11) DNA. Black arrowheads represent the DNA-protein complex and white arrowheads represent unbound probe. ‘*’ represents a band which was seen in some experiments which we suspect is a POP-1 degradation product.Helper sites are important for binding of POP-1 to (PDF)Click here for additional data file.Figure S2K08D12.3 locus. (A) Probes derived from genomic sequences of 3 genes identified in a genome-wide search for one HMG site - two Helper sites clusters. (B) The long probe derived from a region upstream of the K08D12.3 gene showed robust binding to POP-1 (lanes 1–3). A shorter probe lacking the first Helper site has weaker binding (lanes 4–6). Two other clusters were negative for binding to POP-1 (lanes 7–12). A ceh-22b probe was used as a positive control (lane 13). Black arrowheads represent the DNA-protein complex and white arrowheads represent unbound probe.Secondary screen for putative WREs using EMSA reveals strong binding of POP-1 to a HMG-Helper site cluster from the (PDF)Click here for additional data file.Figure S3ceh-22b WRE probes. EMSA analysis of the ceh-22b WRE probe containing two Helper sites (lanes 1–5) and a probe where these motifs are mutated (lanes 6–10). Recombinant wild-type or C-clamp mutant POP-1 (400 & 800 ng/reaction) was added where indicated. Either probe was used at 1.5 femtomoles/reaction. A dramatic reduction in binding was observed in the C-clamp mutants  or when wild-type POP-1 was incubated with the Helper site mutant probe (lanes 7 & 8).Helper sites are important for binding of wild-type and C-clamp mutant POP-1 to (PDF)Click here for additional data file.Figure S4POPHHOP reporter construct. (A–F) DIC images with GFP fluorescence overlay. The reporter is expressed with a high penetrance during the L1 stage, in the int9 cells, the tail neurons and VC neurons (A) and during L3 stage in DTCs (B). During the L1 stage, expression was occasionally seen in muscle cells (C), seam cells (D–G) and QL daughters (E–F). Scale bar = 10 µm.Expression pattern of the (PDF)Click here for additional data file.Figure S5Expression of human Lef1 and Lef1-C-clamp chimera in wing imaginal discs. (A) Cartoon of human LEF1 and LEF1-C-clamp fusion showing the β-catenin binding domain (green), the HMG domain (red), the basic tail (orange), the C-clamp (blue), a linker (gray) and the V5 epitope (hatched box). Immunoblot showing the expression levels in dissected wing discs from two lines (A and B) of V5 tagged Lef1 (*) or the Lef1-C-clamp chimera (**).(PDF)Click here for additional data file.Figure S6pha-4 TlSS showing the putative HMG (red) and Helper sites (blue). B) Genomic sequence of egl-8 showing clusters i and ii with HMG (red) and Helper (blue) sites identified in the intronic regions of egl-8. The flanking exon sequences are highlighted in green.HMG-Helper site clusters in putative POP-1 targets. A) Genomic sequence of a region upstream of the (PDF)Click here for additional data file.Figure S7egl-18, which was used for a transcriptional fusion reporter Putative HMG and Helper sites in a known Wnt target. Genomic sequence of a region upstream of the TlSS of (PDF)Click here for additional data file.Table S1Mutations introduced in the HMG or Helper sites for reporter constructs and EMSA experiments. For each motif the wild-type sequence is shown in the first row with mutant substitutions (lower case) in the second row for either reporter gene constructs (mutT) and/or EMSAs (mutE).(DOCX)Click here for additional data file.Table S2Score Matrices used for HMG and Helper computational search using target explorer (DOCX)Click here for additional data file.Table S3List of 19 hits from a genome-wide search for HMG-Helper site clusters. Hits contained at least two Helper and one HMG site within 50 bp. The cutoffs for Helper and HMG sites were 5.51 and 6.69, using the weighed matrices shown in (DOCX)Click here for additional data file.


Drosophila, the polycomb repressive complexes (PRC) associate with defined polycomb response DNA elements (PREs). In mammals, however, the mechanisms underlying polycomb recruitment at targeted loci are poorly understood. We have used an in vivo approach to identify DNA sequences of importance for the proper recruitment of polycomb proteins at the HoxD locus. We report that various genomic re-arrangements of the gene cluster do not strongly affect PRC2 recruitment and that relatively small polycomb interacting sequences appear necessary and sufficient to confer polycomb recognition and targeting to ectopic loci. In addition, a high GC content, while not sufficient to recruit PRC2, may help its