Escherichia coli is achieved mainly by the action of two regulators, Pch and Ler, which are encoded by horizontally transferred elements. Although Ler has been shown to activate transcription by counteracting H-NS silencing, the mechanism for Pch is poorly understood. We show here that Pch activates the LEE1 promoter and also enhances the Ler-mediated activation of other LEE promoters. Transcriptional activation was completely dependent on repression by the H-NS/StpA/Hha/YdgT complex, indicating that Pch-derived activation was achieved by alleviating H-NS-mediated silencing. Expression of pch reduced the binding of H-NS at LEE1 promoter and altered the nucleoprotein complex. Furthermore, in vitro reconstruction of the protein-DNA complex on LEE1 promoter DNA confirmed the exclusive effect of Pch on H-NS binding. These results demonstrated that Pch is another anti-silencing regulator and a modulator of H-NS-containing nucleoprotein complexes. Thus, the anti-silencing mechanism plays a key role in the coordinated regulation of virulence genes in EHEC.The horizontally transferred chromosomal segments, which are the main source of genetic diversity among bacterial pathogens, are bound by the nucleoid protein H-NS, resulting in the formation of a nucleoprotein complex and the silencing of gene expression. The de-silencing or activation of virulence genes necessary for the colonization of enterohemorrhagic  The acquisition of external genetic information by bacteria is one of the dynamic forces underlying the emergence of genetic diversity, allowing an organism to adapt to a novel environment. The sequencing of a variety of bacterial genomes has revealed strain- and subspecies-specific genomes as well as the common background genome that characterizes a species. The genome of pathogenic bacteria is often distinguished from that of non-pathogenic strains of the same species by the presence of virulence genes on pathogen-specific genomic islands. Such genomic segments commonly reside in mobile genetic elements, such as plasmids, phages, conjugative transposons, and integrons . The horSalmonella Typhimurium and Escherichia coli, it has been shown that the silencing of laterally acquired genes is mediated by selective binding at AT-rich regions of the nucleoid protein H-NS [While the incorporation of exogenous genes is important for bacteria to acquire new abilities to adapt and survive in changing environments, the controlled expression of these exogenous genes is essential for maintaining the organism’s physiological activity. In ein H-NS –7. Becauein H-NS –10.E. coli (EHEC) is a human pathogen that causes a broad spectrum of illnesses, ranging from mild diarrhea to severe diseases, such as hemorrhagic colitis and hemolytic uremic syndrome. EHEC belongs to a group of A/E pathogens that are defined by their ability to form a histopathological feature known as attaching and effacing (A/E) lesions [LEE1 transcription, including the ler gene. The expression of many genes at different loci on the chromosome are controlled in coordination with the LEE genes, through the action of Pch and Ler [Enterohemorrhagic  lesions . A/E les lesions . The gen lesions . The tra and Ler .E. coli K12 common backbone regions. The binding sites on virulence genes are found at the coding region as well as at downstream and upstream sequences. Ler has been shown to activate transcription through its anti-H-NS activity [LEE1 promoter, on which Ler functions as an autorepressor by forming a DNA loop at the promoter region [E. coli (EPEC), PerC, have been shown to activate the LEE1 promoter [LEE1 promoter seems different between EHEC and EPEC, since transcription of LEE genes in EPEC is dependent on PerC only under the specific conditions, such as static in the presence of CO2 [pch genes are essential for the expression of LEE genes and EHEC virulence [perC gene of EPEC, in which LEE expression is unaffected by the absence of the perC gene[LEE1 promoter of EHEC is activated in E. coli K-12 background only in the presence of pch gene [E. coli K-12 in the absence of PerC [The Pch/Ler-activated genes are divided into two classes, L1 and L2, according to their dependency on Ler for their transcriptional activation: the transcriptional activation of genes in class L1 requires Ler and Pch, while those in class L2 are less dependent on Ler and can be activated by Pch alone. Ler is activity , 18–22, r region . On the promoter , 24, 25 e of CO2 , 16, 26.perC gene. In addipch gene , while t of PerC .LEE1 promoter. The LEE1 promoter was activated by Pch only when the promoter activity was repressed by H-NS and other related proteins. LEE and other virulence loci of EHEC were bound by H-NS/StpA, but the occupancy of H-NS/StpA was reduced by the expression of pch in addition to ler. Furthermore, the binding of H-NS to LEE1 promoter DNA was shown to be inhibited by Pch. These findings supported a mechanism in which the activation of gene expression was achieved by reducing H-NS/StpA binding by competitive binding of Pch.In this study, we explored the role of Pch in LEE expression and the effects on H-NS-containing nucleoprotein complex formation at the LEE1 operon, which includes the ler gene, was positively regulated by Pch, which is encoded on other prophage-like elements. Although growth in DMEM enhanced the expression of LEE genes, compared with growth in LB, the level of LEE gene expression in DMEM nevertheless could have been lower than its level when fully activated. Because the optimal conditions for the full expression of LEE genes and other LEE-associated genes are currently unknown, we attempted to achieve the full expression of these genes artificially by inducing a high level of the positive regulator PchA, which also increases ler expression, in wild type of EHEC O157 Sakai. Transcription of the pchA gene was increased by placing the gene downstream of an IPTG-inducible tac promoter and by adding IPTG to the growth medium. The promoter activity increased with increasing IPTG concentrations of up to 25 μM  without Ler and Pch, while the activity increased in the presence of Ler to 76-fold that of the basal level. In contrast, Pch alone showed a slightly positive effect on the promoter activity . In the presence of both Ler and Pch, the promoter activity was enhanced and reached a much higher level than Ler alone . The results confirmed that full activation of the LEE promoters by Ler occurred in cooperation with Pch.To explore the positive effect of Pch on LEE gene transcription, we measured the promoter activity of the ch genes . The LEEE. coli possesses an additional H-NS paralog, StpA, and H-NS homologs, Hha and YdgT, and the involvement of these proteins in gene silencing of subset of genes has been suggested[LEE1 promoter of EHEC is activated by Pch without Ler and is negatively regulated by H-NS and Hha [LEE1 promoter in the presence or absence of the pch gene was compared in a series of deletion mutants for genes encoding H-NS and its paralogs and homologs. Since hns gene seems to be essential for growth of EHEC, we choose to make a set of mutations in E. coli K-12 strain W3110. A plasmid, pTB101-pchA or pTB101 (for vector control), was introduced into W3110, which does not possess ler gene, and its derivatives harboring pLux-PLEE1, and luciferase activity was measured. In the W3110 wild type , the promoter activity remained at a low level without Pch, although the activity was enhanced 110-fold in the presence of Pch even at a basal level expression without IPTG  mutant of EHEC, grown in DMEM. However, little to no obvious changes in the H-NS-binding profiles were observed (data not shown). Then, we used medium containing 6 μM IPTG to determine the effects of Pch and Ler on the binding of H-NS/StpA to the chromosome of EHEC pch mutant harboring Ptac-pchA, which is hereafter called the Pch+ strain. Induction of pchA expression resulted in simultaneous enhancement of production of Ler as well as PchA in EHEC.To explore the effects of Pch and Ler production on H-NS binding, we first compared the H-NS-binding profiles of wild-type EHEC and a pch and ler expression (Pch-). Decreased binding of H-NS was also observed at regions containing non-LEE effector genes belonging to both regulatory classes L1 and L2 [espJ-tccP, nleA-nleM1  was almost the same as in the EHEC strain lacking both regulators . After incubation of immobilized LEE1 promoter DNA on magnetic beads with crude protein extract, the beads were isolated and washed. Proteins bound to immobilized LEE1 promoter DNA were extracted and separated by gel electrophoresis, and major proteins were then identified by LC-MS/MS. Although many of major proteins observed in gel were ribosome subunit proteins, several types of nucleoid proteins, such as IHF, HU, and Fis, were found in both preparations . From the analysis for the LEE1 promoter of EPEC, It was suggested that PerC acts as a derepressor as well as a transcriptional activator. In the report the transcription of the LEE1 promoter was shown to be enhanced by the presence of perC gene in the hns deletion mutant [hns mutant the presence of the pchA gene also enhanced the LEE1 promoter activity but additional deletion of the stpA gene completely abolished the positive effect of the pchA gene. The results indicated that positive effect observed in the hns mutant is the results of counteraction against repression by the remaining gene products in the mutant such as StpA and Hha. Therefore, the horizontally acquired factor Pch could modify the nucleoprotein complex formed with H-NS and its related proteins, resulting in the de-silencing of virulence genes.Our results also suggested that the binding of Pch inhibited the binding of H-NS at specific local sequences, thus blocking the formation of the H-NS-DNA complex and preventing gene silencing. In EPEC pression . Though n mutant . Our resTo acquire new traits and to incorporate them into the virulence regulatory system, a strategy to counteract the host transcription-repression mechanism would be efficient because a strict upstream activation sequence for regulatory factors is not necessary. The global regulatory system consisting of Pch and Ler provides a flexible, easy-to-expand capability for bacteria to adapt to new environments or to evolve advanced infectious strategies.hns-FLAG derivatives of EHEC O157 Sakai were constructed using the methods and plasmids of Datsenko and Wanner [hns and/or stpA, hha, and ydgT were constructed by P1 transduction, using the corresponding mutants in our stock as donors [tac-ler was constructed by replacing the Ptac promoter DNA with the upstream sequence of the ler gene on pWKS-ler. Bacteria were grown overnight in LB, diluted 100-fold, and then incubated in DMEM (Sigma) at 37°C with shaking to 0.9–1.0 OD600. To examine the effects of IPTG on the expression of LEE genes, bacteria were grown overnight in LB, diluted 100-fold, and then grown in DMEM containing 0–400 μM IPTG at 37°C with shaking to 0.9–1.0 OD600.EHEC O157 Sakai (RIMD 0509952)  and its d Wanner . Derivats donors . pWKS-Pt600 of the culture, and samples prepared from similar numbers of cells were analyzed by immunoblotting. The proteins were separated by SDS-polyacrylamide (12% or 10%) gel electrophoresis (SDS-PAGE) and were transferred onto an Immobilon membrane (Millipore). The proteins were detected with antibodies specific for EspB, Tir [Bacteria were collected from cultures by centrifugation, and the cell pellet was dissolved in SDS sample buffer. The concentration of each sample was normalized to the ODspB, Tir , or DnaKLEE1-gfp operon fusion plasmid pACYC-PLEE1-gfp or the PLEE1-lux operon fusion plasmid pLux-PLEE1 [LEE1 operon. Briefly, bacteria were grown overnight in LB and were diluted 100-fold with DMEM containing various amounts of IPTG. For fluorescence, at the sampling time points, an aliquot of culture was removed, and the bacteria were collected by centrifugation, washed with PBS, and suspended in PBS. The OD600 was measured, and the intensity of the fluorescence was measured by an FP-6200 (Jasco). The fluorescence intensity was normalized to the OD600 value. The average of three independent experiments was presented. For luciferase activity, at the sampling time points, 800 μl was removed to measure the OD600, and 100 μl was obtained to measure the luminescence intensity, using a TD-20/20 luminometer (Turner Biosystems). The luciferase activity was calculated by dividing the luminescence intensity by the OD600. The average and standard error were calculated from the results of three experiments.The Pux-PLEE1  was usedThe adherence assay was performed as previously described , with a 600 = 0.7–0.8) at 37°C. Formaldehyde  was added to 10 ml of the culture, and the mixture was incubated at room temperature for 20 min. To terminate the crosslinking reaction, glycine  was added, and the mixture was incubated at room temperature for 5 min. Bacteria were collected by centrifugation and were washed first with TBS and then twice with washing buffer , were suspended in 1 ml of lysis buffer  and were stored at -20°C until use.Chromatin immunoprecipitation and DNA microarray analysis were performed as described by Oshima et al. (2006) and by Abe et al., (2008) with slight modifications. Derivatives of the EHEC O157 Sakai strain were grown in DMEM with or without 6 μM IPTG to logarithmic phase , and then lysed by sonication using a UCD-200 Bioruptor  at 4°C with 15 30-sec pulses at 30-sec intervals. The cell debris was removed by centrifugation at 15,000 rpm for 30 min. An 800-μl aliquot of the supernatant was mixed with Protein G-conjugated magnetic beads (Dynabeads) bound to anti-FLAG  and incubated at 4°C overnight. The beads were washed three times with washing buffer and then with TE. The protein-DNA complex bound to the beads was released into 100 μl of elution buffer  by heating at 65°C for 20 min. The DNA fragments that were cross-linked to proteins were released by incubating the eluate with proteinase K (1 mg/ml) at 42°C for 2 h and then at 65°C for 6 h, followed by purification using a Qiagen PCR Clean-up kit (Qiagen) in 200 μl of TE. The recovered DNA fragments were amplified, terminally labeled, and hybridized with the EHEC O157-tiled microarray, as previously described [http://www.ebi.ac.uk/microarray-as/ae/) under accession numbers E-TABM-819 and E-TABM-818.The raw intensity data (.CEL files) were processed using In Silico Molecular Cloning Array Edition software (In Silico Biology) for analysis and visualization. The signal intensities were obtained by subtracting the signal intensities of the mismatch probes from those of the perfect-match probes, and probes with negative values were excluded from further analysis. To compensate for differences among the probes caused by different probabilities of DNA disruption and hybridization efficiency, the signals were normalized by dividing them by the signal intensities from the DNA samples obtained before the beads were added. The binding sites were identified with In Silico Molecular Cloning Array Edition software (In Silico Biology) by selecting a region with continuous 10 or more signal ratios  greater than the threshold (1.0). The resolution of the experiments are around 50 bp, because average density of DNA probe is 50bp and DNA fragments used for the experiments are ~200bp after sonication. The reproducibility and specificity of the binding of H-NS to several sites were confirmed by ChIP-PCR. The raw data (.CEL format) from the ChIP-chip experiments were deposited in the ArrayExpress database (4)2Fe(SO4)2 and 0.8 mM EDTA) and 2.5 μl of 20 mM sodium ascorbate were placed on the side wall of the tube. Treatment was started by short centrifugation and incubation for 1, 2 or 4 min at room temperature until the addition of 5.5 μl of 0.1 M thiourea and 0.5 μl of 0.2 M EDTA to stop the reaction.The H-NS-DNA complex, prepared as described above, was treated with hydroxyl radicals as follows, and then the DNA was decrosslinked and purified as described above. Fifty microliters of the samples was placed at the bottom of Eppendorf tubes, and 2.5 μl of Fe(II) EDTA solution . Crude protein extract was prepared from W3110 harboring pTB101 or pTB101-pchA, and binding to immobilized DNA was performed as previously described [Immobilized escribed . The proescribed .t-test under the condition of two-tails/two-sample unequal for all analysis. Data are presented as means with standard error of mean (S.E.M). All experiments were repeated for reproducibility and the representative data was shown in figures.The statistical analysis was calculated using the build-in mathematical function of Excel (Microsoft). Difference between two groups was identified using the Student’s S1 FigμM), growth were monitored by measuring OD600.After dilution of overnight culture 100-fold with LB containing various amount of IPTG Click here for additional data file.S2 Fig600) at sampling for promoter assay. Promoter activity shown in 600. For example, wild type was measured at 4h, and the hns stpA double mutant was measured at 6h. B. Growth of wild type and mutants of W3110. After dilution of overnight culture 100-fold with LB, growth were monitored by measuring OD600. W3110 (square), hns stpA double mutant (circle), hns stpA hha ydgT quadruple mutant (triangle)A. Representative of cell density (OD(PDF)Click here for additional data file.S3 FigThe H-NS, Pch, and Ler binding for four representative Pch/Ler-regulon gene loci is shown. The rows are the same as in (PDF)Click here for additional data file.S4 FigThe H-NS, Pch, and Ler bindings for five representative Pch/Ler-regulon gene loci are shown. The rows are the same as in (PDF)Click here for additional data file.S5 Figpch mutant, which does not express either pch or ler, and the pch-expression strain, which expresses both pch and ler. Affected: the H-NS binding sites with decreased occupancy in the pch expressing strain. B. Size distributions of H-NS binding sites.A. G+C content of H-NS binding sites. The effect of the expression of Pch and Ler on H-NS occupancy was determined by comparing H-NS binding profiles taken from the (PDF)Click here for additional data file.S6 Figpch and ler expression (Row 2), EHEC expressing Ler but not Pch (Row 3), and EHEC expressing both Pch and Ler (Row 4). Rows 5 and 6 show Ler and Pch binding, respectively. The bottom row shows the ORFs in the E. coli K12 common chromosome (blue) and the ORFs in the LEE and other laterally transferred elements (red).A. H-NS binding profiles on the whole EHEC chromosome. Graphs on the three outer circles show H-NS binding to the chromosome of the EHEC Sakai strain. Graphs on the fourth and fifth circles represent Ler binding and PchA binding from previous data . The radial blue bars indicate the relative value of occupancy. The sixth circle shows the G+C content. The seventh circle shows the EHEC chromosome, including the positions and prophages or prophage-like elements. B. H-NS binding profiles in the LEE of EHEC O157 Sakai and its surrounding region. The top row shows the G+C content. The next three rows show the H-NS binding of EHEC deficient in (PDF)Click here for additional data file.S7 FigLEE1 promoter were detected by PCR. For control, uncharged Dynabeads protein G was used for precipitation. Samples were as input (i), precipitates with control Dynabeads (-), and precipitates with Dynabeads TALON (+).After crosslinking the H-NS-DNA complexes were precipitated with Dynabeads TALON. DNA segments corresponding to (PDF)Click here for additional data file.S8 Fighha mutant, the ydgT mutant or the hha ydgT mutant for 90 min. Unattached bacteria were then removed, and the cells were incubated for another 3 h. Microcolonies were visualized by Giemsa staining (A). The frequency of appearance of microcolonies is presented as the number of microcolonies per cell, calculated from a total of 5 microscopic sights and the average of three independent experiments for each strain (B).Caco-2 cells were infected with wild-type EHEC, the (PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.


Open chromatin is implicated in regulatory processes; thus, variations in chromatin structure may contribute to variations in gene expression and other phenotypes. In this work, we perform targeted deep sequencing for open chromatin, and array-based genotyping across the genomes of 72 monozygotic twins to identify genetic factors regulating co-twin discordance in chromatin accessibility.We show that somatic mutations cause chromatin discordance mainly via the disruption of transcription factor binding sites. Structural changes in DNA due to C:G to A:T transversions are under purifying selection due to a strong impact on chromatin accessibility. We show that CpGs whose methylation is specifically regulated during cellular differentiation appear to be protected from high mutation rates of 5′-methylcytosines, suggesting that the spectrum of CpG variations may be shaped fully at the developmental level but not through natural selection. Based on the association mapping of within-pair chromatin differences, we search for cases in which twin siblings with a particular genotype had chromatin discordance at the relevant locus. We identify 1,325 chromatin sites that are differentially accessible, depending on the genotype of a nearby locus, suggesting that epigenetic differences can control regulatory variations via interactions with genetic factors. Poised promoters present high levels of chromatin discordance in association with either somatic mutations or genetic-epigenetic interactions.Our observations illustrate how somatic mutations and genetic polymorphisms may contribute to regulatory, and ultimately phenotypic, discordance. Open chromatin provides access for a wide spectrum of DNA binding proteins to regulate transcription, DNA repair, recombination, replication, and so on. As such, open chromatin profiling has been used to identify the genomic locations of various regulatory regions, including promoters, enhancers, insulators, silencers, replication origins, and recombination hotspots -4. The bChromatin accessibility has been a focal point in the studies exploring the intersection of genetics and epigenetics. Meanwhile, coupling between chromatin accessibility and underlying genetic polymorphisms renders chromatin status a heritable feature . In a reWhile monozygotic (MZ) twins are useful for studying epigenetic differences caused by differential environmental exposure, DNA methylation is the only epigenetic mechanism that has been studied in depth with regard to MZ discordance -17. A reIn this work, we investigated co-twin chromatin discordance and the associated genetic factors. We first sought to obtain the full spectrum of somatic and genetic single nucleotide variations that underlie open chromatin. To this end, we compared the patterns of somatic mutations  with those of genetic polymorphisms . In addition, we attempted to characterize genetic-epigenetic interactions by finding genetic polymorphisms that influence within-pair differences in chromatin accessibility. Thus, we performed quantitative trait loci (QTL) mapping for quantitative within-pair chromatin differences across twin pairs.We performed high-quality, in-depth open chromatin sequencing , SNP calling (HapMap genotyping and 1000 Genomes sequencing), and population (Yoruba)  confirmeThe concept of ‘variability genes’ was suggested based on the finding that the Kidd blood group locus is associated with within-pair differences in the total cholesterol level when serum lipid levels were examined across twin pairs with different genotypes . Therefocis) associations were identified for 1,325 chromatin loci  and Korea Advanced Institute of Science and Technology (KAIST). Written informed consent was obtained from all individuals. Peripheral blood mononuclear cells were extracted from 36 pairs of MZ twins, aged 30 to 60 years, who were discordant for immunological traits mostly involving allergic symptoms  . Tags weX1 - X2|, was also obtained, wherein X1 and X2 indicate chromatin accessibility in each sibling of a twin pair. While this provides a direct measure of chromatin differences, it can cause a bias toward highly accessible chromatin sites such as active promoters or strong enhancers. Therefore, we normalized this measure as |X1 - X2|/(X1 + X2) and used the normalized difference particularly when comparing different regulatory states. Discordant chromatin sites were determined based on effect size  or based on variation analysis .For the quantification of chromatin accessibility, the open chromatin peaks identified from each of the 72 samples were merged into meta-peaks using BEDTools . For eacP value from Fisher’s exact test lower than 1 × 10-3. For