Taxus, but the molecular mechanism has not yet been resolved. To establish the jasmonic acid signalling pathway of Taxus media, based on the gene of the jasmonic acid signalling pathway of Arabidopsis thaliana, sequence analysis was performed to isolate the jasmonic acid signal from the transcriptome, a transcriptional cluster of pathway gene homologs and the full length of 22 genes were obtained by RACE PCR at 5′ and 3′: two EI ubiquitin ligase genes, COI1-1 and COI1-2;7 MYC bHLH type transcription factor ; 12 JAZ genes containing the ZIM domain; and MED25, one of the components of the transcriptional complex. The protein interaction between each were confirmed by yeast two hybridization and bimolecular fluorescence complementation based on similar genes interaction in Arabidopsis. A similar jasmonate signaling pathway was illustrated in T. media. All known paclitaxel biosynthesis genes promoters were isolated by genome walker PCR. To investigate the jasmonate signaling effect on these genes’ expression, the transcription activity of MYC2, MYC3 and MYC4 on these promoters were examined. There are 12, 10 and 11 paclitaxel biosynthesis genes promoters that could be activated by MYC2, MYC3 and MYC4.Paclitaxel is an important anticancer drug. The phytohormone jasmonic acid can significantly induce the biosynthesis of paclitaxel in  Taxus biomass, and its biosynthesis and regulation have been studied [Taxus, but the molecular mechanism has not yet been resolved [Paclitaxel is an important anticancer drug, which is mainly produced by chemical semi-synthesis by using paclitaxel, which is biosynthesized in Pacific yew. Due to its complex molecular structure, its raw materials have been dependent on  studied . The phyresolved ,3,4.The biosynthetic pathway of paclitaxel is very complex, involving at least 19 steps of enzyme-catalysed reactions, and the related enzymes and synthetic pathways have not been fully resolved. The synthetic pathway can be divided into the following four steps: taxane ring synthesis; side hydroxylation, acetylation and phenylacetylation modification; epoxidation modification at C5 position and oxidation at C9; and side chain at C13 position addition [Taxus cells [The precursor of taxadiene ring biosynthesis is derived from isopentenyl pyrophosphate (IPP) and dimethyl propylene pyrophosphate (DMAPP), the synthesis, mainly through the plastids of us cells ,6,7. AftThe known modification process is first hydroxylation of the taxane-45), 11(12)-diene at the 5α position. This is the rate-limiting step for paclitaxel synthesis [, 11(12)-The biosynthesis step of paclitaxel is very complicated, and it is very difficult to directly increase the expression of the biosynthetic pathway gene to increase the paclitaxel content. As the analysis of all synthetic pathways is not complete, the metabolic pathways cannot be precisely regulated to control the metabolic flux. In this case, for such a complex metabolic pathway, a better choice for regulating genes in the synthetic pathway is to regulate upstream transcription factors, thereby comprehensively regulating the expression of genes in the synthetic pathway . It is iTaxus are being closely studied [Transcription factors regulating Taxol biosynthesis in  studied ,3,4. PreIn the transcription factor-related studies in Taxus, no relevant research results of jasmonic acid signaling pathways in model plants have been used. There is also no systematic and in-depth discussion of the signaling pathway of jasmonic acid in Taxus. Li et al. used high-throughput sequencing to study the transcriptome of jasmonic acid treatment for 16 h, and obtained an AtJAZ-like gene induced by jasmonic acid, and a similar AtMYC2 gene, but no in-depth study . In the T. media was obtained by sequence comparison analysis and RACE PCR. It includes sequences similar to the important genes of the jasmonic acid signalling pathways such as AtCOI1, AtMYC2, AtJAZ and AtMED25 . The interaction between JAZ and MYC transcription factors and other genes was studied, and the jasmonic acid signalling pathway in Taxus was confirmed. The above work further studied the regulation of the promoter of the paclitaxel synthesis pathway gene by the MYC transcription factor, and elucidated the regulation mechanism of the transcription factor of the jasmonic acid signaling pathway on the biosynthesis pathway of paclitaxel.In this study, high-throughput sequencing was used to obtain transcription factors after an early induction with jasmonic acid. In our previous study, the sequence of several genes related to the paclitaxel synthesis pathway and jasmonic acid signaling pathway were obtained by sequencing analysis of the transcriptome and expression profile of Taxus. Based on this, the full-length sequence of jasmonic acid signalling pathway-related transcription factors in We also found that some of the genes involved in the paclitaxel synthesis pathway were highly expressed by jasmonic acid at 0.5 h, similar to the transcription factors of the jasmonic acid signalling pathway and the expression of self-synthesis-related genes, but faster. Expression of anthocyanin synthesis-related genes regulated by the jasmonic acid signalling pathway was noted at three hours . This inArabidopsis protoplasts. Regulation of the promoter of the paclitaxel biosynthetic pathway gene and the transcription factor mutants with application value were designed, which lays the foundation for the future construction of a high-yield paclitaxel-based Taxus cell line.To analyse the mechanism of jasmonic acid signalling pathway specifically regulating the paclitaxel synthesis pathway gene, 14 promoters of paclitaxel synthesis pathway gene were isolated, and five MYC transcription factors were verified by fluorescence activity assay in Arabidopsis AtJAZ1 gene and the eight JAZs with reference to the Tm_ZK transcriptome, and the threshold of the E value was set to 0.01. Finally, 16 transcription cluster sequences which may be JAZ were obtained, as shown in At least eight JAZ-containing ZIM domains were obtained in the differentially expressed genes by the transcriptome sequencing. For comprehensive detection of JAZ that may be contained in the transcriptome, BLAST analysis was performed using the full length of the For the 16 possible JAZs, specific primers were designed and the full length of the gene was obtained by RACE PCR at the 3′ and 5′ ends. Three of the transcription clusters comp60304_c0, comp64513_c0, and comp68543_c0 failed to obtain a full-length sequence. The other 13 obtained the full length sequence, and wherein comp70749_c0 was a non-coding sequence. The remaining 12 transcription clusters obtained the encoded amino acid sequence—see Homology analysis and domain analysis in the amino acid sequence were performed see , and theThe primers were designed to amplify the 12 full-length genes, and the cDNA was used as a template for PCR. The bands obtained from the cloning were sequenced to obtain 18 sequences. Among the 18, JAZ1, JAZ2, JAZ9, JAZ11 and JAZ12 all contained varying cleavages. There were three in JAZ2, and the other had two coding sequences  dissolved in ethanol was added to 2 g cells that were suspension cultured in 50 mL of medium 14 days after subculture. Samples were collected after 0, 0.5, 3, and 24 h MeJA treatment using a Büchner funnel. Three biologically independent experiments were performed and a total of 12 samples were obtained. Total RNA was isolated from each of the samples as described before [g for 15 min. The supernatant was added to an equal volume of isopropanol to precipitate the total RNA. The total RNA was purified using an RNeasy Mini kit  according to the manufacturer’s instructions.The The full length sequence of the 5′ and 3′ ends of the gene was obtained using the GeneRacer Kit with SuperScript III RT kit based on Invitrogen’s RACE PCR technology  using high quality RNA extraction. First, the extracted RNA is dephosphorylated, and then the enzyme is used to remove the 5′ end cap of the RNA, and then the known RNA linker sequence is ligated. Using this as a template, the first strand of the cDNA is synthesized by the oligo(T) used as a locking primer to bind the the 3′ end ploy(A) tail of mRNA. Design specific primers for nested PCR at the 5′ and 3′ ends, respectively, clone the obtained DNA fragments, analyse the sequence of the coding region after sequencing, and after splicing, both stop codons and reach the longest coding region to determine the full length sequence at both ends.Based on Clontech’s MATCHMAKER LexA Two-Hybrid System, JAZ and other related genes are ligated into a pLexA vector containing a LexA promoter binding domain (BD), and another gene for detecting interaction is linked to transcriptional activation domain(AD) of the pB42AD vector. The above two plasmids were then transferred to the lacZ-expressing active EGY48 yeast strain containing LexA control using the Frozen-EZ Yeast Transformation Kit of Zymo Research. After overnight culture in SD/–His/–Trp/–Ura liquid medium, transfer 5–20 μL of yeast culture solution to SD/Gal/Raf/–His/–Trp/–Ura/ for activity detection. In X-gal + BU salts solid medium, the appearance of blue spots was observed after four days of culture to evaluate the interaction between lacZ activity and protein.Agrobaterium tumefaciens by electroporation. After induction with acetosyringone for two days, the two plasmids were simultaneously transferred into the tobacco leaves of the 4-week culture by injection, and then the fluorescence signal of the injection site on the leaves was observed using a laser confocal microscope.The sequences of the two genes detecting the interaction were ligated into p35SnYFP and p35ScYFP, respectively, and the fusion protein was formed with the C-terminus and the N-terminus of the yellow fluorescent protein YFP, respectively. The assembled plasmids were then transferred to GV3101 T. media was extracted using the Plant Genomic DNA Purification Kit . Based on Clontech’s Universal Genome Walker Kit , genomic DNA was digested with four different blunt-end restriction enzymes DraI, EcoRV, PvuII, and StuI, followed by ligation to the linker. Primers were designed for two rounds of nested PCR by known coding region sequences and linker sequences, and the obtained DNA bands were cloned, sequenced, spliced and analysed to obtain the promoter sequences of the corresponding genes.To obtain the promoter of the paclitaxel biosynthetic pathway gene, the genome of Arabidopsis protoplast transformation method [Arabidopsis protoplasts were transferred by a PEG transformation method, and a plasmid containing a GAL4 promoter-controlled transcription factor was simultaneously transformed. The plasmid also contained the reporter gene for the LUC fluorescent protein in the plasmid and the plasmid for the continuous expression of the REN fluorescent protein internal reference. The fluorescence intensity changes of the two fluorescent proteins were detected using the Promega Dual Luciferase Reporter Assay system kit (Cat. E1910) , and the activity of the transcription factors in the plant cells was analysed by comparing the changes in the fluorescence intensity ratio.To analyse the transcriptional activity, the transcription factor was transferred to a plasmid 35SGAL4DB containing a GAL4DB domain in a plant cell to form a plasmid expressing a fusion of the GAL4DB domain and a transcription factor. Based on the mature n method , the AraThe promoter of the paclitaxel biosynthetic pathway gene was inserted into the coding region of the LUC fluorescent protein in the 0800 plasmid to construct a plasmid, which contained the reporter gene with promoter-controlled LUC fluorescent protein and the continuous expression of REN fluorescent protein as internal reference. At the same time, the transcription factor to be detected is inserted into another plasmid 62sk, and the transcription factor can be continuously expressed in the plant by 35S regulation. The two plasmids were co-transformed into protoplasts by PEG transformation. The fluorescence intensity of the two fluorescent proteins was simultaneously detected using the Promega Dual Luciferase Reporter Assay system (Cat. E1910) kit, and the regulation of the transcription factor on the paclitaxel biosynthesis pathway promoter was analyzed.Arabidopsis model, the jasmonic acid signalling pathway of T. media was isolated and identified. The interaction between related proteins was analysed, and the molecular mechanism of the jasmonic acid signalling pathway in Taxus was constructed. Further analysis of the activation or inhibition of the promoter of paclitaxel biosynthesis pathway gene by transcription factors such as MYC2 confirmed the regulation of MYC2 and other transcription factors on the biosynthesis pathway of paclitaxel. Based on this design, the transcriptional activity of MYC2 and MYC3 was not affected by the JAZ inhibition function, which laid a foundation for the construction of a high-yield paclitaxel variant Taxus cell line.In this study, based on the jasmonic acid signalling pathway of the Taxus has a corresponding jasmonic acid signalling pathway, and it directly regulates the expression of paclitaxel biosynthesis pathway genes through MYC transcription factors, and JAZ and non-transcriptionally active MYC transcription factors comprehensively regulate the expression of related genes. Based on the signalling pathway, a variant MYC transcription factor was constructed to activate transcription without inhibition by JAZ protein, and was the basis for constructing a taxol-producing Taxus cell line.