local spreading. We discuss the importance of PRC2 recruitment over Hox gene clusters in embryonic stem cells, for their subsequent coordinated transcriptional activation during development.Polycomb group (PcG) proteins are essential for the repression of key factors during early development. In  Hox genes are essential for the proper organization of structures along the developing vertebrate body axis. These genes must be activated at a precise time and their premature transcription is deleterious to the organism. Early on, Hox gene clusters are covered by Polycomb Repressive protein Complexes (PRCs), which help keep these genes silent. However, the mechanism(s) that selectively recruit PRCs to these particular genomic loci remains elusive. We have used a collection of mutant mice carrying a set of deletions inside and outside the HoxD cluster to try and detect the presence of any DNA sequence of particular importance in this mechanism. We conclude that a range of low affinity sequences synergize to recruit PRCs over the gene cluster, which makes this process very robust and resistant to genetic perturbations. Polycomb) was characterized genetically as a repressor of Drosophila homeotic genes via epigenetic mechanisms such as X-inactivation trithorax families (trxG), displaying opposing functions, was shown to maintain Hox gene expression during the entire life of DrosophilaPolycomb group (PcG) proteins are essential for proper development of most eukaryotic organisms. The founding member . PRC2 carries a methyl-transferase activity that methylates the histone H3 tail at lysine 27, a mark largely associated with gene silencing. While the initial deposition of this post-transcriptional modification is carried through by PRC2, PRC1 maintains this methylated status and compacts chromatin, largely, though not solely Drosophila, PcG group proteins are recruited to chromatin through the recognition of polycomb response elements (PREs). These DNA segments, which were generally identified by forward genetics, must satisfy three criteria: (1) they must bind polycomb when randomly inserted into the genome, (2) they must be able to induce a H3K27me3 domain and (3) they must repress a reporter construct, when associated with them Drosophila PREs are approximately 1.5 kb long in average and are usually located in proximal promoter regions. This is the case of the PREs associated with either engrailedHedgehogpolyhomeoticIn in vivo several kilobases away from their target genes and their identification may be biased by genome wide chromatin immunoprecipitation (ChIP) studies. Recently indeed, it has become clear that only a fraction of polycomb enriched regions are direct targets of PRC, whereas others are indirect, via chromatin looping polycomb enriched regions may not always reflect the genuine presence of polycomb at a particular locus. Instead, they may derive from the three-dimensional organization of the genome, which along with the technology employed, may lead to false positives. Furthermore, although high throughput studies have shown that the binding profiles of polycomb proteins correlate with both the transcription start sites (TSSs) and stalled RNA PolII However, PREs have also been mapped Pho ortholog YY1 Pho and PRC remains controversial In mammals, the understanding of the general mechanism (if any) accounting for the recruitment of PRC2 is lacking too. PRC2 recruitment has been associated with the presence of binding sites for the Hox gene clusters  cells, including the four clusters . In thisHox loci by using an in vivo approach, based on the large number of genomic re-arrangements associated with the HoxD locus Drosophila Bithorax complex where a 300 kb large domain of trimethylated histones involves only few PREs, we show that the mouse HoxD locus implements a mechanism that can compensate for large and systematic deletions within the target DNA interval. These results indicate that PRC2 recruitment at this locus must rely upon a range of cooperating binding sites, rather than upon a few nucleation sites. By using isolated transgenes, we also show that in this particular context, CpG islands (or a high GC content) are not the prime factors in PRC2 recruitment, despite their potential importance for local spreading.We have addressed the question of polycomb recruitment at in vivo, we used a genetic approach of the mouse HoxD gene cluster, which is a main target of Pc silencing in ES cells and adult tissues To study the recruitment of polycomb complexes and the resulting H3K27me3 histone modification HoxD cluster did not seem to affect