the identification of genetic polymorphisms (SNPs), we employed the germline variant-calling functionality of VarScan to analyze variants that were not significantly different between twin siblings . Variant calls were filtered when >90% of the supporting reads in each sample came from only one strand in order to remove alignment-related artifacts. We considered autosomal genomic positions that were covered by at least one read in all of the 72 samples. To be identified as a polymorphism, a minimum allele frequency of 1% among all the reads across the samples was required. Association mapping was conducted between the identified SNPs and the normalized accessibility signals across the 72 samples based on simple linear regression by using the scipy.stats module of SciPy. Significant associations were chosen at the FDR of 0.05 with P value adjustment by using the fdrtool package of R.Somatic mutations were defined as within-pair single nucleotide differences. We employed the method suggested by VarScan ,39 for tP-value threshold. Sixty-six percent of the individual genotypes at individual loci were reproducible  was isolated from the peripheral blood cells of one sibling from each twin pair and genotyped on the Affymetrix Genome-Wide Human SNP array 6.0. Low-concentration genomic DNA was amplified prior to genotyping according to the manufacturer’s protocol (QIAGEN). The Bayesian Robust Linear Modeling using Mahalanobis Distance (RLMM) algorithm was used to perform genotype calling of 516,188 SNPs . SNP imp00 ng wasComplementary base pairs were concatenated by ‘:’  and dinucleotides were connected by ‘p’ . For the typing of mutations and polymorphisms arising at different dinucleotides, we extracted three consecutive bases centered on each of the identified variants (mutations or polymorphisms) using BEDTools  and counTotal RNA was extracted from lymphoblastoid cells using RNeasy Mini Kit  according to the manufacturer’s instructions. The yield and purity of the extracted RNA were evaluated by A260/A280 measurement and agarose gel electrophoresis. Gene expression levels were measured for the 72 MZ twin samples on the Illumina Human HT-12 chip. The raw intensities were log2 transformed and normalized by using the VST-quantile normalization method of the lumi R packages (v.14.01).in vivo TF binding were obtained from the ENCODE Transcription Factor Binding tracks. All data available for the GM12878 cells were generated either by the Hudson Alpha Institute for Biotechnology (HAIB) or by Stanford/Yale/USC/Harvard (SYDH). We first discovered regions enriched for TF binding by using the peak finding functionality of the Homer package .bp: base pair; ChIP: chromatin immunoprecipitation; FAIRE: formaldehyde-assisted isolation of regulatory elements; FDR: false discovery rate; MZ: monozygotic; QTL: quantitative trait locus; SNP: single-nucleotide polymorphism; TF: transcription factor; TFBS: transcription factor binding site.The authors declare that they have no competing interests.KK and HJB performed the data analyses and drafted the manuscript. JS, KL, and MY carried out the FAIRE-seq, genotype array, and expression array experiments. KK participated in the FAIRE-seq experiments. HJB participated in the genotype array and expression array experiments. SCK assisted the data analyses. KP, SBC, and JKC conceived the study. SBC and JKC supervised the study and wrote the manuscript. All authors read and approved the final manuscript.Information of the MZ twin FAIRE-seq data.Click here for fileClinical information of the MZ twins used in this work.Click here for fileDensity plot for genome-wide correlation coefficients of chromatin accessibility between twin siblings and between unrelated individuals.Click here for fileThe number of open chromatin regions we identified that overlap with peaks identified from the public FAIRE-seq and DNase-seq data for the GM12878 lymphoblastoid cells. Our FAIRE regions were extended 10 to 200 bp before overlapping. Approximately 83% of the FAIRE regions that we found to overlap with the public FAIRE data were confirmed by the public DNase I data.Click here for fileList of sequencing variants whose genotype is confirmed by Affymetrix SNP array 6.0.Click here for fileList of sequencing variants whose genotype is in conflict with the Affymetrix SNP array 6.0 genotype. Red/blue coloring indicates cases in which a sequencing variant calling matches an array variant calling in terms of genotype identity irrespective of homo-/hetero-zygosity.Click here for fileWithin-pair differences in chromatin accessibility as a function of the distance between the mutation and the center of the chromatin region.Click here for fileWithin-pair differences in chromatin accessibility according to the number of mutations per base pair as an estimate of the density of mutations relative to the size of the open chromatin region.Click here for fileObserved-to-expected ratios of the substitution frequency of TFBS mutations and polymorphisms that were associated with chromatin discordance and inter-individual variation, respectively.Click here for fileThe relative enrichment of dinucleotides in TFBSs. The ratio of the number of the specified dinucleotides in TFBSs to the number in the surrounding chromatin regions was divided by the ratio for all the different dinucleotides.Click here for fileThe relative enrichment of dinucleotides in TFBSs. The ratio of the number of each dinucleotide in TFBSs to the number in the whole genome was divided by the ratio of the number of all the different dinucleotides in TFBSs to the number in the whole genome.Click here for fileDifferential methylation levels between B lymphocytes and other cell types (embryonic stem cells (ESC), hematopoietic progenitor cells (HPC), and neutrophils) for each TFBS CpG. The average of cytosine methylations in TFBSs was obtained and plotted.Click here for fileThe frequency of different types of polymorphisms arising in TFBSs as identified in this work (dark blue) and in the previous work by Degner et al. [ (sky blue). Only changes from the reference homozygote were considered.r et al.  (sky bluClick here for fileThe frequency of TFBS dinucleotides containing polymorphisms identified in this work (dark blue) and in the previous work by Degner et al. [ (sky blue).et al.  , hematopoietic progenitor cells (HPC), and neutrophils) were calculated.r et al. , observeClick here for file


Francisella tularensis is a Gram-negative bacterium whose ability to replicate within macrophages and cause disease is strictly dependent upon the coordinate activities of three transcription regulators called MglA, SspA, and PigR. MglA and SspA form a complex that associates with RNA polymerase (RNAP), whereas PigR is a putative DNA-binding protein that functions by contacting the MglA-SspA complex. Most transcription activators that bind the DNA are thought to occupy only those promoters whose activities they regulate. Here we show using chromatin immunoprecipitation coupled with high-throughput DNA sequencing (ChIP-Seq) that PigR, MglA, and SspA are found at virtually all promoters in F. tularensis and not just those of regulated genes. Furthermore, we find that the ability of PigR to associate with promoters is dependent upon the presence of MglA, suggesting that interaction with the RNAP-associated MglA-SspA complex is what directs PigR to promoters in F. tularensis. Finally, we present evidence that the ability of PigR (and thus MglA and SspA) to positively control the expression of genes is dictated by a specific 7 base pair sequence element that is present in the promoters of regulated genes. The three principal regulators of virulence gene expression in F. tularensis therefore function in a non-classical manner with PigR interacting with the RNAP-associated MglA-SspA complex at the majority of promoters but only activating transcription from those that contain a specific sequence element. Our findings reveal how transcription factors can exert regulatory effects at a restricted set of promoters despite being associated with most or all. This distinction between occupancy and regulatory effect uncovered by our data may be relevant to the study of RNAP-associated transcription regulators in other pathogenic bacteria. Francisella tularensis are found ubiquitously at promoters including those they do control and those they do not. Furthermore, we present evidence that these regulators—the RNA polymerase-associated SspA family members MglA and SspA, and the putative DNA-binding protein PigR—exert their coordinate regulatory effects only at promoters that contain a small DNA sequence element. Our findings reveal how transcription factors can associate with many promoters but only exert regulatory effects at a few. They also have implications for how SspA family members and other RNAP-associated transcription regulators might exert their effects in other pathogens.Most transcription regulators are found only at those promoters they control. Here we show that the most prominent regulators of virulence gene expression in  Francisella tularensis is a Gram-negative bacterium and the aetiological agent of tularemia, a disease that can be fatal in humans [F. tularensis to cause disease is dependent principally upon its ability to grow within macrophages [F. tularensis are those located on the Francisella pathogenicity island (FPI), which are thought to have been acquired through horizontal transfer [n humans . This pan humans . The abirophages ,3–5. Protransfer –8. Genestransfer ,10, thertransfer ,11,12.F. novicida) that works in concert with MglA and SspA by contacting the RNAP-associated MglA-SspA complex directly [F. tularensis [Expression of the genes on the FPI is dependent upon the coordinate activities of three regulators –18. Two directly –18. MglAdirectly –17. The larensis ,17,20.According to the current view for how MglA, SspA, and PigR control the expression of virulence genes, PigR functions like a classical transcription activator, binding specifically to a DNA sequence present at the promoters of regulated genes; thus, contact between DNA-bound PigR and the RNAP-associated MglA-SspA complex would stabilize the binding of RNAP to those promoters that contain a PigR binding site ,18. HoweF. tularensis. We also demonstrate that PigR requires MglA (and thus presumably the MglA-SspA complex) in order to specifically associate with promoters. Finally, we present evidence that the promoters of PigR-regulated genes contain a specific sequence motif that is both necessary and sufficient for PigR-mediated control. Our findings reveal that the most prominent regulators of virulence gene expression in F. tularensis are found at essentially all promoters but only positively control those that contain a specific sequence element.Using chromatin immunoprecipitation followed by high-throughput DNA sequencing (ChIP-Seq), we show that PigR, MglA, and SspA are present at virtually all detected promoters in F. tularensis. To do this, we determined the locations of the β′ subunit of RNA polymerase (RNAP) on the F. tularensis chromosome using ChIP-Seq. To immunoprecipitate the β′ subunit of RNAP, we constructed a strain of F. tularensis LVS in which the chromosomal copy of the rpoC gene was modified to encode β′ with a vesicular stomatitis virus-glycoprotein (VSV-G) epitope tag fused to its C-terminus , and another strain of LVS that synthesized σ32 with a VSV-G tag fused to its C-terminus (LVS σ32-V). As a control we also constructed a strain that synthesized HipB, a predicted site-specific DNA-binding protein [70-V strain identified 333 promoter regions, of which 277 (83.2%) overlap with the locations of promoters defined by determining the location of the β′ subunit of RNAP in cells grown in the presence of rif  of which were intergenic and 86 (15%) of which were intragenic  tag fused to its C-terminus [pigR had been altered such that it specified PigR with a VSV-G epitope tag fused to its C-terminus (LVS PigR-V); and another in which the native chromosomal copy of sspA had been altered such that it specified SspA with a VSV-G epitope tag fused to its C-terminus (LVS SspA-V).Having determined the locations of promoters in terminus . We alsoF. tularensis and not just at the promoters of regulated genes  is present at the majority of promoters, it appears to only positively regulate the expression of a