Hypoestrogenism in women is strongly associated with menopause and it can lead to lipid disorder, which predisposes people to premature cardiovascular disease. However, the mechanism of lipid disorder remains unclear. Sterol regulatory element-binding protein 2 (SREBP2) is the key transcription factor regulating cholesterol metabolism. We hypothesize that estrogen regulates SREBP2 transcription through an estrogen response element (ERE) in the SREBP2 promoter region.2) for 24 h. Then, SREBP2 expression was determined via real-time PCR and immunofluorescence. The expressions of the SREBP2 downstream target genes HMGCR and LDLR were determined via real-time PCR. Lipid secretion in the culture media of HepG2 cells was measured using ELISA. Through bioinformatics analysis, we identified high-scoring ERE-like sequences in the SREBP2 gene promoter. Chromatin immunoprecipitation analysis was used to confirm the ERE. DNA fragments of the putative or mutated ERE-like sequence were synthesized and ligated into pGL3-basic plasmid to construct the SREBP2 promoter luciferase reporter systems. SREBP2-Luciferase (SREBP2-Luc), SREBP2-Mutation (SREBP2-Mut) and the blank control were transfected into hepatic cell lines. Luciferase activities were measured using the dual-luciferase reporter assay system. Chromatin immunoprecipitation analysis and the luciferase reporter assay were repeated in human hepatoma cells (HuH-7).Human hepatoblastoma cells (HepG2) were treated with dose-dependent concentrations of estradiol (E2 dose-dependently increased the expression of SREBP2 in HepG2 cells and that the increased levels were blocked when treated with an estrogen receptor-alpha antagonist. Additionally, E2 increased both HMGCR and LDLR expression and lipid secretion in HepG2 cells. Notably, we identified a functional ERE in the SREBP2 gene promoter, to which E2 could specifically bind and induce transcription.We found that EAn ERE was identified in the SREBP2 gene promoter. It mediates the regulation of SREBP2 expression by estrogen in hepatocytes. This study provides a mechanism to link cardiovascular disease with estrogen. Effective methods to prevent cardiovascular diseases are essential, since they are major causes of morbidity and mortality around the world . ExampleA large number of epidemiological studies have shown that determinants of cardiovascular disease include behavior, environmental factors and heredity factors . Risk faThe incidence of cardiovascular disease increases sharply in females after menopause. Studies have shown that this can be mainly attributed to lipid disorder, vascular stability destruction and blood pressure increase, which result from a series of pathological changes caused by decreased estrogen levels –11.As an important steroid hormone, estrogen mainly regulates estrogen-sensitive genes via the classical pathway: estrogen receptor α (ERα) binds to estrogen response elements (EREs) to regulate gene transcription . EstrogeSterol regulatory element-binding proteins (SREBPs) are a family of key nuclear transcription factors that can regulate lipid metabolism by controlling the expression of a series of enzymes required for the synthesis of endogenous cholesterol, triacylglycerol, fatty acid and phospholipid . NuclearThe aim of this investigation was to evaluate whether SREBP2 is regulated by estrogen and to further understand the regulatory pathway. Lipid metabolism mainly occurs in the liver, so we focused on hepatocytes to study this mechanism.2 in high-glucose phenol red Dulbecco’s modified Eagle medium  supplemented with 10% fetal bovine serum  and 100 U/ml streptomycin and penicillin (Gibco-BRL). After the cells were 40–50% confluent, the medium was replaced with phenol red-free DMEM (Gibco-BRL) supplemented with 1% charcoal-stripped FBS (Gibco-BRL). 17β-estradiol , which is the most active form of estrogen oestra-1,3,5(10)-tiene-3, 10− 5 mol/l; Tocris Bioscience) for 24 h. No intervention was used as a blank control.Approximately 10Subsequently, the cells were fixed in 4% paraformaldehyde (Sigma) for 30 min at room temperature and then permeabilized with 0.25% Triton X-100 (Sigma) in PBS for 20 min at 25 °C. The fixed and permeabilized cells were blocked in 1% BSA and incubated overnight at 4 °C with a 1:100 dilution of anti-SREBP2 antibody (Abcam). Then the cells were incubated with a 1:200 dilution of Alexa Fluor 594-labelled mouse anti-rabbit IgG (Invitrogen) for 2 h. The nuclei were stained with 0.125 μg/ml DAPI for 15 min.http://rsb.info.nih.gov/ij/). The intensity was calculated using the mean grey value.We analyzed the slides with an Olympus BX51TF fluorescence microscope (Olympus Corporation), with excitation/emission at 530/580 nm for the red fluorescence and 358/461 nm for DAPI. Finally, we analyzed the images using Image-J software from the United States National Institutes of Health (http://rsat-new.ccb.sickkids.ca/) to analyze the sequence of the SREBP2 gene promoter to find high-score ERE-like sequences. HepG2 and HuH-7 cells were treated with 10− 7 mol/l E2 for 24 h and then cross-linked according to the Millipore EZ-ChIP Assay Kit protocol (Millipore).We used regulatory sequence analysis tools , which respectively recognize the sequences C^TCGAG and GGTAC^C. Then, they were ligated into the pGL3-basic plasmid (Promega). After that, we completed construction of the luciferase reporter systems of the SREBP2 promoter: SREBP2-Luciferase (SREBP2-Luc) and SREBP2-Mutation (SREBP2-Mut).The putative ERE-like sequence was GCATTCGCTCCGAGGCCGCGGGGGGAGGGACCTCACTATGCAAATCTGAGCTGCTGATCGATGACGCGCCATCACCCCACGCACCGCTTCGCTCGCCCATTGGCTGAGATGAGCCTGGTCCCATTGACAACAAACAGGGGGGCGCGCGGCCTGGAGGCGGGGCCGCAGGGGGCGCGGGCTGGGGCGGGGGAATCCCGCCCCGCC.The mutated ERE-like sequence was GCATTCGCTCCGAGGCCGCGGGGGGAGGGACCTCACTATGCAAATCTGAGCTGCTGATCGATGACGCGCCATCACCCCACGCACCGCTTCGCTCGCCCATTGGCTGAGATGAGCCTCCCAGCATTGACAACAAACAGGGGGGCGCGCGGCCTGGAGGCGGGGCCGCAGGGGGCGCGGGCTGGGGCGGGGGAATCCCGCCCCGCC.− 7 mol/l E2 was added for an additional 24 h. Luciferase activities in the cell lysates were measured using the dual-luciferase reporter assay system (Promega) according to the manufacturer’s instructions. After normalization to the Renilla luciferase activity, luciferase values were calculated.HepG2 and HuH-7 cells were cultured in 6-well plates for 24 h in phenol red-free DMEM supplemented with 1% charcoal/dextran-treated FBS. Using Fugene HP transfection reagent (Roche Applied Science), the luciferase reporter plasmid containing the SREBP2 promoter and the pRL-TK reporter plasmid  were co-transfected into the cells. After 24 h of transfection, 10p < 0.05.The 2-tailed Student’s t test was used to evaluate the statistical significance of the difference between two groups. One-way ANOVA and Turkey’s post hoc tests were used to evaluate the statistical significance of the difference between more than two groups. All statistical analyses were carried out using SPSS 16.0. The results were recorded as the means ± SEM and were considered significantly different at 2  treatment.Furthermore, EThe promoter sequence of the SREBP2 gene was analyzed using regulatory sequence analysis tools to identify the putative EREs. The bioinformatics analysis showed that there were multiple possible ERE-binding regions in the SREBP2 promoter. The high-scoring ERE-like sequences were: GGTCCcatTGACA (− 88 ~ − 76), GATGAcatGGACA (− 1510 ~ − 1498) and GCACAcctCGGCC (− 1675 ~ − 1663).2 through an ERE. Different primers were used to amplify the high-scoring ERE-like sequences in the promoter of SREBP2. The results show an ERE sequence at − 88 ~ − 76 of the SREBP2 promoter . Luciferase activities were measured after treatment with E2 Fig. a to dete2 could promote SREBP2 expression in HepG2 cells. Through a bioinformatics analysis of the sequence of the SREBP2 gene promoter, we predicted that there were multiple ERE-like sequences in this region. Furthermore, a functional ERE was identified in the SREBP2 promoter.In this study, we found that ESREBPs belong to a transcription factor superfamily that can activate a series of enzymes involved in lipid biosynthesis and absorption, such as LDLR, HMGCR, HMG-CoA synthase and squalene synthase. Therefore, SREBPs play a key role in the lipid metabolic pathway , 23. SREIt has been reported that inflammatory cytokines can aggravate cholesterol accumulation in hepatocytes by disturbing SREBP2 regulation, and that SREBP2 expression inhibition can significantly increase cholesterol accumulation . In addiAberrant accumulation of lipids in the arterial wall can lead to atherosclerosis, which plays an important role in the progression of cardiovascular disease . Lipid d2 could regulate SREBP2 expression. This was validated by SREBP2 expression inhibition after treatment with the ERα antagonist ICI. The expression of SREBP2 mRNA significantly increased after treatment with 10− 7 mol/l and 10− 5 mol/l E2 compared to 10− 9 mol/l E2, which is in the physiological range for premenopausal women [2 treatment in hepatic cell lines. Therefore, our results reveal that E2 may regulate lipid metabolism by affecting SREBP2 expression, and thereby relates to cardiovascular diseases.In this study, we found that Eal women . FurtherLipids are mainly synthesized in the liver , so we uEstrogen can exert multiple-pathway regulatory effects by means of acting on its receptors . ClassicThere has been evidence of the regulation of SREBP gene transcription as a mechanism to alter SREBP levels. There are binding sites for the thyroid hormone receptor in the 5′-flanking sequence of SREBP2 . Strikin2 treatment, the activity of the plasmid containing this ERE sequence was significantly higher than that of the blank control or mutant sequence.We used a luciferase activity assay to analyze the activity of this ERE sequence and found that after E2 could directly regulate SREBP2 expression via an ERE in its promoter.In our study, a functional ERE (GGTCCcatTGACA) was identified in the − 88 ~ − 76 region of the SREBP2 promoter for the first time. The sequence of ERE in the SREBP2 gene promoter is similar to that of typical EREs, which are elements with palindrome structure. The common sequence is 5′-GGTCAnnnTCACC-3′ (where n represents any nucleotide) and contains 13 inverted repeat base pairs and 3 bases that can be randomly replaced. However, in the genes regulated by estrogen, only a few contain the canonical ERE sequence and the majority of them have a variation of the common ERE sequence , 40. ThuOur findings demonstrate that estrogen directly activates SREBP2 gene expression. An ERE, one binding site for ERα, has been identified in the 5′-flanking sequence of SREBP2. It suggests that estrogen may have an effect on lipid metabolism by regulating the SREBP2 promotor. Our results provide a basis for cardiovascular disease prevention and treatment (Fig.