the binding profiles of PRC proteins when assessed by ChIP-qPCR ) in vivo had no effect upon gene expression within the HoxD cluster del(12)). Here again, we did not score any modification of the H3K27me3 profile throughout the gene cluster. We next scanned the entire locus with a set of adjacent deletions, including either Hoxd8, Hoxd9, Hoxd10, Hoxd11, Hoxd12 or Hoxd13 and found no significant change in polycomb-mediated silencing (data not shown), using H3K27me3 as a proxy for PcG occupancy  and Del(9-12)  . These tH3K27me3 , suggestHox gene cluster were of particular importance to set up a platform for recruiting PRC2. We used a large deletion (del(1-10)), where two-thirds of the anterior part of the cluster were removed including its most anterior gene Hoxd1. Again, the remaining mini-cluster was able to compensate for this significant trimming and the H3K27me3 pattern over the remaining loci was nearly identical to that found in wild type conditions d11Lac), did not significantly affect the presence of these epigenetic marks over the remaining 5′ located Evx2 gene , even though the latter displayed massive amounts of H3K27me3 over the anterior part of the cluster , del(13) and del(10-13), which share the same breakpoints but in various configurations. In this set of deletions, the reconstitution of three different neighborhoods did not modify the methylation patterns.This result indicated that each piece of the gene cluster is rather independent in its ability to recruit PRC2, regardless of what would happen over the neighboring loci. The poor impact of the neighboring sequences upon the coverage of any given HoxD. This intergenic region ‘i’ is over 13 kb long and maps between Hoxd4 and Hoxd8. Its deletion (del(i)) results in a further concentration of genes by bringing Hoxd1, Hoxd3 and Hoxd4 closer to the centromeric (posterior) side including Hoxd8 to Hoxd13. del(i) did not show any clear difference in the H3K27me3 profile over the remaining parts of the gene cluster (del(i)).Single gene deletions did not markedly change the relative distance between transcription units and hence they may not affect PRC2 recruitment, should several PREs locate near each gene and synergize. We thus modified the distance between two genes by excising the longest gene free DNA segment within i to produce a cluster with a 26 kb large gene-free domain inside. Intriguingly, the duplicated configuration did not exhibit any change on region i, except for a weak gain of signal over the duplicated region suggesting that both copies are covered by H3K27me3. The flanking DNA segments, however, displayed the same H3K27me3 profiles as in wild type brains (del(i), dup(i)) indicating that the mechanism recruiting PRC2 over the mouse HoxD cluster can compensate for modifications in the distance between transcription units, emphasizing once more the robustness of this process.We next looked at the effect of introducing a gene free region within the cluster such as to increase the distance between neighboring genes. We duplicated region HoxD cluster, which contain numerous cis-acting sequences. We deleted a 230 kb large piece of DNA, from eight kb upstream Evx2 to a breakpoint located within the flanking centromeric gene desert (del(R1-R5)-d9Lac) HoxD gene cluster per se, it removed the border of the H3K27me3 domain and hence it reconstituted a neighborhood between heavily H3K27 tri-methylated nucleosomes and nucleosomes not methylated at all.In the absence of any strong and discrete signal for PRC2 recognition and nucleation within the cluster itself, we asked whether Pc proteins may be targeted by elements localized within the regulatory landscapes flanking the HoxD cluster in the developing brain, nor did it induce any leakage over the centromeric DNA from the gene desert (del(R1-R5)-d9Lac). Therefore, as previously reported The deletion of this ‘epigenetic border’ did not elicit any loss of H3K27me3 marks over the HoxA, HoxC and HoxD clusters. In the case of HoxD, the Evx2 gene is found ca. 10 kb upstream Hoxd13 on the opposite strand. This gene, which is covered by H3K27me3 marks and locates close to the epigenetic border, was however not removed in the del(R1-R5)-d9Lac deletion. Therefore, we analyzed a second deletion, ca. 260 kb large, with the same upstream breakpoint into the gene desert (see above), but with a telomeric breakpoint located between Hoxd10 and Hoxd11. In this del(11-R5)-d9Lac mutant, the entire posterior part of the HoxD cluster was removed including Hoxd11, Hoxd12 and Hoxd13 as well as the Evx2 transcription unit and the epigenetic