fraction of the corresponding genes. We therefore reasoned that PigR might function as an activator at specific promoters through recognition of a specific sequence element.F. tularensis. To do this we quantified specific candidate transcripts in both wild-type LVS cells and in cells of a LVS ∆pigR mutant strain using Nanostring (see PigR response element (PRE) is depicted in To search for a conserved sequence motif in the promoters of genes that are regulated by PigR we first tested whether certain genes previously shown to be regulated by MglA and SspA were also regulated by PigR in ring see . Using Mring see , or both70, σ32, or the β′ subunit of RNAP (in cells grown in the presence of rif) as determined by ChIP-Seq. This gave us transcription start sites with high confidence for 110 promoters, including 3 of the 11 putative promoter regions used to initially identify the PRE through MEME . Consistent with the idea that these differences are due to a decrease in the activity of the iglA promoter, cells of the reporter strains containing mutations in the -10 element that are predicted to decrease the activity of the promoter exhibit dramatically reduced lacZ expression . These findings demonstrate that the PRE is sufficient to confer on a promoter the ability to respond to PigR, and by inference, the ability to respond to MglA and SspA.Having established that conserved base pairs within the PRE are important for expression of a PigR-regulated gene we next asked whether the PRE was sufficient to confer control on a promoter that did not ordinarily contain a PRE. To do this we introduced 3 mutations into the FTL_0361 promoter that generated a consensus PRE 6 bp upstream of the putative -35 element . We thenF. tularensis. We have also found that PigR requires the MglA-SspA complex in order to specifically localize to promoter regions. We infer from this that interaction between PigR and the RNAP-associated MglA-SspA complex directs PigR specifically to promoter regions. Despite their ubiquitous presence at promoters, PigR, MglA, and SspA coordinately control the expression of approximately 5% of known genes and we have uncovered the molecular basis for this specificity. In particular, we have identified a 7 bp sequence element that we have called the PRE (the PigR response element), located approximately 6 bps upstream of the putative -35 element of promoters that are positively regulated by PigR/MglA/SspA. The PRE is both necessary and sufficient to confer control by PigR. Our findings indicate that although PigR, MglA, and SspA are present at essentially all promoters, they control the activities of only those promoters that contain a specific sequence element.Using ChIP-Seq we have found that PigR, MglA, and SspA are found at the majority of promoters in 70-containing RNAP holoenzyme, σ70 together with the core subunits of RNAP and SspA were found to co-purify with MglA in LVS in stoichiometric amounts [Finding PigR, MglA, and SspA at promoters but not within transcribed regions suggests that these proteins are associated with the RNAP holoenzyme and likely exert their regulatory effects at the level of transcription initiation. Consistent with the idea that the MglA-SspA complex interacts with the σ amounts .70-dependent promoters in F. tularensis, together with our identification of the PRE, we propose a model for how PigR works in concert with the MglA-SspA complex to positively regulate the expression of a specific set of genes, including many that are required for virulence (70 (i.e. are σ70-dependent promoters), since our ChIP-Seq studies reveal PigR, MglA, SspA, and σ70 are present at many of the same promoter regions. Note also that our model explains only how PigR, together with the MglA-SspA complex, exerts positive effects on gene expression; the small number of genes that are negatively regulated by PigR/MglA/SspA [pigR is the only gene encoding a putative DNA-binding protein that is positively regulated by PigR/MglA/SspA [PigR contains a putative helix-turn-helix motif, suggesting it might exert its regulatory effects through interaction with the DNA ,17. Baseirulence . AccordiglA/SspA –17, may glA/SspA –17.Mycobacterium tuberculosis that has been found to associate with the majority of promoters [E. coli, members of the MarA family of transcription activators are thought to associate with RNAP prior to DNA-binding and to contact their DNA target sites as a pre-assembled activator-RNAP complex [E. coli.Our model specifies that PigR is a DNA-binding protein that associates with RNAP via the MglA-SspA complex prior to promoter binding, and is therefore associated with all promoters, as is supported by our ChIP-Seq data. According to the classical view, transcription activators that bind the DNA and contact RNAP are found only at the promoters of regulated genes and function by first recognizing their respective target sites on the DNA and then, once tethered to the DNA, by contacting RNAP ,22. Howeromoters ,34. Alth complex –37. HoweWe note that although PigR is predicted to be a DNA-binding protein, PigR has yet to be shown to be capable of binding the DNA, and need not necessarily exert its regulatory effects through direct interaction with the PRE. It is formally possible that interaction between PigR and the RNAP-associated MglA-SspA complex may enable some other portion of RNAP, such as the C-terminal domain of one of the α subunits ,38, or pThe virulence genes present on the horizontally acquired FPI are the ones that are most strongly regulated by PigR, MglA, and SspA, and it is tempting to speculate that the limited size of the PRE (at 7 bp) may have facilitated the expansion of the PigR/MglA/SspA regulatory network to include these. Only three mutations were required in order to generate a consensus PRE within the FTL_0361 promoter . More thOur ChIP-Seq studies suggest that PigR interacts with the MglA-SspA complex at the majority of promoters. This raises the possibility that through interaction with the MglA-SspA complex, PigR may modulate the activity of any other regulator that functions through interaction with the RNAP-bound MglA-SspA complex. Indeed, it has been suggested that PmrA, another important regulator of virulence gene expression in Francisella, might function through interaction with MglA and SspA . It is tE. coli, SspA evidently functions as a co-activator of P1 late gene expression by making simultaneous contact with RNAP and the phage-encoded sequence-specific DNA-binding protein Lpa [E. coli or just those driving expression of the P1 late genes. SspA family members have been shown to be important for the virulence of a variety of pathogens [The role of the MglA-SspA complex in positively controlling the expression of virulence genes appears to be to simply serve as a contact site on RNAP for PigR. Evidence for other SspA family members serving as contact sites for transcription activators comes from studies of bacteriophage P1 late gene expression; in tein Lpa . Howeverathogens –47. ServF. tularensis subsp. holarctica LVS and its derivatives were grown at 37°C in either Mueller Hinton (MH) broth (Difco), supplemented with glucose (0.1%), ferric pyrophosphate (0.025%), and Isovitalex (2%), or on cysteine heart agar (Difco) medium supplemented with 1% hemoglobin solution (VWR); when appropriate, kanamycin was used for selection at either 5 μg/ml or 10 μg/ml. Escherichia coli strain XL1-blue (Stratagene) was used for plasmid construction and, when appropriate, kanamycin was used to select for resistance at 50 μg/ml. E. coli containing plasmid pBSK iglA-lacZ, or its derivatives, were grown at 30°C.903 kanamycin resistance gene (Epicentre) driven by the LVS groES promoter. The plasmid pKL01 was generated by first amplifying the last 400 base pairs (bp) of the FTL_1743 locus (rpoC), minus the stop codon, by PCR. The 5′ primer contained DNA specifying a KpnI site upstream of the gene fragment. The 3’ primer included DNA containing a NotI site and one extra base pair, encoding a 3 amino acid alanine linker. The linker is followed by DNA specifying the 11 amino acid vesicular stomatitis virus-glycoprotein (VSV-G) epitope tag, followed by a stop codon and DNA specifying an EcoRI site. The corresponding PCR product was digested with KpnI and EcoRI and cloned into pEX18Kan that had been digested with KpnI and EcoRI, generating pKL01. We largely removed the sacB gene by digesting with MscI and EcoRV and re-ligating the vector together, resulting in pKL02.A modified version of pEX18Kan  was used as the vector for VSV-G tagging integration constructs. We have used pEX18Kan for deletion constructs ,17; it urpoD, rpoH, sspA, pigR, and hipB were generated by amplifying the last 250–400 bp (depending on gene size) of the gene using a 5′ primer containing a KpnI site and a 3′ primer containing a NotI site, which allows each fragment to be fused with DNA specifying the alanine linker and VSV-G epitope tag. Fragments were subcloned into pKL02 that had been digested with KpnI and NotI. Plasmid pKL05 contains the DNA specifying the 3’ end of rpoD and was used to generate strain LVS σ70-V. Plasmid pKL04 contains the DNA specifying the 3’ end of rpoH and was used to generate strain LVS σ32-V. Plasmid pKL08 contains the DNA specifying the 3’ end of pigR and was used to generate strain LVS PigR-V. Plasmid pCS05 contains the DNA specifying the 3’ end of hipB and was used to generate strain LVS HipB-V. Plasmid pKL07 contains the DNA specifying the 3’ end of sspA. Because expression of the putative sspA operon could potentially be interrupted by plasmid integration, pKL07 was modified to contain an outward facing promoter after plasmid integration. To do this, another groES promoter was amplified from LVS genomic DNA and cloned upstream of the sspA gene fragment, into the BamHI and PstI sites, resulting in plasmid pKL13, which was used to generate strain LVS SspA-V.VSV-G tagging integration constructs for iglA-lacZ plasmid  utilizes a ColE1 origin of replication, which is nonfunctional in LVS, contains a kanamycin resistance determinate (aphA1), and contains a transcriptional fusion between the iglA promoter and the lacZ gene [iglA-lacZ plasmid contained two tandem PacI sites, so pMO1, which contains DNA specifying the wild-type iglA promoter with a single PacI site before the lacZ gene, was generated by digesting pBSK iglA-lacZ with PacI and NotI and recloning the wild-type iglA promoter fragment into the plasmid backbone. The pMO1 plasmid was used to generate LVS PiglA-lacZ and LVS ∆pigR PiglA-lacZ. Mutations in the PRE of the iglA promoter were generated using splicing by overlap extension PCR [iglA-lacZ that had been digested with PacI and NotI to replace the wild-type iglA promoter. The pMO2 plasmid contains DNA specifying the iglA promoter containing the PRE mutant 1 and was used to generate LVS PiglA-M1-lacZ and LVS ∆pigR PiglA-M1-lacZ. The pMO3 plasmid contains DNA specifying the iglA promoter containing the PRE mutant 2 and was used to generate LVS PiglA-M2-lacZ. The pMO4 plasmid contains DNA specifying the iglA promoter containing the -10 mutant and was used to generate LVS PiglA-10M-lacZ and LVS ∆pigR PiglA-10M-lacZ.The pBSK acZ gene . The pBSsion PCR . The corF. tularensis LVS genomic DNA using a 5′ primer containing DNA specifying a NotI site upstream of the promoter and the 3’ primer including DNA containing a PacI site downstream of the promoter. The resulting PCR product was digested with PacI and NotI and cloned into pBSK iglA-lacZ that had been digested with PacI and NotI to replace the iglA promoter, generating pMO5. The pMO5 plasmid was used to generate LVS PFTL_0361-lacZ and LVS ∆pigR PFTL_0361-lacZ. Plasmids containing mutations in the FTL_0361 promoter were generated in the same manner as plasmids containing mutations in the iglA promoter. The pMO6 plasmid contains DNA specifying the FTL_0361 promoter containing the PRE and was used to generate LVS PFTL_0361-PRE-lacZ and LVS ∆pigR PFTL_0361-PRE-lacZ. The pMO7 plasmid contains DNA specifying the FTL_0361 promoter containing the -10 mutant and was used to generate LVS PFTL_0361-10M-lacZ and LVS ∆pigR PFTL_0361-10M-lacZ.The promoter region of FTL_0361 was amplified from lacZ reporter integration vectors). Strains containing the correct integration were confirmed by colony PCR, by Western blotting and/or Southern blotting. Strain LVS βʹ-V, which