This repressive characteristic of nuclear Spry2 was relieved in 16HBE14o- by shRNA knockdown, and stable expression of mutants  that do not interact with the VEGF-A promoter HRE region. We conclude that nuclear Spry2 acts as a molecular link which co-ordinates airway and vascular growth of the cardiopulmonary system. This identifies Spry2 as a contributing determinant of design optimality in the mammalian lung.Sprouty2 (Spry2) acts as a central regulator of tubular growth and branch patterning in the developing mammalian lung by controlling both magnitude and duration of growth factor signalling. To determine if this protein coordinates airway and vascular growth factor signalling, we tested the hypothesis that Spry2 links the primary cue for airway outgrowth, fibroblast growth factor-10 (FGF-10), to genomic events underpinning the expression and release of vascular endothelial growth factor-A (VEGF-A). Using primary fetal distal lung epithelial cells (FDLE) from rat, and immortalised human bronchial epithelial cells (16HBE14o-), we identified a nuclear sub-population of Spry2 which interacted with regions of the rat and human VEGF-A promoter spanning the hypoxia response element (HRE) and adjacent 3′ sites. In FDLE cultured at the PO Early studies in l airway  and lateranching . The eviIn lung development, airway branching is induced by mesoderm-derived fibroblast growth factor-10 (FGF-10) binding to the tyrosine kinase receptor, FGFR2b, expressed in cells at the tip of the endodermal airway tube. Downstream signalling through extracellular regulated kinase 1/2 (ERK1/2) and the mechanistic target of rapamycin complex 1 (mTORC1)  induces 22.1N-terminal FLAG and C-terminal Myc dual tagged Spry2 was constructed by sub-cloning full length Spry2 into pCMV-Tag-1  and cysteine to alanine mutations were generated at positions 218 and 221 to assess Spry2-DNA interactions. Non-genome target control (TR20003) and shRNA vectors targeting human Spry2 in pRS were originally purchased from Origene Technologies, Inc.  and kindly supplied to us by Drs P Yusoff and G Guy . Stable Spry2 knockdown in 16HBE14o- cells was achieved with the clone TI336513 (sequence ‘5-CTGAACAGAGACTGCTAGGATCATCCTTC-3’). Source, concentration and species of all antibodies used in this study are given in Supplementary Table 1.2.22 equivalent to the fetal lung in utero (3% O2) to conserve the FGF-10 and HIF signalling pathway context  were isolated from gestation day 19 Sprague Dawley rat lungs at the pseudoglandular/canalicular stage of development and maintained in polarised culture on permeable supports as described . Culture context . HBE sta2.32. Enrichment of Histone 3 (H3) confirmed nuclear lysis.The full protocol is given in Supplementary Fig. 2 with initial lysis procedures were conducted in a hypoxic chamber equilibrated to 3% O2.432P] radiolabelled oligonucleotides containing the HIF response element (HRE) in the rat VEGF promoter (HRE underlined): 5′-TGC ATA CGT GGG CTT CCA CAG GTC-3′. The same sequence with the HRE mutated to adenosines served as a negative control. Nuclear protein binding and mobility shift were performed as before . 1 μg of RNA was reverse-transcribed using a Quantitect Reverse Transcriptase Kit (Qiagen). qPCR was performed in duplicate on Rotor-Gene Q cycler using a Rotor-Gene SYBR Green PCR Kit using Quantitect Primer Assays (Qiagen). β2.7The full protocol including primer sets and chIP validation of Anti-Spry2 (Abcam Ab50317) is given in Supplementary Fig. 2 and Table 2.2.8Genomic DNA from wild-type or stably transformed HBE cells was bisulfite converted using the Epitect Fast DNA Bisulfite Kit (Qiagen) according to the manufacturer's instructions. Bisulfite-converted DNA was adjusted to 50 ng/μl after a clean-up step and this volume was used in conjunction with Epitect High Resolution Melting PCR (Qiagen) to detect differences in DNA methylation at CpG sites located 145 bp 5′ to the VEGF-A transcription initiation site (TIS) or 179 bp 3′ to this locus. Methyl Primer Express® Software v1.0 (Applied Biosystems) was used to select CpG methylation-specific primer sets. MS-HRM was performed on a Rotorgene 6000 (Qiagen) and once each sample had reached the plateau phase of the PCR a high resolution melting protocol was used to expose methylation specific differences in the amplified DNA.2.9Multiple comparisons were conducted using one way ANOVA with post hoc significance determined using Tukey's HSD. Values are mean ± SEM with the number of observations reported in the legend to each figure.33.12) as well as ambient (21% O2) PO2 because fetal development occurs in an oxygen-restricted environment and tissue oxygenation influences total cellular Spry2 stability . Dual tagged Spry2 expressed in HBE resolved in cytosolic, nuclear soluble and chromatin enriched insoluble fractions Immunofluorescence in FDLE revealed that Spry2 nuclear abundance was favoured at 3% O for 6 h A. NucleakDa form Bi–iv. DukDa form Bi–iv. DukDa form Bi–iv. DuWe investigated the difference in nuclear Spry2 protein mass by determining if either form was influenced by phosphorylation, cleavage or palmitoylation   phosphoacetylation at serine 10 and lysine 14 (H3-S10(P)K14(Ac)) . FGF-10 3.22, HIF-1, 2 and 3α proteins were detected in the nuclear fraction of FDLE cells and their abundance was not significantly altered by FGF-10  and CCND-1 (HIF-2α-regulated)  . Spry2 kgulated) E.Fig. 3F3.3We hypothesised that nuclear Spry2 regulates the interaction between HIF-1α and the VEGF-A promoter during FGF-10 exposure and so chromatin immunoprecipitation (chIP) was used to determine if Spry2 interacts with VEGF promoter elements. The commercial Spry2 antibody (Abcam ab50317) was validated for this purpose  and used in combination with chIP grade antibodies against histone 3 , HIF-1α and CBP/p300 (indicator of HAT binding and epigenetic gene activation). Parallel incubation with non-immune IgG served as a negative control. ChIP primers were designed against elements up to 1Kb upstream of the VEGF-A transcription initiation site (TIS) (Supplementary Table 2 and 3.4199WICxxxCxCxxxNxxxxxCxxC221) is approximated in the same region between each of the other human Spry isoforms and is conserved between species (199WICxxxCxCxxxNxxxxxC/AxxC/A221), created stable HBE cell lines and performed ChIP assays using primers designed to amplify −1089 to −882 bp of the human VEGF-A promoter  was performed to search for possible conserved DNA binding elements in the Spry2 protein sequence. This analysis identified limited homology between amino acids 199 and 221 in the Spry2 CRD and a zinc finger RanBP2-type signature -C-x(3)-N-x(6)-C-x(2)-C; Entry Name: ZF_RANBP2_1, ExPASy Accession number PS01358). The corresponding sequence in Spry2 . ChIP w−968 bp, B. Neithe−968 bp, C and D. 3.5Spry2 knockdown and mutation of CRD residues C218 and C221 augment VEGF-A expression suggesting a role in silencing gene activity. CpG island methylation is an important part of this regulation and the region surrounding the transcriptional initiation site (TIS) of the human VEGF-A gene is rich in potential CpG methylation sites A. We theTwo primer sets designed to detect methylated or unmethylated DNA sequences were used to analyse the methylation status of a region spanning −143 to 0 bp upstream of the VEGF-A site (5′VEGF-A TIS containing 17 CpG sites) and a second region spanning +13 to +199 bp downstream to the VEGF-A TIS (3′VEGF-A TIS containing 16 CpG sites). 20 ng of bisulfite converted DNA was amplified through 40 cycles of qPCR followed by HRM. Commercial methylated and unmethylated DNA served as controls.−1 FGF-10-treated and shRNA control samples that corresponded to the position of the 100% methylated control indicating extensive methylation of this region of the VEGF-A promoter. Treatment with FGF-10 or knockdown of Spry2 induced a leftward shift toward the position of the unmethylated control peak  sets the duration of tubular elongation, 2) determines the position of each branch point, 3) re-calibrates branching subroutines at each daughter generation, 4) co-ordinates all of these processes between airway and vascular systems and 5) determines the point at which the branching programme diverges to form the gas exchange surfaces. Here, we report that Spry2, a conserved regulator of branching morphogenesis across species, is present in the nucleus of airway cells derived from the fetal lung, is regulated by the primary inducer of airway outgrowth, FGF-10, and represses the activity of the main inducer of VEGF-A transcript expression, HIF-1α. Using chIP and DNA methylation analysis, we show that Spry2 interacts directly with regulatory regions of the VEGF-A promoter and that its knockdown induces demethylation and subsequent expression of VEGF-A transcripts and protein. Taken as a whole, we argue that Spry2 has a central role to play in the molecular co-ordination of airway and vascular branching periodicity. As such, it is an important contributor to design optimality in the cardio-pulmonary system.The mechanisms which underpin nuclear Spry2 signalling require evaluation. The electrophoretic migration of Spry2 as a doublet is frequently reported in the literature  and a nRepression of vascular signalling by Spry proteins has been described in a number of models of development and disease and underpins the role of these proteins as critical mediators of vascular stability. For example, Spry2 or 4 knockout in mice causes malformation of cardiovascular structures that is lethal at birth and shRNA knockdown of these proteins in a murine model of hind-limb ischemia-reperfusion injury drives angiogenesis following ischemic injury . In lung2+ finger domains and although we did not investigate if Zn2+ was necessary for the Spry2-DNA interaction, it is possible that this site acts as a redox centre that mediates Spry2 interaction with other macromolecules, including DNA. Indeed the high level of conservation of these residues from insects to mammals and between mammalian Spry isoforms suggests that they are critical mediators of Spry2 function. Taken together, however, these results suggest that Spry2 functions as a repressor of VEGF-A expression, possibly by controlling the recruitment of HIF-1α and CBP/p300 transcription co-activators to the VEGF-A promoter.HIF-1α mediates vascular gene expression in the early stages of lung development and so we tested the hypothesis that repression of HIF activity and VEGF-A expression was mediated by interaction between Spry2 and the VEGF-A promoter to alter one of, i) HIF-1α recruitment to the HRE, ii) CBP/p300 histone acetyl transferase recruitment or iii) DNA methylation status. We found that Spry2 interacted to varying extents with regions of the rat VEGF-A promoter spanning −944 bp to −142 bp. Reliable resolution of protein-DNA interactions could not be detected at sites flanking the transcription initiation site (TIS) because the high GC content this region. Notably, however, we observed a FGF-10-responsive interaction in the region of the promoter that spanned the HRE and apparently constitutive interactions in neighbouring downstream regions that include response elements for STAT3 and SP1/SP3. In FDLE cells, the loss of Spry2-DNA interaction that occurred with FGF-10 treatment corresponded with a transient increase in the HIF-1α-binding at this site and also increased VEGF-A expression. This differed in HBE cells, however, where stable knockdown of Spry2 produced similar increases in VEGF-A expression but did not alter HIF-1α or CBP/P300 binding to this region. In FDLE, the CBP/p300 interaction was detected in the neighbouring downstream site and increased with the decline in Spry2 DNA binding. Since STAT3 interacts with this site and is known to modulate HIF-1α signalling and VEGF-A expression  we invesThe 5′ TIS region of the VEGF-A promoter is flanked by GC rich sequences whose methylation may regulate transcription of the VEGF-A gene. We were unable to reliably detect Spry2 interactions in this region using ChIP and so high resolution melt analysis  was used