border.Another mechanism to set the PcG epigenetic borders may involve transcripts encoded by the opposite DNA strand, a feature found in the del(R1-R5)-d9Lac deletion, suggesting that additional transcriptional units encoded by either DNA strands are not necessary for the recruitment of PRC2 at the extremity of the HoxD cluster, nor for the fixation of a sharp epigenetic boundary (del(11-R5)-d9Lac). In both deletions, however, a Hoxd9/Lac transgene was relocated at the breakpoint, raising the possibility that transgenic sequences would interfere with PRC2 recruitment and hence we used a final mutant configuration carrying a ca. 800 kb large deletion including the HoxD centromeric regulatory landscape. This del(Nsi-Atf2) deletion not only removes the 5′ epigenetic border, but also most of the regulatory elements that contact Hoxd genes and impose a chromatin topology to the locus In this configuration, the H3K27me3 profile remained unchanged when compared to the wild type pattern. In particular, the reconstituted epigenetic boundary was similar to that seen with the shorter HoxD cluster towards the new centromeric neighboring sequences did not exceed a 800 bp large interval, which corresponds to twice the average length of the sonicated DNA fragments (data not shown). These results further indicated that the capacity to recruit PRC2 is restricted to Hox genes themselves, without any contribution from the surrounding genomic sequences. To demonstrate this point, we produced a transgenic line containing the Hoxd10 gene, which had inserted into a genomic region of average GC density and poor in H3K27me3 marks. While H3K27me3 was scored on the entire transgene, this histone modification did not spread over flanking nucleosomes, as assessed by ChIP-seq . Therefore, when this transgenic stock was crossed back into a mouse carrying a homozygous deletion of Hoxd10 (TgN/del(10)−/−), the H3K27 trimethylation profile (or the lack thereof-) over Hoxd10 reflected that of the ectopic Hoxd10 copy. The Hoxd10 locus was selected because the CpG island located upstream the promoter (CpG32 from UCSC) could be removed by using FRT sites and the Flip recombinase in vivo, without affecting the transcription start site (TSS). To make sure that no additional CpG islands remained after deletion of CpG32, we deleted another potential short island (CpG26) from our starting transgenic construct.The Hoxd10 null mice to assess their H3K27me3 status in developing forebrains . Similar results were observed when the CpG26 sequences had been removed (TgNd10). Moreover, similar amounts of H3K27me3 were scored when the transcription start site of TgNd10 was deleted, suggesting that the recruitment of PRC2 may be independent of transcription (TgNd10∂TSS) TgNd10∂CpG).Transgenic animals were crossed with a Cre-deleter strain to adjust copy number to one and various transgenes were thus crossed over rebrains . When th removed , TgNd10.Hoxd10 was used, H3K27me3 marks were no longer detected, even though this construct still contained an annotated CpG island (TgNd10∂3) and was globally GC-rich. Of note, a transgene containing the same four kilobases together with exon 1 of Hoxd10 showed no recruitment of PRC2 either (TgNd10∂PREd10), regardless whether or not the TSS was present (TgNd10∂TSS∂PREd10), suggesting that neither the TSS, nor the CpG32 are essential for recruiting PRC2 in this configuration. Mapping the insertion sites did not reveal any correlation between the presence of H3K27me3 on the transgenes and their insertion into either a H3K27me3-rich or a GC-rich DNA region. In fact, transgenes were found integrated at least 500 kb away from H3K27me3-rich spots and into DNA segments with rather average GC contents (data not shown). These experiments thus defined a 1.4 kb large DNA segment, containing exon 2 and the 3′UTR of Hoxd10, which was necessary for the deposition of H3K27me3 marks. This DNA segments is referred to as PREd10 below.However, when a four kb large transgene containing only the 5′ sequence upstream Hox clusters are twice as dense in differentiated tissues than in ES cells  were thus used to assess the H3K27 methylation status of distinct electroporated DNA elements, overlapping with the deleted Hoxd10 DNA segment. Various portions of the TgNd10 transgene were first cloned between two homologous arms (Env) flanking the transgenes, in the hope of comparing random and targeted integration sites. However, homologous recombination events were not found.While H3K27me3 marks covering the ES cells , the extHoxd10 fragment, including the TSS and both exons, or the 1.4 kb