synthesizes the βʹ subunit of RNAP with a C-terminal VSV-G tag, was generated by electroporation of plasmid pKL02 into LVS. Strain LVS σ70-V, which synthesizes the σ70 protein with a C-terminal VSV-G tag, was generated by electroporation of plasmid pKL05 into LVS. Strain LVS σ32-V, which synthesizes the σ32 protein with a C-terminal VSV-G tag, was generated by electroporation of plasmid pKL04 into LVS. Strain LVS SspA-V, which synthesizes the SspA protein with a C-terminal VSV-G tag, was generated by electroporation of plasmid pKL13 into LVS. Strain LVS PigR-V, which synthesizes the PigR protein with a C-terminal VSV-G tag, was generated by electroporation of plasmid pKL08 into LVS. Strain LVS HipB-V, which synthesizes the HipB protein with a C-terminal VSV-G tag, was generated by electroporation of plasmid pCS05 into LVS.Electroporation of integration plasmids into LVS was performed as described . Cells iiglA-lacZ transcriptional fusion and derivatives were integrated at the FTL_0111 iglA locus, as determined by Southern blotting; the probe was synthesized using the PCR DIG Probe Synthesis Kit (Roche), hybridized to digested chromosomal DNA that had been transferred to a positively charged nylon membrane, and detected using CDP-Star (Roche). Strains PiglA-lacZ and LVS ∆pigR PiglA-lacZ, which contain lacZ under the control of the wild-type iglA promoter, were generated by electroporation of pMO1 into LVS and LVS ∆pigR, respectively. Strains LVS PiglA-M1-lacZ and LVS ∆pigR PiglA-M1-lacZ, which contain lacZ under the control of the iglA promoter containing the two mutations in the PRE (PRE mutant 1), were generated by electroporation of pMO2 into LVS and LVS ∆pigR, respectively. Strain LVS PiglA-M2-lacZ, which contain lacZ under the control of the iglA promoter containing three mutations in the PRE (PRE mutant 2), was generated by electroporation of pMO3 into LVS. Strains LVS PiglA-10M-lacZ and LVS ∆pigR PiglA-10M-lacZ, which contain lacZ under the control of the iglA promoter containing two mutations in the -10 element, were generated by electroporation of pMO4 into LVS and LVS ∆pigR, respectively. Strains LVS PFTL_0361-lacZ and LVS ∆pigR PFTL_0361-lacZ, which contain lacZ under the control of the FTL_0361 promoter, were generated by electroporation of pMO5 into LVS and LVS ∆pigR, respectively. Strains LVS PFTL_0361-PRE-lacZ and LVS ∆pigR PFTL_0361-PRE-lacZ, which contain lacZ under the control of the FTL_0361 promoter containing the PRE, were generated by electroporation of pMO6 into LVS and LVS ∆pigR, respectively. Strains LVS PFTL_0361-10M-lacZ and LVS ∆pigR PFTL_0361-10M-lacZ, which contain lacZ under the control of the FTL_0361 promoter containing mutations in the -10 element, were generated by electroporation of pMO7 into LVS and LVS ∆pigR, respectively.Strains containing the groES promoter; the pF plasmid does not contain the pigR gene or DNA encoding the VSV-G epitope tag. Plasmid pF2-PigR-V synthesizes PigR-V under the control of a weakened groES promoter lacking its putative UP-element and was made by replacing sspA in the plasmid pF2-SspA [pigR mutant strain [pigR ∆mglA mutant strain; LVS ∆pigR ∆mglA was created by using the pEX2-∆mglA vector [pigR background, by allelic exchange and confirmed by Southern blotting.Plasmids pF and pF-PigR-V have been described previously  and werepF2-SspA  with DNAt strain , or cellA vector  in the ∆70-V; LVS σ32-V; LVS PigR-V; LVS SspA-V; LVS HipB-V; LVS (as a mock control); LVS containing plasmid pF (as a mock control); LVS ∆pigR containing plasmid pF2-PigR-V; and LVS ∆pigR ∆mglA containing plasmid pF-PigR-V. In order to perform ChIP-Seq on MglA, we used cells of the LVS strain synthesizing MglA with a C-terminal TAP tag (LVS MglA-TAP) at native levels, which has been described previously [600 0.3–0.4), and when indicated, rifampicin (Sigma) was added to a final concentration of 50 μg/mL for 30 minutes before crosslinking. Cells were incubated in a final concentration of 1% formaldeyhyde (Sigma) for 30 minutes, after which glycine (Sigma) was added to a final concentration of 250 mM. ChIP was performed in biological triplicate  with either 40 mL or 80 mL of culture using anti-VSV-G agarose beads (Sigma) for cells synthesizing VSV-G tagged transcription factors or IgG Sepharose beads  for cells synthesizing TAP-tagged MglA essentially as described previously [ChIP-Seq was performed with cells of the following strains: LVS βʹ-V; LVS σeviously . Cells whttp://www.ncbi.nlm.nih.gov/Traces/sra) with the accession number SRP055716.Illumina libraries were constructed with approximately 2 to 160 ng immunoprecipitated DNA using either the TruSeq DNA Sample Prep Kit (Illumina) or the NEBNext Ultra DNA Library Prep Kit for Illumina (NEB), generally following the supplied protocols. In using the TruSeq DNA Sample Prep Kit, adapters were diluted 1:10 before ligation and libraries were gel-purified after 11 cycles of amplification. When using the NEBNext Ultra DNA Library Prep Kit, adapters were diluted 1:10 before ligation and libraries were size-selected using Agencourt AMPure XP beads prior to 12 cycles of amplification. Libraries were quantified using the Quant-iT PicoGreen dsDNA Assay Kit (Life Technologies) and sequenced by Elim Biopharmaceuticals, Inc. , using an Illumina Genome Analyzer Ilx generating 36 bp single-end reads or an Illumina HiSeq 2500 generating 50 bp single-end reads. Sequencing reads have been submitted to the NCBI Sequence Read Archive  and the sequence of the integrated plasmid, if applicable, using bowtie2-2.0.6 [70 or σ32. Tracks were visualized using the Integrative Genomics Viewer (IGV), version 2.3 [For each strain, the reads were mapped to the e2-2.0.6 . Regionse2-2.0.6 . The thrsion 2.3 . Peak ansion 2.3 , and BEDsion 2.3 .Cell lysates were separated by SDS-PAGE on 4–12% or 12% Bis-Tris NuPAGE gels in MES or MOPS running buffer (Life Technologies). Either the iBlot dry blotting system or the XCell II Blot Module (Life Technologies) was used to transfer proteins to either PVDF or nitrocellulose. Membranes were blocked with SuperBlock Blocking Buffer (Pierce) with 0.25% Surfact-Amps 20 (Pierce) for 1 hour to overnight. Membranes were then probed with polyclonal anti-VSV-G  or anti-GroEL  for one hour, washed  and re-blocked for 1 hour. After membranes were incubated with polyclonal goat anti-rabbit  and washed, proteins were detected using SuperSignal West Pico Chemiluminescent Substrate (Life Technologies).∆pigR mutant strain (described in 17) were grown to mid-log in biological triplicate. 1 mL of each sample was pelleted , resuspended in 500 μL Qiagen buffer RLT, frozen on dry ice, and stored at -80°C. Equal amounts of lysate, normalized to OD600, in 4 μL Qiagen buffer RLT and 1 μL water were submitted to the Epithelial Cell Biology Core Facility  for processing using the Nanostring nCounter Prep Station and Digital Analyzer according to the manufacturer’s instructions. For each replicate, total transcript counts were normalized using internal controls with background subtraction, as per manufacturer’s instructions. Transcript abundance was determined by averaging biological triplicates. Criteria indicating a significant change in gene expression are a 2-fold change in transcript abundance and p-value < 0.05 in a two-tailed Student’s t-test.Cells of the LVS wild-type strain and cells of the LVS iglA, pdpA, FTL_0131, FTL_0207, FTL_0449, FTL_0491, FTL_1218, FTL_1219, FTL_1678, FTL_1790). The 400 bp region surrounding the maximal PigR binding site, upstream from 11 genes, excluding coding regions, was searched for a common motif using MEME, version 4.9.1 [Genes with significant changes in expression in cells lacking PigR in comparison to wild-type cells (greater than 3-fold by microarray  or 2-folon 4.9.1 . We have600 0.3–0.4), and RNA was isolated from 10mL of cells in triplicate as described previously [LVS cells were grown in aerated liquid culture at 37°C in supplemented MH to mid-log (ODeviously . RNA-Seqeviously ), the seeviously ), while pdpA and FTL_1219.Primer extension was used essentially as described previously  to deterE. coli consensus sequence. For each promoter, 11 bp of sequence, 5 bp upstream from the -35 element and extending upstream, was submitted to FIMO [For each transcription start site identified by RNA-Seq, putative -10 and -35 elements were predicted based on homology to the  to FIMO  to searcCells were grown to mid-log phase, and β-galactosidase activity was assessed essentially as described previously . Assays S1 Fig70 (orange), σ32 (cyan), MglA (brown), SspA (light pink), PigR (blue), and HipB (dark pink). The region of the chromosome illustrated includes the hipAB operon (hipA is FTL_1125 and hipB is FTL_1126).A representative track illustrating the density of normalized mapped sequencing reads is depicted on the Y-axis after ChIP-Seq of each epitope-tagged factor: β′ (green), β′+ rif (purple), σ(EPS)Click here for additional data file.S2 FigUpper) Western blot probed with antibody against VSV-G tag. (Lower) Western blot probed with antibody against GroEL serves as a loading control. Wild-type LVS cells containing the empty control vector pF (lanes 1–3); cells of LVS ∆pigR containing plasmid pF2-PigR-V (lanes 4–6); cells of LVS ∆pigR ∆mglA containing plasmid pF-PigR-V (lanes 7–9).Abundance of ectopically expressed PigR-V in biological replicate samples used for ChIP-Seq shown in (EPS)Click here for additional data file.S1 Table*Enrichment is determined with respect to the mock control. **Genes are considered to be associated with a ChIP-Seq peak if the translation start site is within 100 bp upstream to 500 bp downstream of the position of maximal enrichment.(XLSX)Click here for additional data file.S2 Table*Enrichment is determined with respect to the mock control. **Genes are considered to be associated with a ChIP-Seq peak if the translation start site is within 100 bp upstream to 500 bp downstream of the position of maximal enrichment.(XLSX)Click here for additional data file.S3 Table*Enrichment is determined with respect to the mock control. **Genes are considered to be associated with a ChIP-Seq peak if the translation start site is within 100 bp upstream to 500 bp downstream of the position of maximal enrichment.(XLSX)Click here for additional data file.S4 Table*Enrichment is determined with respect to the mock control. **Genes are considered to be associated with a ChIP-Seq peak if the translation start site is within 100 bp upstream to 500 bp downstream of the position of maximal enrichment.(XLSX)Click here for additional data file.S5 Table70, and σ32). Promoters are listed multiple times if more than one transcription factor and ChIP-Seq peak was used to define the promoter. The nucleotide location on the genome is reported for each promoter location (“Start” and “End”). The factors used to define each promoter are indicated (“Factor used to define promoter”): βʹ+rif (“Betaprime+rif”), σ70 (“Sigma70”), or σ32 (“Sigma32”). *Each factor listed refers to a particular ChIP-Seq peak for the given factor within the defined promoter region. Factors may be listed more than once for a particular promoter if multiple ChIP-Seq peaks are included in one promoter region. **Nucleotide position of maximal enrichment of ChIP-Seq peak for indicated factor. ***Genes are considered to be associated if the translation start site is within 100 bp upstream to 500 bp downstream of the position of maximal enrichment.There are 581 promoter regions reported (P1-P581), which were identified by ChIP-Seq of one or more factors Click here for additional data file.S6 Table*Enrichment is determined with respect to the mock control. **Genes are considered to be associated with a ChIP-Seq peak if the translation start site is within 100 bp upstream to 500 bp downstream of the position of maximal enrichment.(XLSX)Click here for additional data file.S7 TablepigR on the expression of 100 genes in LVS using Nanostring. The limited gene set used in this analysis includes a representative sample of 19 genes shown previously to be positively regulated by MglA/SspA and PigR [This table presents an analysis of the effects of and PigR –17, but (XLSX)Click here for additional data file.S8 Table*Transcription start site is indicated in uppercase. **Identified PRE motifs are in bold. ***Predicated extended -10 elements are in bold.(XLSX)Click here for additional data file.