Transcriptome analyses show a surprisingly large proportion of the mammalian genome is transcribed; much more than can be accounted for by genes and introns alone. Most of this transcription is non-coding in nature and arises from intergenic regions, often overlapping known protein-coding genes in sense or antisense orientation. The functional relevance of this widespread transcription is unknown. Here we characterize a promoter responsible for initiation of an intergenic transcript located approximately 3.3 kb and 10.7 kb upstream of the adult-specific human β-globin genes. Mutational analyses in β-YAC transgenic mice show that alteration of intergenic promoter activity results in ablation of H3K4 di- and tri-methylation and H3 hyperacetylation extending over a 30 kb region immediately downstream of the initiation site, containing the adult δ- and β-globin genes. This results in dramatically decreased expression of the adult genes through position effect variegation in which the vast majority of definitive erythroid cells harbor inactive adult globin genes. In contrast, expression of the neighboring ε- and γ-globin genes is completely normal in embryonic erythroid cells, indicating a developmentally specific variegation of the adult domain. Our results demonstrate a role for intergenic non-coding RNA transcription in the propagation of histone modifications over chromatin domains and epigenetic control of β-like globin gene transcription during development. HBE, HBG1, HBG2, HBD and HBB) are arranged in the order of their developmental expression in erythroid cells. The locus includes a locus control region (LCR) [HBE gene, which acts as a long-range regulatory element that physically interacts with the active globin genes during transcription [A staggering proportion of the mammalian genome is transcribed. More than half of the transcribed genomic regions in mammalian cells are intergenic regions –3 producon (LCR) , locatedcription , 8.Hox clusters [HBD and HBB. Interestingly, a long-range chromatin interaction between the LCR and this region was recently revealed by 5C analysis of the HBB locus, and analysis of the various naturally occurring deletion of this region, results in defective HBG silencing, supporting the suggestion that the intergenic promoter has regulatory potential. In patients carrying the Corfu deletion, HBG transcription persists in adult erythroid cells where it effectively competes with the adult HBB gene for the activating effects of the LCR [HBG genes in a region implicated in HbF control. This tight correlation between non-coding transcription domains and domains of increased chromatin accessibility suggested that transcription through chromatin might propagate remodelling or histone modifying activities over large regions. This view is supported by findings of histone acetyltransferases (HATs) and methyltransferases associating with the elongating form of RNA polymerase II and the increasing number of studies linking transcription with simultaneous histone modification. However, in other instances the ncRNAs themselves have been implicated in recruiting chromatin modifying activities to chromatin to affect gene expression [Our previous studies  have shoclusters , 12, corclusters –16 at alclusters , 17, conclusters . The anaclusters  encompas the LCR . Analysipression –23. We ppression , 10, sugloxP sites. We show that alteration of intergenic promoter activity results in an adult stage-specific reduction in intergenic transcription and a domain-wide, markedly diminished histone modification profile compared to the normal active locus in transgenic mice, leading to dramatically reduced expression of the adult β-like globin genes.We hypothesized that regulation of chromatin structural domains plays a key role in control of developmental globin gene expression and that intergenic transcription is required for domain remodeling and subsequent gene activation. To discriminate between cause and effect, as well as effects of other potential regulatory sequences in the larger 2.5kb deletion that was previously analyzed, we generated transgenic mice carrying a 213 kb β-YAC transgene with a 300 bp sequence, corresponding to the putative δβ promoter and transcriptional start site of the adult intergenic transcript, flanked by HBD gene in transgenic mice [Apo to Bst11). In addition, the region from –150 to 0 bp contains putative transcription factor binding sites indicative of possible promoter function. We conclude that the region has promoter activity and that a minimal promoter containing the first few hundred base pairs upstream of the start site retains promoter activity in stably transfected cells.We previously showed through primer extension and 5’RACE that intergenic transcripts from the 5’ region of the adult-specific β-globin domain appeared to initiate from a discrete site approximately 3.3 kb upstream of the nic mice . To deteex vivo model of erythroid development. Nucleated cells were isolated from human peripheral blood and induced to differentiate in a two-phase culture system. During the second phase of growth the induction of HBB expression was measured and changes were observed in cell morphology characteristic of erythroid differentiation (not shown). We and others have previously shown that this is a good model for studying the activation of the adult β-globin domain [HBB expression and lack of hemoglobinization at this stage of differentiation. Over time we saw an increase in signal over the putative intergenic promoter which peaked at around day 7 of erythroid differentiation  sites whereas line FX14 contained only a single loxP site located downstream of the δβ promoter. Of the two ‘floxed’ lines we chose FX115 for further study, based on the fact that it contained a single major band containing regions complimentary to all the tested probes verifying the structural integrity of the transgene. This line was bred with a Cre recombinase expressing transgenic line, Deleter Cre [SfiI fragment  which contains the entire globin locus. This line has previously been shown to express the HBB genes at full levels, equivalent to the endogenous mouse Hbb genes [HBE and HBG at high levels in E10.5 embryonic red cells. RNA FISH analysis shows that embryonic expression of HBE and HBG is normal for lines FX115 and Δ115  [HBE and HBG genes is normal and non-variegated in these lines and is not affected by the intergenic δβ promoter deletion, implying position independent transgene expression provided by a fully functioning LCR.To ascertain the effect of the intergenic promoter deletion on globin gene expression we used RNA FISH to study the transcriptional regulation of the globin genes throughout development. Two wild type YAC transgenic lines with equivalent copy numbers were used for comparison. Line FX14 contains 2 copies of the same human beta-globin YAC as the floxed and deleted lines but has only a single bb genes  and has bb genes . Transgeand Δ115  with app signals . This ded (>90%) . Line FXHBB gene was reduced 2-3-fold in the FX101 and FX115 lines in which the δβ intergenic promoter is still present with flanking loxP sites (HBB compared to FX115. Only 5.8% (n = 431) of the erythroid cells were positive for an HBB transcription signal in Δ115, approximately 6-fold lower than FX115 and 15-20-fold lower than the wild type lines. HBB transcription in both wild type lines were similar to the endogenous Hbb locus, in which >90% of β-globin loci are positive for transcription signals [HBB in the intergenic promoter deletion line Δ115 shows that the 300bp sequence contains an element essential for normal high-level expression of the adult β-globin genes. This was confirmed by RT-PCR analysis, which showed that expression of both adult-type beta-genes; HBB and HBD are dramatically reduced in the deletion line compared to the wild type and floxed lines  cells. EGFP expression was measured in neo-resistant, transfected populations by flow cytometry. The wild type δβ intergenic promoter drove consistent, low-level expression of the EGFP reporter in a very high percentage (66%) of stably transfected cells, compared to the potent CMV promoter/enhancer combination that we used as a positive control . The CMVHBB in most erythroid cells is due to an instability of transcriptional activity, i.e. oscillating between the ON and OFF states in every cell, or the result of variegation of expression in which only a subset of cells stably express the HBB transgene, we performed RNA FISH with exonic HBB probes which detect both primary transcripts in the nucleus and processed HBB mRNAs in the cytoplasm[HBB primary transcript signal in the nucleus had spliced HBB mRNA transcripts in the cytoplasm and vice-versa  never switch on or activate the human beta-globin transgene locus [HBD and HBB genes.In order to determine whether the absence of transcription of cytoplasm. We obsersa Figs  and 4D. ne locus . These fHBD and HBB genes failed in the absence of intergenic transcription [HBB and HBD genes, we sought to investigate the effect on histone modifications over this domain. We performed chromatin immunoprecipitations (ChIP) with antibodies directed against histone tail modifications characteristic of active chromatin followed by real time PCR to assess the effect of the deletion on the histone modification profile of the transgene locus in adult erythroid cells.We previously showed that YAC transgenes with a much larger (2.5 kb) δβ promoter deletion resulted in a 2- to 3-fold reduction in general DNase I sensitivity over the entire adult chromatin domain indicating that the normal, stage-specific chromatin opening of the domain surrounding the cription . In mostcription . Having HBD gene, extending over 30 kb downstream to 3’ HS1 which may correspond to the 3’ boundary of the adult domain [HBB transgene locus (264W) [The ChIP data shows that deletion of the δβ promoter results in a dramatic reduction of H3K4 di- and tri-methylation and H3 hyper-acetylation across the entire adult domain , from apt domain . The decs (264W) . ReductiHBE and HBG, implying full LCR function in primitive and early definitive erythroid cells. Normal, high level expression of these genes was confirmed by RT-PCR analysis. These results suggest some form of activation cooperativity or co-dependency occurs between the LCR and active gene domains in the YAC transgene.A surprising finding from these experiments was that the δβ promoter deletion also resulted in a significant reduction in active histone modifications throughout the LCR domain. The LCR was initially described as a dominant control element capable of chromatin activation at any integration site in the genome. Those early studies used constructs with genes directly linked to the LCR rather than at long range and normal domain context. Our results show that the LCR, though fully active in embryonic cells, is not capable of maintaining an active profile in adult erythroid cells in the absence of an active gene domain within the locus. RNA FISH in Δ115 embryonic and early fetal liver cells  shows noHBD and HBB genes. We show that deletion of a 300 bp sequence that contains the δβ promoter results in dramatic alterations to the normal active histone modification profile over a 30 kb downstream region in YAC transgenic mice. As a result, expression of the adult-specific HBD and HBB genes is severely compromised in erythroid tissues. Our data show that intergenic promoter activity is knocked down by insertion of loxP sites flanking the δβ promoter, with a corresponding