long PREd10 were introduced into our iPSdel(Hoxd10) cells, they became H3K27 tri-methylated, in agreement with the results obtained using classical transgenesis. More surprisingly, when the 5′ sequence corresponding to that used in TgNd10∂3 was assayed, H3K27me3 was detected too, in contrast to the results obtained in transgenic mice. We checked the capacity of either the vector backbone, or the PGK-neomycin gene promoter to recruit PRC2, by electroporating the neomycin cassette alone. The PGK promoter is ubiquitous and hence neomycin transcripts were detected in all conditions tested . We verified if this recruitment was influenced by the presence of the DNA homology arms included for a potential recombination at the locus, which contained sequences from both the Hoxd11 and a portion directly 3′ to Hoxd10, which could thus initiate a ‘spreading’ of H3K27me3 marks over the Env-d10∂3 fragment. Accordingly, we electroporated d10∂3 (42% rich in GC) into iPSdel(Hoxd10) without any other surrounding DNA sequences. While H3K27me3 was detected over a multimerized version of d10∂3, this mark was lost after the CRE recombinase had reduced copy number to one (d10∂3-CRE). In contrast, when CpG-island free PREd10 (44% rich in GC) was introduced into iPSdel(Hoxd10), H3K27me3 marks were readily scored after CRE-excision of the multimers (d10-CREd10PRE). PRC1/2 subunits were also detected over this exogenous, randomly integrated sequence, suggesting it contains all proper information necessary for PcG recruitment to ectopic sites  and PREd10-600 (43% rich in GC), which were tested as individual transgenes. Unexpectedly, both fragments were decorated by H3K27me3, when introduced into iPSdel(Hoxd10) cells as single copy  displaying a temporal sequence in the establishment of their segmented body plan systematically show a complete clustering of their Hox gene complement, whereas other animals following different strategies (such as cell lineages) usually have broken Hox clusters or even Hox genes scattered throughout the genome  Hox clusters are amongst the genomic loci with the highest GC content.As for many Drosophila PREs, PREHox gene clusters, they do not rule out their potential importance for the spreading or the re-enforcement of the coverage by PRC2 (see below). The existence of CpG islands devoid of PcG, as well as PcG target DNA devoid of CpG islands, such as in the case of the first described mammalian PRE-like sequence regulating the mouse MafB/Kreisler, support this view. Moreover, sequences unable to recruit PRC when present as single copy transgenes may become H3K27me3 when concatamerized, suggesting that larger stretches of GC-high sequences can artificially recruit PRC, a possible explanation to the discrepancies observed between our results and those of others Here again however, our results do not support a high GC content as the major parameter in recruiting PRC2. We show that DNA segments with a GC content similar to the average of the mouse genome (42%) are still able to properly recruit PcG proteins and the deletion of CpG islands from our transgenic constructs did not abrogate the trimethylation of H3K27. While these results suggest that CpG islands are neither sufficient, nor required, for the tethering of PcG proteins in the context of ad minima model whereby H3K27me3 is deposited on a series of low affinity PRC2 interacting sequences, which work synergistically between themselves and together with GC-rich sequences to confer robust silencing over target genes .Hoxd1 and either the site telomeric to Hoxd8 (del(1-i)) or the site centromeric of to Hoxd4 (del(1-4)). The del(i) line was produced by TAMERE between the loxP telomeric of Hoxd8 and the one centromeric to Hoxd4. Genotyping was performed on individual yolk sacs.All stocks of mice were kept as heterozygous and bred to homozygosity. Lines were all described and can be found in previous publications of the Duboule laboratory. Two additional lines were produced by recombination between the loxP site in the second exon of Chromatin immunoprecipitation followed by quantitative polymerase chain reaction was performed as described in Mouse embryonic fibroblasts were derived from heterozygous crosses of E13.5 embryos using standard protocols. Cells were cultured in standard MEF/ES cell culture conditions. MEF/ES media contained DMEM supplemented with 10% FBS and LIF (ES media only). Isolated MEF lines were first genotyped using embryonic tissues and subsequently confirmed with DNA extraction procedures. Passage three MEFs were used for iPS derivation experiments.Oct4, Klf4 and Sox2 