PIN auxin transport proteins representing an integral part of the machinery that controls hormone distribution. However, unlike the rapidly emerging framework of molecular determinants regulating PIN protein abundance and subcellular localization, insights into mechanisms controlling PIN transcription are still limited. Here we describe PIN2 PROMOTER BINDING PROTEIN 1 (PPP1), an evolutionary conserved plant-specific DNA binding protein that acts on transcription of PIN genes. Consistent with PPP1 DNA-binding activity, PPP1 reporter proteins are nuclear localized and analysis of PPP1 null alleles and knockdown lines indicated a function as a positive regulator of PIN expression. Furthermore, we show that ppp1 pleiotropic mutant phenotypes are partially reverted by PIN overexpression, and results are presented that underline a role of PPP1-PIN promoter interaction in PIN expression control. Collectively, our findings identify an elementary, thus far unknown, plant-specific DNA-binding protein required for post-embryonic plant development, in general, and correct expression of PIN genes, in particular.Directional transport of auxin is essential for plant development, with  PIN proteins involved in cellular efflux as well as in intracellular compartmentalization of auxin, and therefore subject to multifaceted control mechanisms234The plant hormone auxin controls essential developmental processes throughout the life cycle of plants, which to a large extent depend on directional distribution of the growth regulator within the plant body1PIN proteins, influencing direction and rates of polar auxin transport (PAT)3PIN genes in the regulation of auxin distribution. This is indicated by observations linking adjustments in PIN transcript levels to various cues, such as environmental stimuli67910PIN transcriptional control, thereby specifying morphogenetic processes in the course of plant development. This applies to members of the PLETHORA (PLT) family of transcription factors, activity of which has been associated with transcriptional regulation of PIN genes in the control of root morphogenesisPIN transcriptional controlINDETERMINATE DOMAIN (IDD) family of transcription factors, implicated in diverse developmental processes, were found to be required for correct PIN transcription, pointing to an involvement in specifying auxin-controlled morphogenesis in plantsNext to post-translational regulation of XAANTAL2/AGAMOUSLIKE14 (XAL2/AGL14), a MADS-box protein was found to associate with PIN1 and PIN4 loci, acting as a positive regulator of PIN expression in the control of root developmentArabidopsis mutants deficient in BRAHMA (BRM) SWI2/SNF2 chromatin remodeling ATPase, revealed severe deficiencies in root stem cell niche maintenance, associated with a strong reduction in the expression of a subset of PIN genesPIN loci, and genetic analysis positioned BRM and PLT genes in an overlapping pathway, apparently required for accurate control of auxin distribution via control of PIN expressionPIN expression was found to depend on activity of CYTOKININ RESPONSE FACTOR (CRF) and AUXIN RESPONSE FACTOR (ARF) genes. ChIP assays demonstrated that members of each protein family associate with PIN gene promoter regions, evidently contributing to the transmission of hormonal signals in the regulation of PIN transcription17PIN transcription is essential for the regulation of PAT, and dynamics therein.Evidence for a direct involvement in the transcriptional control of PINs has been provided for some regulatory proteins. PIN transcriptional regulators by employing yeast one-hybrid (Y1H) methodologyPIN2 PROMOTER BINDING PROTEIN1 (PPP1), an evolutionary conserved, plant-specific DNA-binding protein of unknown function. In vitro and in vivo evidence is provided, demonstrating PPP1 interaction with PIN promoters, whereas a detailed in planta analysis identified PPP1 as important transcriptional regulator of PIN genes as well as of PAT-controlled developmental processes. Altogether, our results define PPP1 as founding member of a plant-specific family of DNA-binding proteins with an important role in transcriptional regulation of key determinants of plant morphogenesis and growth responses.In this report, we describe another approach aiming at the identification of PIN transcription via PIN promoter binding. In an attempt to identify further regulators we performed a Y1H screen, in which we used PIN2 promoter fragments as baits that were fused to the baker’s yeast HIS3 reporter genePIN2 ATG start codon, we identified three independent cDNA clones, each corresponding to locus At5g08720. This suggested binding of the protein to the PIN2 promoter fragment and therefore the corresponding gene was named PIN2 PROMOTER BINDING PROTEIN 1 (PPP1).A few regulators of gene expression have so far been demonstrated to influence PPP1 cDNAs. BLAST searches demonstrated that PPP1 represents a single copy locus and structure/domain prediction (http://www.expasy.org/) resulted in identification of conserved motifs (PPP1 contains two copies of a putative lipid-binding domain (aa 99-245 and aa 337-479), related to a domain found in members of the START superfamily (http://www.ncbi.nlm.nih.gov/Structure/cdd/cddsrv.cgi?uid=176942). This domain has been implicated in transport, binding and/or sensing of various hydrophobic compounds in pro- and eukaryotes202122PPP1 by calculating residue compactness describing the structural complexity of an individual residue in the context of 3-D protein fold and local secondary structure elementsPPP1 ranging from residue 664 to 697 that could signal nuclear localization of the protein (http://nls-mapper.iab.keio.ac.jp/cgi-bin/NLS_Mapper_form.cgi). No additional, characterized domains were predicted, but sequence entries related to PPP1 were found in genomes of higher plants, fern and moss as well as of green algae belonging to the charophytes. In contrast PPP1 orthologs were not found in the genomes of non-plant organisms, indicating that PPP1 has evolved plant-specifically  with purified, recombinant GST-tagged PPP1 (GST-tPPP1), to test for DNA-binding of PPP1 in vitro. By using EMSA combined with deletion analysis, we first limited PPP1-binding to a promoter fragment of 40 nucleotides within the 176 bp PIN2 fragment originally used in the yeast one-hybrid screen. Site-directed mutagenesis within this 40 bp fragment identified a DNA stretch of 16 bp as necessary for PPP1-binding in vitro , which might reflect conservation of a DNA-binding motif involved in transcriptional control of PIN genes.  together with HIS3 under control of a minimal yeast promoter plus a PIN2 promoter fragment  conferred growth on medium lacking histidine (PPP1 (PIN2p’::HIS). We then used the PIN1 promoter, containing a sequence motif related to the original PPP1 DNA-binding site, as another bait in the Y1H system, which indeed gave rise to efficient yeast growth under selective conditions , and similar results were obtained for yeast ADE2 when expressed by a minimal promoter . The corresponding mutant PIN2pm::PIN2:VENUS and a wild type PIN2p::PIN2:VENUS construct were transformed into the agravitropic eir1-4 (pin2) null allele and resulting transformants were analyzed for rescue of eir1-4 root gravitropism defects, as this assay represents a reliable read-out for expression and functionality of the PIN2 gene product25Our analysis of eir1-4 PIN2p::PIN2:VENUS control lines, 18% (n = 22 lines tested) exhibited subtle defects in root curling, but no obvious alterations in reporter expression. In addition, 9% (n = 22) showed agravitropic root growth, which coincided with a loss of PIN2 reporter signals, possibly as a result of incorrect integration or silencing of the transgene. In eir1-4 PIN2pm::PIN2:VENUS lines we observed defects in root growth at a higher frequency, indicated by small aberrations in root waving when grown on the surface of solid medium inclined at an angle of 60° . We performed whole mount in situ RNA hybridization experiments on young seedlings (2-3 DAG) and found PPP1 expression in the root meristem, throughout cell division and elongation zones . The corresponding PPP1p::GUS transgenic lines exhibited GUS-activity during vegetative and reproductive growth stages in a range of tissues, with signals most pronounced in vasculature and in proliferating tissue, including lateral root primordia and young leaves . Upon transient expression in onion epidermis cells, we detected nuclear localization and additional, weaker signals in the cytoplasm for both constructs  expressed in frame with genomic  signals . These rppp1-411) and SAIL_175_B03 (ppp1-476), which contain insertions within the PPP1 coding region, and found that both lines failed to produce adult homozygous progeny  and expression analysis resulted in identification of transgenic lines, exhibiting down-regulation of PPP1 transcription, but less pronounced than in ppp1-411 , implying that reduced transcription could arise as a consequence of diminished PPP1 binding to the PILS5 promoter. On the contrary, neither PIN3 nor PIN7 exhibited altered transcription in ami-ppp1, despite the fact that promoters of both genes contain the 7-nunclotide consensus sequence  in the transmission of cytokinin signals on PIN transcriptionPIN loci has not been demonstrated until very recently17Defects in the regulation of PPP1 belongs to a small group of proteins, so far demonstrated to associate with promoters of PIN genes, which makes it a likely regulator of PIN expression. However, unlike additional PIN promoter-interacting proteins characterized to date, which were demonstrated to bind to well-characterized conserved DNA binding motifs, no such motif has been identified for PPP1. In silico analyses led to identification of a 7-nucleotide sub-motif within the PPP1 DNA-binding site from the PIN2 promoter, which was found to be enriched in the promoter region of additional genes. Assuming that these motifs are recognized by PPP1 as well, it might specify its role in transcriptional regulation. The outcome of our Y1H experiments and expression assays performed with PIN1 and PILS5, both characterized by a copy of the 7-nucleotide motif in their promoters, is consistent with this hypothesis. In addition, transcript levels of additional loci, lacking this DNA motif, remained unaffected in ami-ppp1, pointing to a certain amount of specificity in PPP1-target site interaction. On the contrary, expression of PIN3 and PIN7, which both contain the 7-nucleotide motif in their promoters, did not markedly respond to PPP1 downregulation. Moreover, we observed PPP1-dependent activation of yeast reporter genes, expressed by minimal promoters lacking this motif, overall arguing for limited PPP1 DNA-binding specificity. At this moment, we can only speculate about the biological significance of these