knock down of adult gene expression. It is not clear why the inserted loxP sites cause this knockdown of promoter activity. One possibility is that the upstream loxP site disrupts a critical cis element needed for full intergenic promoter activity. A search for potential transcription factor binding sites indicates that the upstream loxP sequence disrupts a potential E-box binding site and could potentially bind TAL1 and/or E47. Transgenic line FX14, which has only one loxP site in the downstream position, does not suffer from a decrease in intergenic promoter activity, further implicating the upstream site. However, deletion of the region containing this putative binding site results in a doubling of the number of transfected cells that express the EGFP reporter gene in stably transfected K562 cells  and the δ-globin gene (HBD), including the HBBP1 pseudogene (ψβ-globin) and the BGLT3 non-coding gene that is between HBG2 and HBBP1, contribute to long range interaction [cis-regulatory element that is well downstream of HBBP1 pseudogene, but upstream of the HBD gene.Intergenic transcription could also potentially play a role in nuclear positioning of the beta-globin locus. Studies on the mouse β-globin locus have shown that activation of the locus during erythroid differentiation is LCR independent and involves re-positioning of the locus away from centromeric heterochromatin in correlation with histone hyperacetylation , 46. Theeraction , 52 Our ex-vivo erythroid precursors from patients with a naturally occurring deletion of the δβ promoter helps to illustrate this point [HBD gene. Unlike the situation in transgenic mice, the deletion does not lead to a general shut down of the affected globin locus and LCR. Instead we found that intergenic transcripts appeared to initiate from an intergenic promoter upstream of the fetal-specific HBG genes. This correlated with an open chromatin structure over the HBG genes and the downstream HBB gene, and high-level transcription of both HBG genes and HBB in the adult cell environment. It is tempting to speculate that binding sites for BCL11A or the PYR repressor complex, factors implicated in HBG silencing, are deleted in the larger Corfu mutation [HBG silencing.A note of caution must be added to these studies since the locus under study is a transgene. Incongruent results have often been obtained when comparisons are made between mutations in the human β-globin transgene locus and seemingly similar mutations in the endogenous mouse β-globin locus. Part of this may be due to the well-documented fact that human transgenes in mouse erythroid cells do not exactly recapitulate the regulatory pattern of the endogenous locus. Our analysis of is point . The Cormutation , 54, whi6 cells/ml in phase one media: Stemspan medium (Stemcell Tech), 1μg /ml cyclosporin A (Sigma), 20 ng/ml IL-3 (Peptrotech), 20 ng/ml IL-6 (Peptrotech) and 50 ng/ml stem cell factor (Miltenyi Biotech). These were incubated for 24 hours at 37°C; non-adherent cells were transferred to a new flask and incubated for a further 6 days at 37°C in 5% CO2. The cells were washed in PBS and then seeded at 1x105 cells/ml in phase two media: Stemspan medium, 2U/ml EPO (Espex), 5 ng/ml IL-3, 2x10-6 M dexamethasone (Sigma), 10−6 M β-estradiol (Sigma) and 20 ng/ml stem cell factor. Cells were collected at different time-points of phase two growth to monitor erythroid differentiation and extract RNA using Tri-reagent (Sigma). Differentiation was assessed using qRT-PCR  and May-Grunwald morphological staining.Samples of venous blood (50 ml) were collected from informed and consenting volunteers using EDTA vacuette tubes and spun on Histopaque-1077 (Sigma) to isolate the mononuclear cells. Following six PBS platelet removal washes these were resuspended at 1x10Microarrays were designed by Nimblegen Roche to cover the β-globin locus at high resolution. 100 ng of RNA was treated with DNase (Sigma) before being converted to cDNA using the Transplex complete whole transcriptome amplification kit (Sigma) as per the manufacturer’s guidelines. The cDNA was RNase (Sigma) treated and cDNA precipitated and labelled using the Nimblegen Expression Array kit using the manufacturer’s protocol. The labelled cDNA was hybridised to a single chamber in a Nimblegen 12plex microarray slide following the manufacturer’s guidelines. The slide was washed and scanned at 3 μm resolution using an InnoScan 700 Microarray Scanner and TIFF images were generated using Mapix ver 5.1 software. The TIFF images were aligned and converted into probe intensity values using the Nimblegen DEVA software with background correction and uploaded onto UCSC genome browser.To confirm the peak of transcription over the δβ-promoter region in human erythroid cells quantitative RT-PCR was performed; cDNA was first generated from 150 ng of DNase-treated (Sigma) RNA using the High Capacity RNA-to-cDNA kit (Applied Biosystems). Quantitative PCR was carried out on the BioRad CFX96 QPCR machine using IQ SYBR green supermix (Bio-Rad), 0.5 μM concentration of each primer and 0.9 μl of cDNA. No-RT controls and no-template controls verified the lack of genomic or PCR contamination respectively (not shown). Standard curves for the U-amplicon and D-amplicon were generated by amplifying genomic DNA at a range of concentrations and used to quantify the relative levels of transcription in the region. The ratio of downstream over upstream signal was used to estimate the changing transcriptional pattern during erythroid differentiation.Primer sequences:5’-ATGCCAATGTGGGTTAGAATG-3’Downstream-F (forward primer): 5’-CATACCATGTGGCTCATCCTC-3’Downstream–R (reverse primer): 5’-TTAAATGCCAGTGCTCTCCAC-3’Upstream-F (forward primer): 5’-CAGAAGGAAACTAGGATGTGTCC-3’Upstream-R (reverse primer): For strand-specific RT-PCR of the D and U amplicons cDNA was generated from 150 ng of DNase-treated RNA using the GoScript Reverse Transcription System (Promega) with either the Downstream-F or Downstream-R primer. PCR was then performed using Downstream-F and Downstream-R primers. To assess the RNA integrity an additional cDNA reaction was performed by using random-primers in the RT step.loxP sites upstream and downstream of the minimal δβ intergenic promoter. Briefly, a gene targeting vector was generated by inserting loxP sites at the BseRI and ClaI restriction sites of the 2.5 kb BglII restriction fragment . Transgribed in . After s5’-CTTGAGTGCATTGACAAAATTACC-3’ and 5’-CAGACTTGGACCATGACGGTG-3’. The PCR products were cloned into pGEM-T easy (Promega) and the sequence of the resulting plasmids was verified. The δβ promoter fragments were released by EcoRI/ClaI (264W) or EcoRI/SalI (FX115 and Δ115) digestions, blunted with T4 DNA polymerase (New England Biolabs) and ligated into vector pEGFP-C1  at the SmaI site. Plasmids were verified by sequencing.50 ng of mouse genomic DNA from lines 264W, FX115 and Δ115 were amplified by PCR with primers 7 cells were transfected by electroporation with 50 μg of linearized plasmid DNA at 350 V, 1000 μF in 500μl of 1x Hepes-Buffered Saline. Plasmid pEGFP-N1 (Clontech), expressing EGFP under the control of a CMV promoter, was also used as a positive control. After transfection cells were transferred to non-selective medium for 24 hours followed by 14 days in medium with 600 μg/ml G418. EGFP expression was analysed by FACS performed on a FACSCalibur. Background was determined by analysing non-transfected cells and percentage of green cells as well as mean fluorescence intensity was determined using the CellQuest software.Cells were grown in RPMI 1640 supplemented with 10% foetal calf serum and Penicillin/Streptomycin. 5×10RNA FISH was performed as previously described . CocktaiHBE intron 2 probes:Dinitrophenol-labelled human 5’-AATCTTGAGGACTTTCCCAATCAACTTGCT-3’5’-GTTCTAACATCAAGCTCGACCCATGATTTC-3’5’-CCTTCACTTGGTCACAATACTGATGGGAAA-3’5’-CTGACCTCAAACTGTTCCAAGGTTTGTGCC-3’HBG intron 2 probes:Digoxigenin-labelled human 5’-CGACCTGGACTTTTGCCAGGCACAGGGTCC-3’5’-TCACTCCCAACCCCAGTATCTTCAAACAGC-3’5’-GCATCTTTTTAACGACCATACTTGTCCTGC-3’5’-ACAGAGCTGACTTTCAAAATCTACTCCAGC-3’HBB intron 2 probes:Dinitrophenol-labelled human 5’-TTCCACACTGATGCAATCATTCGTCTGTTT-3’5’-TGTGTACACATATTAAAACATTACACTTTA-3’5’-ATTAGCAATATGAAACCTCTTACATCAGTT-3’5’-AGTAATGTACTAGGCAGACTGTGTAAAGTT-3’Hbb-b1 intron 2 probes:Dinitrophenol-labelled mouse 5’-TCAACACTCCACACACAGTCATGGAGACTG-3’5’-GATATCAGGATGGGAAGTAAATAACCAGCT-3’5’-AAACAAACGTAGATCAAAGAAGAAGAAATG-3’5’-AGCTATGAGAAGAAACAGGGACATATCTTC-3’HBB exon 3 probes:Digoxigenin-labelled human 5’-TTTAATAGAAATTGGACAGCAAGAAAGCG-3’5’-AGGCCCTTCATAATATCCCCCAGTTTAGTA-3’Dinitrophenol-labelled probes were detected with Texas Red-labelled antibodies ; Digoxigenin-labelled probes were detected with FITC-labelled antibodies . Slides were examined on an Olympus BX41 epifluorescence microscope. To determine the percentage of cells with human transgene signals, a minimum of 300 loci were counted for each data point.Total RNA was isolated from adult anaemic spleen 5 days after phenylhydrazine injections  using thThe following primers were used for PCR amplification of cDNA:HBD gene, intron 2 forward primer: 5’-TGGGGATCAGTTTTGTCTAAGATTTGGG-3’HBD gene, intron 2 reverse primer: 5’-GCGGAGAAGAGGTAGGCAGATACATGC-3’HBB gene, intron 2 forward primer: 5’-GACGAATGATTGCATCAGTGTGGAAGTC-3’HBB gene, intron 2 reverse primer: 5’-TGCGGAGAAGAAAAAAAAAGAAAGCAAG-3’5’-TCATCTGGGATTTTGGGGACTATGTC-3’intergenic RT3B forward primer: 5’-CAGACTTGGGAATTGGGATTATACAGGC-3’intergenic RT3B reverse primer: 5’-CTTGTGTTCACGACTGACATCACCG-3’intergenic RT4 forward primer: 5’-GGAGACAAATAGCTGGGCTTCTGTTG-3’intergenic RT4 reverse primer: 5’-GAGTTTGATGACTCTCAGGACAATGCAG-3’mouse carbonic anhydrase II forward primer: 5’-AGATGAGCCCCAGTGAAAGTGAAAC-3’mouse carbonic anhydrase II reverse primer: 7 cells/ml in ice cold RSB buffer , 0.1% Triton X-100, 0.5 mM DTT, 0.1 M sucrose, 0.1 mM PMSF, 5 mM Na butyrate, supplemented with protease inhibitor cocktail (Sigma). The cells were homogenised in a cold B-type Dounce homogeniser and diluted with an equal volume of the same buffer with 0.25 M sucrose. The suspension was layered onto a sucrose cushion of 0.33 M sucrose, 5 mM MgCl2, 10 mM Tris pH 8.0, 0.5 mM DTT, 0.1 mM PMSF, 5 mM Na butyrate. This was then centrifuged at 800 × g for 5 min at 4°C to pellet nuclei. Chromatin was digested with micrococcal nuclease generating DNA predominantly mononucleosomal in length. ChIP was carried out using the following rabbit polyclonal antibodies: anti-trimethyl-histone H3 (K4) (Abcam), anti-dimethyl-histone H3 (K4) (Upstate Biotechnology), anti-acetyl-histone H3 (K9/K14) (Upstate Biotechnology).Histone modification profiles across the human β-globin locus were assessed by native chromatin immunoprecipitation  and perfT method, Bound/Input = 2(Input Ct–Bound Ct) [The representation of multiple sequences across the human β-globin locus in the Input and Bound fractions was determined by quantitative real-time PCR. Real-time PCR was performed in an ABI PRISM 7000 Sequence Detection System using SYBR green PCR Master Mix (Applied Biosystems). All PCR reactions were carried out in duplicates using 3 ng Input or Bound DNA as template. For primer sequences, see Miles et al. . The ratound Ct) . Data weS1 FigGγ-globin; HBG1 = fetal Aγ-globin; HBBP1 = ψβ-globin pseudogene; HBD = adult δ-globin; HBB = adult β-globin) following labelling and hybridisation of RNA extracted from primary erythroid cells at different phases  of phase II culture. The results shown are representative of five biological replicates.The line below the microarray data is a schematic diagram of the human β-globin locus. Gray boxes represent globin genes; the white box represents the locus control region (LCR); and the vertical line represents the location of the δβ promoter region (see (PDF)Click here for additional data file.