were cloned and separated by bacterial 2A sequences, in a single lentiviral backbone (3F). Virus was produced in 293T cells using FuGENE HD transfection reagent  and ultracentrifuged. Induced pluripotent (iPS) stem cells were derived following standard protocols SSEA1, Nanog, Oct4 and Sox2 by immunohistochemistry and western blot, standard protocols), a non-aberrant chromosome count  and re-establishment of bivalent domains .Human 6. Electroporated cells were plated on 10 cm dishes coated with DR4 resistant feeders. G148 selection (200 µg/ml) (Sigma G8168-10ML) was started 24 hours after electroporation and was continued until individual colonies were picked and genotyping. CRE treatment of iPS cells was done as follows: 3×105 cells were plated overnight and transduced with a PGK-CRE lentiviral construct at MOI 100. Individual colonies were picked 5 days post-transduction, expanded and genotyped.Electroporation of induced pluripotent stem cells was performed using an Amaxa Nucleofector I and the Lonza mouse embryonic stem cell kit (Lonza VPH-1001). Briefly, 25 µg of DNA were digested overnight, phenol-chloroform purified and resuspended in 10 µl H2O. Media was changed 4 hours before electroporation. Cells were washed twice with Mg(2+)-Ca(2+)-Free PBS, trypsinized and aliquoted to 2×10HoxD cluster were spotted with 25-mer oligonucleotides at 15b bp resolution . Fragmentation, labeling and hybridization of ChIPed DNA were done following standard protocols.Affymetrix custom-made tiling arrays covering two megabases surrounding the mouse Rps9) were used to derive mean values. Primers are given in Cells were first disrupted and homogenized using a Polytron (kinematic) before RNA was extracted using the RNeasy Microkit . qRT-PCR was performed with SYBR Green. Two biological replicates, processed in triplicates and normalized to a housekeeping gene  from Affymetrix. Data was exported as plain text using a log2 or −10log10 scale for the signal, respectively the p-value. Files were visualized in RChiV, an in-house developed genome browser, which takes into account the deleted segment and normalizes the signal using a sliding window approach.Figure S1HoxD cluster in mutant configurations. (A) ChIP-qPCR profiles of PRC1  and PRC2 (Ezh2 (middle panel) and Suz12 (lower panel)) over the HoxD cluster. The wild type values of six genes (from Evx2 to Hoxd3) found within the H3K27me3 domain are used as positive controls (black). Lnp and Mtx2 are found outside of the H3K27me3 domain and are thus used as negative controls (black). Different mutant configurations are color coded and specified on the top (del(10), del(10-11), del(9-12), del(10-13), del(11-R5)). dN stands for HoxdN. NA refers to the absence of the given DNA segment in the specified allele. NI refers to mutant alleles which where not included in the experiment (Suz12 on both del(10-11) and del(11-R5)).Binding profiles of PRC1 and PRC2 over the (TIF)Click here for additional data file.Figure S2HoxD cluster and flanking gene deserts. (B) H3K27me3 profiles of wild type and deleted animals. Genotypes are specified on the left.Effect of large deletions upon the H3K27me3 profiles. (A) Wild type genomic landscape of the murine (TIF)Click here for additional data file.Figure S3Hoxd10 specific probe . A wild type sample (+/+) was used as control (right panel). The positions of both the restriction sites for PvuII and HindIII and the probe used for southern blot are depicted on the wild type profile in Southern blot of transgenic animals. Southern blot using a robe see . Restric(TIF)Click here for additional data file.Figure S4del(10) embryo .H3K27me3 profiles in pluripotent and terminally differentiated cells. A large (A) or focused (B) view of wild type H3K27me3 profiles from differentiated cells dissected from the embryonic brain (top) compared to pluripotent cells derived from a (TIF)Click here for additional data file.Figure S5Hoxd genes in various constructs eletroporated in iPS cells carrying a deletion of Hoxd10. Lnp is located outside the HoxD cluster and is used as a control for active genes, while Hoxd13 is used as a control for silent genes. Clones and culture conditions are color coded and specified on the top. G418 stands for the presence (+) or absence (−) of the antibiotic. Vector refers to a control cell line.H3K27me3 and RNA profiles in various cell lines. ChIP-qPCR and mRNA expression of control and (TIF)Click here for additional data file.Table S1List of the primers used for RT-PCR, either for ChIP experiments (top) or for RNA dosage (bottom).(DOC)Click here for additional data file.