observations, since cis-acting requirements for PPP1 DNA-binding and transcriptional control are not entirely explained. For example, it cannot be excluded that additional components, absent in yeast and in vitro experiments, confer specificity to PPP1-DNA interactions. Protein-protein interaction and complex formation have been demonstrated to define DNA binding activities, crucial for the transcriptional regulation of a wide range of target genes3940PPP1-interacting factors, together with a genome-wide documentation of PPP1 DNA-binding sites should enable an in-depth characterization of binding specificities and activities of this so far unknown DNA-binding protein.PPP1 in the regulation of PIN2 transcription comes from analysis of PIN2mp::PIN2:VENUS, mutagenized in its PPP1 in vitro binding site. Although only incomplete conservation of DNA motifs appears to be required for PPP1 binding, mutations within this PIN2 promoter fragment resulted in stochastic variations or even a total loss of gene expression, at a frequency considerably higher than in controls. This suggests that the PPP1 binding site in the PIN2 promoter is essential for specifying or maintaining the expression status of the reporter gene in vivo. The extensive variability in VENUS signals observed in the different eir1-4 PIN2mp::PIN2:VENUS lines would be consistent with a general function of PPP1, ensuring stable expression of PIN2 and presumably a range of additional loci, including further auxin transport proteins such as PIN1. A related model has been put forward for Arabidopsis BRM chromatin remodeling ATPase, with a rather broad spectrum of targets, amongst which expression control of PIN genes appears to play a key roleBRM was found to cause reduced expression of PIN genes, together with severe developmental aberrations, resembling defects associated with ppp1 loss-of-function lines. Notably, patchy PIN2-VENUS signals that we observed are a characteristic feature of reporter genes that underwent somatic gene silencing events. This might argue for an involvement of the PPP1 DNA-binding motif in controlling the epigenetic status of the PIN2 promoter region. If true, then chromatin association of PPP1 could have an active function in transmission or maintenance of epigenetic signatures required for the correct expression of target genes, analogous to BRM. Clearly, genome-wide identification of PPP1 targets, together with an in-depth analysis of ppp1 loss-of-function lines will aid the analysis of PPP1 and its role in the regulation of gene expression.Indirect evidence for a requirement of PPP1, demonstrated an essential role for the corresponding gene product. However, the factual growth arrest, occurring a few days after germination, makes these ppp1 alleles rather inaccessible to an instructive phenotypic characterization. Analysis of ami-ppp1 plants on the other hand, established a link to the control of auxin-dependent growth and development. This is emphasized by ami-ppp1 growth defects, resembling phenotypes of auxin-related mutants as well as by a reduction of PIN1 and PIN2 transcript levels, which is consistent with PPP1 modulating auxin distribution via transcriptional control of PIN genes. Conversely, the strong phenotypes of the likely ppp1 null alleles do not necessarily reveal connections to auxin signaling. Limited DNA-binding specificity, which is indicated by our experiments, suggests a broader range of PPP1 targets, presumably affecting expression of Arabidopsis loci unrelated to auxin transport or signaling, which offers a straightforward explanation for the severe defects associated with the ppp1 knockout alleles. Partial rescue of ppp1-411 root growth defects caused by constitutive overexpression of PIN1 however, is in agreement with a function of PPP1 as a fundamental, positive regulator of PIN genes, shaping intercellular auxin distribution and plant development.Phenotypes that co-segregated with two distinct T-DNA insertion lines disrupted in PPP1 targets, distinct phenotypes of leaky ami-ppp1 alleles, reflecting aberrations in auxin-related processes, appear unexpected to some extent. Similar observations have been made upon interference with basic cellular processes, such as endocytic sorting or translational control, disturbance of which repeatedly resulted in auxin-related growth defects4243PPP1, acting as a pleiotropic regulator in plant development, with its function as a positive regulator of PIN transcription contributing to distinct aspects of auxin-controlled morphogenesis.When assuming a broad range of PPP1 might influence gene expression. Circumstantial evidence for a role of PPP1 as part of regulatory protein interaction networks comes from work published in recent years45PPP1 interaction with type-A ARABIDOPSIS RESPONSE REGULATORS (ARR), which act as repressors of transcriptional responses in cytokinin signaling46PPP1 and plant U-box–type E3 ubiquitin ligase PUB22 that is essential for pathogen-associated molecular pattern-(PAMP)-triggered responses in ArabidopsisPPP1 contains two START-domains that form structurally conserved cavities predicted to interact with hydrophobic binding partnersPPP1 are currently not known, it is tempting to speculate about a role for PPP1-ligand interaction with respect to its function in transcriptional regulation. Studies that will characterize PPP1 START-domains and its interaction partners should help to obtain insights into the function of this plant-specific DNA-binding protein.At present we can only speculate about mechanisms by which Arabidopsis cDNA library fused to the GAL4-activation domain (pACT2)19PIN2 promoter restriction enzyme-cut fragments has been employed for identification of promoter binding proteins, namely EcoRV (nt. -2159) - BglII (nt. -1685); BglII (nt. -1685) - BalI (nt. -1429); BalI (nt. -1429) - NruI (nt. -1175); NruI (nt. -1175) - PmlI (nt. -1002); PmlI (nt. -1002) - BamHI (nt. -572); BamHI (nt. -572) - XbaI (nt. -329). PPP1 cDNA clones were obtained, when using the PmlI (nt. -1002) – NruI (nt. -1175) PIN2 promoter fragment as bait cloned into pINT16 yeast transformants were screened both at RT and at 30 °C. For transactivation analysis the PPP1 cDNA was cloned into pGBKT7 (Clontech), and expressed in yeast strain PJ69-4APIN1 promoter region was cloned into pMW#2 using Gateway cloningPPP1 was tested in strain YM4271 on SC medium, lacking histidine and leucine and complemented with 20 mM 3-AT.The yeast one-hybrid system described in Ouwerkerk and Meijer (2001) was used to screen an PPP1 fragment was cloned into pGEX4T-2, expressing a truncated version of PPP1 ranging from amino acid residue 113 to 642. E. coli BL21-DE3 cells were used for heterologous expression of the recombinant protein. GST:tPPP1 was purified by binding to a gluthathione-sepharose matrix  essentially as describedPIN2 promoter fragments as describedFor electrophoretic mobility shift assays (EMSAs) an EcoRI-XhoI 3, 2 mM MgSO4, 2 mM Ca(NO3)2, 250 mM KPO4, 70 μM H3BO3, 14 μM MnCl2, 500 nM CuSO4, 1 μM ZnSO4, 200 nM Na2MoO4, 10 μM NaCl, 10 nM CoCl2, 50 μM FeSO4; pH adjusted to 5.7; supplemented with 1% (w/v) agar and 1% (w/v) sucrosePIN2p::GUS, DR5::mRFP, 35S::PIN1 and eir1-4 (SALK_091142) have been described previously252931PPP1 artificial microRNA construct was designed using the web tool on the website of the Weigel laboratory by using oligonucleotides I-ppp1-ami 5′-GATATGTCATAACGACCTGCTGGTCTCTCTTTTGTATTCC-3′; II-ppp1-ami 5′-GACCAGCAGGTCGTTATGACATATCAAAGAGAATCAATGA-3′; III-ppp1-ami 5′-GACCCGCAGGTCGTTTTGACATTTCACAGGTCGTGATATG-3′; IV-ppp1-ami 5′-GAAATGTCAAAACGACCTGCGGGTCTACATATATATTCCT-3′ (weigelworld.org)PPP1p::GUS was generated by PCR amplification of a PPP1 promoter fragment ranging from nt. -1112 to nt. -1 relative to the predicted PPP1 start ATG by using primers 5′-AAGTCGACTTCTACCGTTGAATTCTCACAGAT-3′ and 5′-AAGTCGACTAGCGAAAGGTGTTCGACGAA-3′. The resulting fragment was cloned into pPZP-GUSPPP1, driven by the 35S-promoter, the PPP1 cDNA lacking the stop codon was amplified using the oligonucleotides 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTTATGTCAGTGAGCAAGTTTCCACATCTC-3′ and 5′-GGGGACCACTTTGTACAAGAAAGCTGGGTCATATTGAACCCAATTGATATCAAGATCTTT-3′. The amplified PCR fragment was recombined by a BP reaction into pGEM-Teasy containing the Gateway recombination sites, creating pGEM-Teasy-PPP1 min. pGEM-Teasy-PPP1 min was subsequently used in a LR reaction with the destination vector pK7FWG2, thus creating pK7FWG2-PPP1 min. For generating the N-terminal GFP fusion driven by the 35S-promoter, the PPP1 cDNA lacking its start codon and containing the stop codon was amplified using oligonucleotides 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTTTCAGTGAGCAAGTTTCCACATCTCTCT-3′ and 5′-GGGGACCACTTTGTACAAGAAAGCTGGGTCTCAATATTGAACCCAATTGATATCAAGATCT-3′. The amplified PCR fragment was recombined by a BP reaction into pGEM-Teasy containing the Gateway recombination sites, creating pGEM-Teasy-PPP1plus. pGEM-Teasy-PPP1plus was subsequently used in a LR reaction with the destination vector pK7WGF2. For generation of PIN2pm::PIN2:VENUS we performed site directed mutagenesis on PINp::PIN2:VENUSeir1-4. T2 pools of randomly picked primary transformants were analyzed for fluorescent signals on a Leica binocular and a Leica SP5 Confocal Laser Scanning Microscope (CLSM). In total, we screened 38 eir1-4 PIN2pm::PIN2:VENUS T2 populations that exhibited a 3:1 segregation for the transgene. Root growth responses were determined on seedlings germinated on vertically oriented nutrient plates. For ami-ppp1 lines, 1.5% agar plates were used at an inclined angle of 60°, as these conditions make the ami-ppp1 root phenotype more apparent.Plants were grown on plant nutrient agar plates  and SAIL_175_B03 (ppp1-476), were from the Nottingham Arabidopsis Stock Centre  and genotyped by using oligonucleotides 5′-CCACCCATTCTTGTAATGGC-3′, 5′-GCATGAGATTCGTGAGCAG-3′ for ppp1-411 and 5′-CCTAAGTAACCAATGCAATGAGTGCA-3′, 5′-GAATGTTCTTACTGATTATGAACGA-5′ for ppp1-476. For complementation we introduced a genomic T-DNA cosmid clone harboring the entire PPP1 locus into PPP1/ppp1-476 plantsppp1 mutation. A similar setup was used for complementation analysis of ppp1-411, but a PPP1p::cPPP1 T-DNA construct was used instead. DR5-mRFP, CYCB1;1:GUS and PIN2p::PIN2:VENUS were introduced into ppp1-476 by crossing with heterozygote PPP1/ppp1-476. Analysis was performed in F2 and F3 generations.The in situ hybridizationPPP1 cDNA fragment ranging from nucleotide 1711 to 1989 was used for probe synthesis. The fragment was amplified and subsequently cloned into pGEM-T Easy (Promega). Subsequently, T7- and SP6-specific primers were used to synthesize anti-sense and sense probe, respectively.For whole-mount Whole-mount immunofluorescence was performed as describedStandard conditions were used for GUS staining with adaptation of