Nitric oxide (NO) is a key signaling molecule in all kingdoms. In plants, NO is involved in the regulation of various processes of growth and development as well as biotic and abiotic stress response. It mainly acts by modifying protein cysteine or tyrosine residues or by interacting with protein bound transition metals. Thereby, the modification of cysteine residues known as protein S-nitrosation is the predominant mechanism for transduction of NO bioactivity. Histone acetylation on N-terminal lysine residues is a very important epigenetic regulatory mechanism. The transfer of acetyl groups from acetyl-coenzyme A on histone lysine residues is catalyzed by histone acetyltransferases. This modification neutralizes the positive charge of the lysine residue and results in a loose structure of the chromatin accessible for the transcriptional machinery. Histone deacetylases, in contrast, remove the acetyl group of histone tails resulting in condensed chromatin with reduced gene expression activity. In plants, the histone acetylation level is regulated by S-nitrosation. NO inhibits HDA complexes resulting in enhanced histone acetylation and promoting a supportive chromatin state for expression of genes. Moreover, methylation of histone tails and DNA are important epigenetic modifications, too. Interestingly, methyltransferases and demethylases are described as targets for redox molecules in several biological systems suggesting that these types of chromatin modifications are also regulated by NO. In this review article, we will focus on redox-regulation of histone acetylation/methylation and DNA methylation in plants, discuss the consequences on the structural level and give an overview where NO can act to modulate chromatin structure. S-Nitrosylated nuclear proteins have been identified using the biotin switch technique, which labels S-nitrosylated proteins with a biotin linker allowing detection, purification, and identification of these proteins  oxidize arginine to citrulline, thereby releasing NO. Although NOS-like activities have been measured in chloroplasts and peroxisomes of higher plants , NO syntar pores . Moreoveproteins . In mammproteins . Nuclearbidopsis .Pseudomonas syringae in Arabidopsis leaves, and is required for SAR induction in tobacco  make it highly multifunctional. Whereas some studies report toxic and harmful action of NO species, such as cell death , damage  tobacco . Moreove tobacco . Althoug2 into ortho carbons of aromatic ring of tyrosine residues that leads to the formation of 3-nitrotyrosine , cells of multicellular organisms are identical. However, each cell differs from others through the differences in gene expression pattern that might occur in a temporally and spatially-dependent manner. Genes might be silenced/switched off and switched on again only when they are required. Such an activation/inactivation can be regulated through a direct control of regulatory elements on gene promoters. Moreover, over the last few decades, it was found that affecting the accessibility of the DNA by modification of the chromatin structure, is also a key regulator of transcription.Genetic material of all eukaryotic organisms has to be packed into the nucleus to prevent it from becoming damaged. Since the length of eukaryotic DNA is far greater than the diameter of a nucleus, it has to be organized in a very tightly packaged structure, known as chromatin. The core subunit of chromatin is an octamer, which is composed of two copies of the histone proteins, H2A, H2B, H3, and H4, which are positively charged and enable an electrostatic interaction with negatively charged DNA. 145–147 bp of DNA are wrapped around a histone complex forming repeating nucleosomal units, which are connected with each other by short DNA fragments called “linker DNA.” Linker histone H1 is located between the nucleosomes and stabilizes chromatin structure, resulting in highly condensed 30 nm fibers .Chromatin structure in eukaryotic organisms is very dynamic, and can be changed during growth and development and in response to environmental stimuli. Chromatin marks are able to induce chromatin remodeling and therefore to control important molecular processes such as gene transcription, replication, repair, and recombination . DNA metHistone modifications play an important role in the regulation of chromatin structure and in subsequent gene transcription. N-terminal histone tails, which are exposed outside the nucleosome may interact with neighboring nucleosomes and therefore manipulate the chromatin structure . HistoneArabidopsis , HDT2 (HD2B), HDT3 (HD2C), and HDT4 (HD2D). These consist of an N-terminal domain that has a conserved pentapeptide MEFWG region, which is part of a gene repression activity  strongly diminished the abundance of these histone mark  class disease resistance proteins and the transcription factors WRKY27, WRKY53, TGA2 and TGA5  are sensitive to oxidation; however, the physiological function of this modification is still not understood . Treatmeone mark . Since, and TGA5 . Plant pand TGA5 . WRKY trbidopsis . WRKY53 nse gene . In sum,nse gene .Arabidopsis HDA6). Additionally, Cys325 of Arabidopsis HDA6 is highly conserved within the plant HDa6 and HDA19. The cysteine residues, which are targeted by NO in human HDA2 (Cys262 and Cys274) are located within the region, which is conserved in plant HDA6 and HDA19  based on the available crystal structure of HDA2 revealed a strikingly similar 3D-fold of these proteins, where these two conserved cysteines are located close to the substrate binding site at the same positions , responsible for CHH methylation at pericentromeric heterochromatin  and DEMETER-like protein 2 (DML2)   are diffa2noa1-2 . PRMT1b,a2noa1-2 . Anothersplicing . PRMT5 ia2noa1-2  and posia2noa1-2 . Regardia2noa1-2 .via differential expression of DNA-methyl modifiers. However, modulation of their activity by NO-based post-translational modifications cannot be excluded.NO-dependent changes in DNA-methylation have been described in rice plants exposed to 0.5 mM NO donor sodium nitroprusside . The treArabidopsis plants  3, KDM5, JMJD6, and Putative-Lysine-Specific Demethylase (PKDM) 11, and PKDM13 subfamilies present in plants, animals, and fungi. Other subfamilies are detected only in plants and animals (PKDM12) or in animals and fungi but not in plants (KDM2 and KDM4). PKDM7-9 are plant-specific groups. The existence of Jumonji C-containing histone demethylases in plants suggested that at least some might be regulated by NO, similar the ones described in mammals.Methylation of histones and DNA can be regulated on two main levels–on the level of methyltransferases and de-methylases, which are catalyzing the methylation/de-methylation reactions, and on metabolite level. S-Adenosylmethionine (SAM) is the major methyl group donor in the cell. DNA and histones are subject to methylation by specific SAM-dependent methyltransferases. Each transfer of a methyl-group generates S-adenosylhomocysteine (SAH), which is cleaved into homocysteine and adenosine (Ado) by S-adenosylhomocysteine hydrolase (SAHH) . It is kFor example, AtSAHH1 gene resulted in reduced cytosine methylation and release of transcriptional gene silencing  SAHH. This modification decreased the catalytic activity of SAHH (In rocesses . S-Nitrostigated . Beside  of SAHH . The resIn sum, there are different levels, where NO can affect chromatin modulation, for instance transcription and activity of chromatin modifiers or the supply of methyl group donors or methylation inhibitors (summarized in Finally, the gained knowledge could be subjected to genetic/epigenetic engineering or classical breeding to improve plant traits permanently.AA-K, ER and CL wrote the review article. CL coordinated the completion of the article.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The importance of transcription factors (TFs) and epigenetic modifications in the control of gene expression is widely accepted. However, causal relationships between changes in TF binding, histone modifications, and gene expression during the response to extracellular stimuli are not well understood. Here, we analyze the ordering of these events on a genome-wide scale in dendritic cells in response to lipopolysaccharide (LPS) stimulation.Stat1−/− cells.Using a ChIP-seq time series dataset, we find that the LPS-induced accumulation of different histone modifications follows clearly distinct patterns. Increases in H3K4me3 appear to coincide with transcriptional activation. In contrast, H3K9K14ac accumulates early after stimulation, and H3K36me3 at later time points. Integrative analysis with TF binding data reveals potential links between TF activation and dynamics in histone modifications. Especially, LPS-induced increases in H3K9K14ac and H3K4me3 are associated with binding by STAT1/2 and were severely impaired in While the timing of short-term changes of some histone modifications coincides with changes in transcriptional activity, this is not the case for others. In the latter case, dynamics in modifications more likely reflect strict regulation by stimulus-induced TFs and their interactions with chromatin modifiers.The online version of this article (10.1186/s13059-018-1524-z) contains supplementary material, which is available to authorized users. Epigenetic features, such as histone modifications and DNA methylation, are thought to play a crucial role in controlling the accessibility of DNA to RNA polymerases. Associations have been found between histone modifications and both long-term and short-term cellular processes, including development, heritability of cell type identity, DNA repair, and transcriptional control , 2. For Transcription factors (TFs) are key regulators in the control of epigenetic changes , 11. DurAs sentinel cells of the innate immune system, dendritic cells (DCs) are well equipped for detecting the presence of pathogens. Lipopolysaccharide (LPS), a component of the cell wall of Gram-negative bacteria, is recognized by DCs through the membrane-bound Toll-like receptor 4 (TLR4), resulting in the activation of two downstream signaling pathways . One patStat1−/− cells. Together, these results suggest that stimulus-induced dynamics in a subset of histone modifications reflect the timing of activation of stimulus-dependent TFs, while others are more closely associated with transcriptional activity.Here, we present a large-scale study of short-term changes in histone modifications in mouse DCs during the response to LPS. We focused on the timing of increases in histone modifications at promoters and enhancers, relative to the induction of transcription and to TF binding events. We observed that LPS stimulation induced increased levels of H3K9K14ac, H3K27ac, H3K4me3, and H3K36me3 at LPS-induced promoters and enhancers. Surprisingly, we observed clearly distinct patterns: accumulation of H3K9K14ac was early (between 0.5 and 2 h after stimulation), regardless of the timing of transcriptional induction of genes. Accumulation of H3K36me3 was late and spreads from the 3′ end of gene bodies towards the 5′ end, reaching promoters at later time points (between 8 and 24 h). H3K4me3 accumulation was later than that of H3K9K14ac (between 1 and 4 h) and was more correlated with transcriptional induction times. Integrated analysis with genome-wide binding data for 24 TFs revealed possible associations between increases in H3K9K14ac and H3K4me3 and binding by RelA, Irf1, and especially STAT1/2. LPS-induced accumulation of H3K9K14ac and H3K4me3 was severely impaired in To elucidate the temporal ordering of stimulus-induced changes in transcription and chromatin structure, we performed chromatin immunoprecipitation experiments followed by high-throughput sequencing (ChIP-seq) for the following histone modifications in mouse DCs before and after LPS stimulation: H3K4me1, H3K4me3, H3K9K14ac, H3K9me3, H3K27ac, H3K27me3, H3K36me3, and similarly for Pol2   and 34,079 enhancers , possibly reflecting differences between DC- and macrophage-specific enhancers and the molecular processes that define these cell types.Recent studies using the same cell type and stimulus showed that most changes in gene expression patterns were controlled at the transcriptional level, without widespread changes in RNA degradation rates , 26. We LPS-induced promoters were less frequently associated with CpG islands (57%) than stably expressed promoters  or at stably active promoters.However, the accumulation of histone modifications showed more varying patterns Fig. –g. IncreIncreases in H3K4me3 were concentrated between 1 and 4 h after stimulation Fig. . In contFinally, H3K36me3 was only induced at later time points (between 8 and 24 h), regardless of transcriptional induction times of promoters , TFs with pervasive binding even before stimulation , and E2F1, which has been shown to be recruited by NF-κB through interaction with RelA .Together, these results suggest a strong correlation between increases in activation-associated histone modifications and LPS-induced binding by RelA, IRF1, STAT1, and STAT2.The relative timing of LPS-induced TF binding events and increases in histone modifications can reflect potential causal relationships. Particularly, many LPS-induced promoters show increases in H3K9K14ac between 0.5 and 2 h after LPS stimulation Fig. , and we We observed a particularly strong coincidence in timing between STAT1 binding and increases in H3K9K14ac Fig. . Of the Similar to H3K9K14ac, we observed a general increase in H3K4me3 around STAT1 binding sites Fig. , betweenSimilar patterns were observed for enhancers and promoters bound by STAT2 2 h after stimulation  cells, LPS-induced type I IFN production, activation of the JAK-STAT pathway, and activation of STAT1 and STAT2 target genes are severely impaired [Trif−/− and MyD88−/− DCs, we defined a set of TRIF-dependent genes  showed increases in H3K9K14ac and H3K4me3 in WT but not in KO cells .RT-qPCR and ChIP-qPCR experiments in WT, Ifit1 and Rsad2, and accumulation of H3K9K14ac and H3K4me3 at their promoters , these time frames appear to be non-coincident with the timing of induction of transcription.In our dataset, we roughly observed three patterns of modifications. The first was early induction of H3K9K14ac, which occurs mainly in the first 2 h after stimulation. A second pattern consisted of increases in H3K4me3 and H3K27ac, roughly coinciding with induction of transcription. Finally, a third pattern consisted of changes in H3K36me3, occurring only around 8–24 h after stimulation. Although H3K4me3 is widely used as a marker for active genes, the functional role of this modification is still unclear. For example, the deletion of Set1, the only H3K4 methyltransferase in yeast, resulted in slower growth than in wild type but otherwise appears to have only limited effects on transcription . Other sTnf, Il6, and Il12b has been reported, resulting in enhanced TF and Pol2 recruitment after subsequent TLR4 activation [Since the induction of remodeling appears to occur specifically at LPS-induced genes, it is likely that histone modifiers are recruited by one or more LPS-activated TFs to specific target regions in the genome defined by the binding specificity of the TFs. In this model, primary response regulators could control immediate stimulus-induced changes in transcription and histone modifications, while the later “waves” could depend to different degrees on 1) the process of transcription itself, (2) subsequent activation of secondary regulators, and (3) the presence of other histone modifications Fig. . This fitivation . These p the proctivation . Such “nAlthough many studies have compared histone modifications before and after stimulation, most lack sufficient time points and resolution to allow analysis of temporal ordering of changes. One recent study in yeast reported results that are partly similar to ours : specifiOur time series ChIP-seq data and analysis present a first genome-wide view of the timing and order of accumulation of histone modifications during a stress response in mammalian immune cells. The stimulus-induced accumulation of H3K9K14ac, H3K4me3, H3K27ac, and H3K36me3 followed distinct patterns over time. Integrative analysis suggests a role for STAT1/2 in triggering increases in H3K9K14ac and H3K4me3 at stimulus-dependent promoters and enhancers. Differences in interactions between histone modifiers, TFs, and the transcriptional machinery are likely causes for the different patterns of dynamics in histone modifications.Stat1-deficient mouse was described previously [Bone marrow cells were prepared from C57BL/6 female mice and were cultured in RPMI 1640 supplemented with 10% of fetal bovine serum under the presence of murine granulocyte/monocyte colony-stimulating factor  at the concentration of 10 ng/mL. Floating cells were harvested as bone marrow-derived dendritic cells (BM-DCs) after 6 days of culture with changing medium every 2 days. The cells were stimulated with LPS  at the concentration of 100 ng/mL for 0, 0.5, 1, 2, 3, 4, 6, 8, 16, and 24 h and were subjected to RNA extraction or fixation. Murine IFN-β was purchased from Pestka Biomedical Laboratories and was used to stimulate the cells at the concentration of 1 × 10^2 unit/mL. TRIF-, IRF3-, or IFNR-deficient mice have been described previously –53. Stateviously .For each time point, thirty million BM-DCs were stimulated with LPS and subjected to fixation with addition of 1/10 volume of fixation buffer . The cells were fixed for 10 min at room temperature, and immediately washed with PBS three times. ChIP and sequencing were performed as described . Fifty microliters of lysate after sonication was aliquoted as “whole cell extract” (WCE) control for each IP sample. Antibodies used were Pol2 , H3K4me3 , H3K9K14ac , H3K36me3 , H3K9me3 , H3K27me3 , H3K4me1 , and H3K27ac .One million BM-DCs were stimulated with LPS for indicated times and subjected to RNA extraction by using TRIzol (Invitrogen) according to the manufacturer’s instruction. RNAs were reverse transcribed by using RevaTra Ace (Toyobo). The resulting cDNAs were used for qPCR by using Thunderbird SYBR master mix (Toyobo) and custom primer sets  samples using Bowtie2 (version 2.0.2), using the parameter “very-sensitive,” against the mm10 version of the mouse genome . ProcessWe predicted peaks for each time point using MACS (version 1.4.2) , using eIn a next step, we counted the number of reads mapped to each region at each time point for both the IP samples and WCE control samples. Using these counts, we performed a read count correction, as described by Lee et al. . BrieflyFinally, the corrected read counts were converted to reads per kilobase per million reads (RPKM) values (using read counts and the lengths of each region) and normalized using quantile normalization, under the assumption that their genome-wide distribution does not change substantially during each time series. The normalized RPKM values were converted to reads per million reads (ppm) values.TSS-seq data for BM-DCs before and after stimulation with LPS was obtained from the study by Liang et al.   for each window size. For overlapping regions with significantly high read counts, the region with the lowest associated FDR was retained.In order to remove potentially noisy TSSs, we removed TSSs that were located within 3′ UTRs and TSSs located > 50 kb upstream of any known gene. For the remaining TSSs, we used a simple model . Together, TSS-seq-based TSSs and Refseq-based TSSs resulted in a total of 24,416 promoter regions.CpG-associated promoters were defined as those having a predicted CpG island (from the UCSC Genome Browser Database) in the region − 1 to + 1 kb surrounding the TSS . Other pEnhancers were defined based on the signals of H3K4me1 and H3K4me3. First, we collected all genomic regions with significantly high levels of H3K4me1 see the “” sectionEnhancers were naively assigned to the nearest promoter (TSS-seq based or Refseq-based) that was < 150 kb separated from it (center-to-center). For 30,448 enhancers (89%), a promoter could be assigned.Genome-wide binding data (ChIP-seq) is available for mouse DCs before and after stimulation with LPS, for a set of 24 TFs with a known role of importance and/or high expression in DCs  (GEO accPromoters (region − 1 to + 1 kb around TSS) and enhancers (entire enhancer region or region − 1 to + 1 kb around the enhancer center for enhancers < 2 kb in size) were considered to be bound by a TF if they overlapped a ChIP-seq peak with a significantly high peak score. New binding events by a TF at a region were defined as time points with a significantly high score where all previous time points lacked significant binding.In order to analyze induction times of increases in histone modifications and Pol2 binding, we defined the induction time of a feature as the first time point at which a significant increase was observed compared to its original basal levels (at 0 h). Significant increases were defined using an approach similar to methods such as by DESeq and voom , 62, whidx, f, sx, f, and mx, f represent the dispersion, standard deviation, and median of feature f in region x over the 10 time points of the time series, respectively. Fitting a second-order polynomial function on the log as a function of log for all promoter and enhancer regions, we obtained expected dispersion values in function of median ppm value .The values of all histone modifications, Pol2, RNA-seq, and TSS-seq reads  were collected for all promoters (region − 1 to + 1 kb) and enhancers (entire enhancer region or region − 1 to + 1 kb around the enhancer center for enhancers < 2 kb in size). For each feature , we calculated the median and standard deviation in ppm values for each region, over the 10 time points. Dispersion was defined as follow:For the analysis of induction times of H3K9K14ac, H3K4me3, and H3K36me3 at enhancers in function of their pre-stimulation basal levels . For each of the thus obtained 1413 LPS-induced promoters, the transcription induction time was defined as the first time point for which Zx, TSS − seq, t ≥ 2 was observed. Unchanged promoters were defined as those promoters having absolute values of Zx, TSS − seq, t < 1 for all time points, leading to 772 promoters.LPS-induced promoters were defined using TSS-seq ppm values. LPS-induced promoters should have Trif−/− mice and Myd88−/− mice.RNA-seq data for mouse BM-DCs treated with LPS were obtained from the study by Patil et al.   and Bowtie2 (version 2.0.2) , 64. MapTrif−/− RNA-seq data and in the Myd88−/− RNA-seq data. TRIF-dependent genes were defined as genes for which the fold-induction was more than five times lower in the Trif−/− data than in WT, leading to 141 TRIF-dependent genes  treated with LPS , including TSS-seq, and ChIP-seq (H3K9K14ac and H3K4me3) data as described above. Data was processed in the same way as the original time series dataset see the “” sectionWe used Fisher’s exact test to evaluate the significance of differences between induced and non-induced promoters and enhancers Fig. , the sigAdditional file 1:Contains supplementary results, supplementary methods, supplementary tables, and supplementary figures. (PDF 5904 kb)