concentrations for potassium ferricyanide and potassium ferrocyanide in the staining buffer (0.2 and 0.5 mM)5 μg of DNA were delivered into onion epidermal cells using gold particle bombardment. Gold particles  were coated with DNA according to the manufacturer’s directions. Particles were bombarded into onion epidermal cells using a Biolistic PDS-1000/He system (Bio-Rad) with 1100 psi rupture discs under a vacuum of 28 inHg. After bombardment, the cells were allowed to recover for 16–24 h on agar plates at 22 °C in the dark, after which positive cells were identified using a Leica MZ16F UV-binocular equipped with a GFP filter set. Subsequently, GFP positive cells were analyzed with a Zeiss Axio Imager M2 confocal microscope using 488 nm excitation and 495–565 nm emission wavelengths.EIF4a and TUB) using the Livak methodWhole RNA of seedlings (5 DAG) was extracted using the innuprep Plant RNA kit  from which cDNA was synthesized using the iScript cDNA synthesis kit (Biorad). qRT-PCR analysis was performed using a Biorad CFX96 Real time system with the IQ SYBRgreen super mix (Biorad) according to manufacturers’ recommendations. qRT-PCR was carried out in 96-well optical reaction plates heated for 3 minutes to 95 °C to activate hot start Taq DNA polymerase, followed by 40 cycles of denaturation for 10 seconds at 95 °C, annealing for 30 seconds at 55 °C and extension for 30 seconds at 72 °C. Expression levels were normalized to the expression levels of 2 household genes (http://www.wwpdb.org/). From the 3D coordinate files, distances between amino acids A and B were extracted and scored as a function of amino acid types . Additionally, the primary sequence distance between residues A and B was taken into account. To describe the spatial neighborhood of the two amino acids in the 3D structure of the entire protein (e.g. the way the two amino acids are embedded in the 3D fold), the pairwise distance distributions were transformed from cartesian space (distance rAB) to topological space (dAB). dAB reflects differential structural neighborhood properties of two amino acids  and can be used to predict topological information (compactness parameter) and local secondary structure elements. Further information is provided in Konrat (2009)In brief, the calculation of meta-structural parameters is based on statistical distributions of 3D atomic coordinates extracted from the Protein Data Bank (PDB) database .


Pluripotent stem cells (PSCs) represent a unique kind of stem cell, as they are able to indefinitely self-renew and hold the potential to differentiate into any derivative of the three germ layers. As such, human Embryonic Stem Cells (hESCs) and human induced Pluripotent Stem Cells (hiPSCs) provide a unique opportunity for studying the earliest steps of human embryogenesis and, at the same time, are of great therapeutic interest. The molecular mechanisms underlying pluripotency represent a major field of research. Recent evidence suggests that a complex network of transcription factors, chromatin regulators, and noncoding RNAs exist in pluripotent cells to regulate the balance between self-renewal and multilineage differentiation. Regulatory noncoding RNAs come in two flavors: short and long. The first class includes microRNAs (miRNAs), which are involved in the posttranscriptional regulation of cell cycle and differentiation in PSCs. Instead, long noncoding RNAs (lncRNAs) represent a heterogeneous group of long transcripts that regulate gene expression at transcriptional and posttranscriptional levels. In this review, we focus on the role played by lncRNAs in the maintenance of pluripotency, emphasizing the interplay between lncRNAs and other pivotal regulators in PSCs. The term long noncoding RNAs (lncRNAs) refers to a heterogeneous class of RNA polymerase II (Pol II) transcripts greater than 200 nucleotides in length with no evident protein-coding capacity , 2. TheyEven though only a part of lncRNA transcripts have been mechanistically characterized, several studies have shown the participation of lncRNAs in different processes related to normal physiology and/or disease , 6. As t cis-acting (on neighboring genes) and trans-acting (on distant loci) lncRNAs can exert either repressive or promoting activities on target genes by coordinating protein and RNA interactions [ cis-acting species, the enhancer-associated ncRNAs (eRNAs) are functional transcripts participating in many programs of gene activation. In particular, they play fundamental roles in targeting chromatin-remodeling complexes to specific promoters and to assist the formation of chromatin loops [The intracellular localization of lncRNAs is predictive of their mode of action , 10. Usuractions –14. Baseractions , nuclearractions –17. As aractions , 13. Amoin loops . Using ain loops . The expβ-site APP-cleaving enzyme 1 BACE1-AS antisense RNA, which binds to BACE1 mRNA inducing its stabilization. By regulating BACE1 expression, the noncoding RNA plays a role in controlling the boundaries between physiology and pathology driving Alzheimer's disease pathology [Some other lncRNAs are localized in the cytoplasm, where they can regulate gene expression through base-pairing complementary regions on target RNAs. In human, several cytoplasmic lncRNAs transactivate Staufen1-mediated mRNA decay by duplexing with 3′UTRs via Alu elements . Anotherathology . Base-paathology . In thisathology , 23. Finathology , 25, who in vitro counterpart of the pluripotent epiblast of the blastocyst and constitute a useful system to study the molecular mechanisms at the basis of pluripotency. A group of transcription factors (TFs), comprising OCT4, NANOG, and SOX2, has been proposed as the core regulatory circuitry in ESCs [β/BMP signalling, which is a crucial pathway for the choice between maintenance of pluripotency and differentiation [Embryonic Stem Cells (ESCs) are the in ESCs . These a in ESCs . It is n in ESCs –30. One  in ESCs . Among ontiation . In ESCsntiation . Other mntiation . In this in vitro disease modeling.ESCs have represented for a long time the only system to model human early development. More recently, the Nobel Prize-awarded derivation of induced Pluripotent Stem Cells (iPSCs) provided an alternative source of pluripotent cells . iPSCs cAs for protein-coding genes and miRNAs , PluripoAs in the case of mouse ESCs, K4-K36 domains analysis allowed the initial identification of a characteristic set of lncRNAs genes expressed in human ESCs . This liIncreasing evidence points to a crucial role for lncRNAs in the maintenance of ESC self-renewal (pluripotency), thus preventing their differentiation. In a large-scale functional study, the individual knockdown of more than 90% of lncRNAs (out of 147 tested) caused a significant perturbation of the transcriptome, often resulting in the loss of mESC pluripotency . Interes in trans at genomic loci encoding for factors involved in differentiation and development [The challenge now is to dissect the molecular mechanisms underlying the functions of these ESC lncRNAs. Mechanistically, nuclear lncRNAs may exert their function by binding and regulating the activity and/or target specificity of chromatin-modifying factors. It has been shown that ESC lncRNAs interact with all classes of histone modifiers , as well as other chromatin-associated proteins . This iselopment . These gelopment   factors, including mammalian MLL complexes, positively regulate transcription via the H3K4me3. This activity is required to maintain pluripotency in ESCs. In particular, the WDR5 member of the MLL complex directly interacts with the core transcriptional regulatory circuitry and its depletion causes loss of self-renewal . By takiechanism . lincRNAechanism . This evechanism , suggest Hoxa6 and Hoxa7 are involved in the specification of mesoderm derived tissues and organs [ Hoxa6 and Hoxa7 genes by recruiting MLL to chromatin [ Hoxa6 and Hoxa7 culminates in the expression of genes involved in early germ layer specification in differentiating mESCs. Another interesting example of lncRNA involved in mESC differentiation is pRNA, which is localized in the nucleolus [The interplay between lncRNAs and Trithorax complexes may also direct specification towards specific cell fates upon ESCs differentiation. The homeotic genesd organs , 54. Berhromatin . MIRA-meucleolus . In Plurucleolus ). Chromaucleolus . InteresIn addition to nuclear ESC lncRNAs, on the other side of the coin, fewer examples exist for cytoplasmic lncRNAs that regulate pluripotency. During the initial steps of the reprograming process, cells initiating their conversion to pluripotency must elude inhibitory hurdles, such as cell cycle arrest, senescence, and apoptosis, raised by p53 activation by the overexpression of the reprogramming factors . Thus, a cis by recruiting Dnmt1, Dnmt3a and Dnmt3b on its promoter. Although the cited work was mainly focused on myogenic differentiation, it is tempting to speculate that a similar regulatory mechanism might play a role in pluripotent cells as well. Dppa2 is highly enriched in pluripotent cells and activation of endogenous Dppa2 during late steps of reprogramming specifically marks the small subset of cells that will achieve full pluripotency, in which Dppa2-mediated induction of Nanog transcription is a crucial event [More recently, Bao and colleagues  showed tal event . Therefo in vitro platforms to model human disease and to test drugs in preclinical studies. Such potential applications, however, are subordinated to a deep understanding of the molecular mechanisms underlying pluripotency.Pluripotency is a unique property of ESCs and iPSCs, which are the only cell types able to undergo indefinite self-renewal and differentiation into derivatives of the three germ layers. Pluripotent cells therefore represent both ideal candidates for dissecting the mechanisms of early embryonic development and potential therapeutic tools for regenerative medicine. Patient-specific iPSCs also provideAn orchestra of transcription factors, chromatin regulators, signaling transducers, miRNAs and lncRNAs play coordinately in pluripotent cells. Each of them cannot be considered a solo player. Complex networks and feedback loops exist, which comprise members of each class of regulatory factors. A huge increase of transcriptome-wide analyses, facilitated by recent advancements in next-generation sequencing technologies, uncovered a universe of long noncoding transcripts. While there is no general consensus on the extent of the global impact of lncRNAs on the regulation of cell identity and differentiation, few examples in which selected lncRNAs have been more deeply analyzed exist. As discussed in this review, at least a subset of known lncRNAs are as important as previously defined “core transcription factors” in the context of pluripotent cells . The pau