Microarray and DNA-sequencing based technologies continue to produce enormous amounts of data on gene expression. This data has great potential to illuminate our understanding of biology and medicine, but the data alone is of limited value without computational tools to allow human investigators to visualize and interpret it in the context of their problem of interest.We created a web server called SHOE that provides an interactive, visual presentation of the available evidence of transcriptional regulation and gene co-expression to facilitate its exploration and interpretation. SHOE predicts the likely transcription factor binding sites in orthologous promoters of humans, mice, and rats using the combined information of 1) transcription factor binding preferences (position-specific scoring matrix (PSSM) libraries such as Transfac32, Jaspar, HOCOMOCO, ChIP-seq, SELEX, PBM, and iPS-reprogramming factor), 2) evolutionary conservation of putative binding sites in orthologous promoters, and 3) co-expression tendencies of gene pairs based on 1,714 normal human cells selected from the Gene Expression Omnibus Database.www.garuda-alliance.org), where it joins other popular gadgets such as the CellDesigner pathway analyzer and the Reactome database, as part of analysis workflows to meet the growing needs of molecular biologists and medical researchers. SHOE is available from the following URL http://ec2-54-150-223-65.ap-northeast-1.compute.amazonaws.comSHOE enables users to explore potential interactions between transcription factors and target genes via multiple data views, discover transcription factor binding motifs on top of gene co-expression, and visualize genes as a network of gene and transcription factors on its native gadget GeneViz, the CellDesigner pathway analyzer, and the Reactome database to search the pathways involved. As we demonstrate here when using the CREB1 and Nf-κB datasets, SHOE can reliably identify experimentally verified interactions and predict plausible novel ones, yielding new biological insights into the gene regulatory mechanisms involved. SHOE comes with a manual describing how to run it on a local PC or via the Garuda platform (https://www.youtube.com/watch?v=qARinNb9NtEA video demonstration of SHOE can be found here: The online version of this article (10.1186/s12864-018-5101-3) contains supplementary material, which is available to authorized users. The analysis of gene regulatory regions is a centrally important problem in biology. Many experimental –3 and coIdeally a computer program could automatically and reliably combine all available evidence, but this has not yet been achieved. While many excellent motif discovery tools, such as CONSENSUS , the GibSHOE takes a different approach; it tries to provide human biology experts with an interactive, visual presentation of the available evidence to facilitate the exploration and interpretation of transcription regulation analysis results. We demonstrate the SHOE analytical workflow by analyzing the CREB1 and Nf-κB datasets.SHOE consists of a server application and front-end interface. The server application and its database run on Vagrant. Vagrant is virtualization software that enables the setting up of a software environment on any platform such as Windows or OS X. The server application is implemented in PHP 5, which is one of the most well-known Web application languages. The SHOE server connects and stores analysis data in a MySQL database. Some of the back-end programs are implemented in C language, Perl, and shell script. The client-side application is written in HTML and JavaScript.http://www.celldesigner.org). Garuda is an open platform that provides a framework to connect, discover, and navigate through different applications in the fields of biology and medicine [GeneViz is a network visualization application created for SHOE. It shows multiple graphs of genes and transcription factors in one window. Users can compare common or different elements on these graphs and search for genes on them. GeneViz is written in JavaScript and is seamlessly integrated with SHOE, which can easily import data from it. SHOE also provides a gadget for the Garuda platform implemented in Java and installed as a plugin for CellDesigner  by using the SEARCH local alignment program ; the exeTo evaluate the degree to which an observed region in an alignment of the three species should contribute to our belief that it is part of a conserved region, we adopted an estimate of the likelihood ratio of observing the region in an alignment of orthologous promoters versus that of observing the region in an alignment of unrelated promoters.ortho table. In the same manner, 1,000 non-orthologous triples were randomly selected, and their promoters of length 5,000 nt were repeatedly aligned. After 1,260 non-orthologous three-species alignments  were obtained with ClustalW, the frequency of each possible alignment column was observed and stored as a random alignment frequency in a table.To obtain this estimate, first, we randomly selected 1,000 orthologous triplets  and repeatedly aligned their orthologous promoters of length 5,000 nt. After 835 orthologous three-species alignments  were obtained with ClustalW, the frequency of each alignment column was observed and stored in an orthologous alignment frequency table, i.e., an We refer to the obtained alignment column frequencies as the “ortho” alignment and “random” alignment frequencies, respectively. Using those column frequency tables, we define the multiple alignment (MA) scores using the following formula:c is the probability of the observed pattern in each column , and m is the length of the alignment region, which for our application is equal to the motif length m.where m to evaluate the degree of similarity between the identified conserved sequence and known transcription factor binding sites, SHOE uses its motif-scoring module, which calculates similarity scores with matrices in public databases such as Transfac32 (492 matrices) [After multiple alignment scores have been calculated for the human-mouse-rat regions of length atrices) , Jaspar atrices) , HOCOMOCatrices) , ChIP-seatrices) , PBM (30atrices) , and iPSatrices) , which wThe PSSM-related score is calculated using the following formula:pseudocount = 1, and m is the motif length.where To represent the scores as positive values, the PSSM score is subtracted from 10.Since SHOE identified putative TF binding sites via two sources of information , we decided to apply the Pareto-optimal front method to optimize this multi-objective solution . The proi,yi), where xi is the MA score and yi is the PSSM score, is calculated byThe Pareto number of  , which iwhere K is the number of genes with a match to motif m in the gene set of interest, N is the total number of genes in the gene set, and p0 is the frequency of the same motif in a background set of genes.http://cellmontage.cbrc.jp/) [Considering that genes with similar transcriptional profiles might be affected by the same transcriptional mechanism, we calculated the Pearson correlation coefficients for all gene pairs in the datasets based on 112 “Eigen cell” synthetic expression profiles available from the Cell Montage web site (brc.jp/) .For the reader’s convenience, here we briefly outline whom those Eigen cells were computed for. First, 5000 human cell profiles from the Gene Expression Omnibus database  were clahttp://chip-atlas.org) we selected 1000 human promoters (1000 nt length) as the target genes of the CREB1 transcription factor and 1000 human promoters (1000 nt length) as the target genes of the Nf-κB1 transcription factor. With predicted target genes, gene-interaction maps have been visualized in CellDesigner , ribosomal protein L23 (RPL23), and ribosomal protein S29 (RPS29) are part of the conserved structures or pathways in the DAVID database. RPL23 and RPS29 , which is involved in apoptosis, is induced by Nf-κB in mice . HIF1A iMany Nf-κB target genes are part of the immune system such as the ICAM1 gene. qPCR analysis identified ICAM1 as a target of Nf-κB , and Nf-SHOE predicted another three genes that have not yet been associated with Nf-κB in mice and rats but have been predicted by SHOE: TRIP10, STRN, and GABBR1. Also, several transcription factor candidates were found to potentially co-regulate these genes together with Nf-κB.http://www.uniprot.org).TRIP10 (CIP4), which is involved in insulin signaling and actin reorganization, has been identified as a potential target gene of Nf-κB. TRIP10 (CIP4) is required for the translocation of GLUT4 to the plasma membrane  since enhancer regions are less conserved comparing to promoters, they can have weaker scores in multiple sequence alignment, thus being left out, as in the case of ATP5D gene, when both sides of peak are very well conserved but peak itself is missed because of weaker multiple alignment similarity; b) motifs in peaks are more corrupted/(having more mismatches) with consensus sequence of known binding site, as it is observed by SHOE ; c) some peaks are distantly allocated from the TSS , which means that more sensitive, i.e. partial sequence search directly on those regions might be more appropriate. Despite the above considerations are not included in current SHOE methodology, SHOE is successfully finding ChiP-seq locations in a range of cases thus increasing the confidence of the results that will augment the interest of the user to the software.Through case studies we demonstrate the utility of SHOE in visualizing and exploring potential regulatory interactions involving TFs. The analysis made by SHOE further emphasizes the evolutionary importance of CREB in stress-regulated responses and the regulation of antioxidant enzymes. Moreover, for the example of Nf-kB, 10 out of 13 predicted target genes had evidence in the literature supporting the predictions. Not only known but also novel potential target genes in the insulin pathway and the nervous system were identified, which may give insight into new pathways and how Nf-kB is involved in their regulation. For 11 out of 33 transcription factors, there were studies showing interactions with Nf-kB, portraying SHOE as a strong predictor of not only target genes but also potential transcription factors that might co-regulate target genes. Analysis on the MAPK pathway using the SHOE method have been discussed in a previous study . By idenSHOE is connected to such tools as the CellDesigner pathway editor and analyzer , PercellWe also are considering adding the option to consider tissue-specific data such as ChIP-seq binding data and epigenetic data such as DNA methylation and histone modification.Project name: SHOE: Interactive visual tool for promoter analysis.http://ec2-54-150-223-65.ap-northeast-1.compute.amazonaws.comProject home page: https://www.youtube.com/watch?v=qARinNb9NtEProject demo: Operating system(s): Windows / macOS / Linux.Programming language(s): C, Perl, PHP, JavaScript.Other requirements: Vagrant, VirtualBox.Restrictions for use by non-academics: None.Additional file 1:Figure S1. Pareto front score optimization on SHOE. A) Illustration of the conflict between two scores (MA score and PSSM score) and the concept of dominance; B) Visualization of several groups of scores using Pentachlorophenol response dataset (PCP), PCP_raw (red) denotes random score zone, PCP_ma (purple) denotes zone where MA score is high, PCP_pssm (blue) is the zone where the PSSM score is high, and PCPtrimmedLog (green) is the zone where a trade-off between two scores is taken. C) Example of the analysis with 30 top Pareto fronts with each type of motif shown in different shape and color. (PDF 360 kb)Additional file 2:Figure S2. Trade-off on the number of genes in the dataset basing of motif similarity threshold to the consensus when Pearson correlation thresholds |r| ≥ 0.0 and |r| ≥ 0.6 are applied. (PDF 20 kb)Additional file 3:Figure S3. Visualization of ChiP-seq peaks from ArrayExperss database idenyified in SHOE predictions. A) Demosntrates two genes from overexpressed in mouse liver in Diabet 2 condition; B) Demonstrate TNF genes of mTOR human pathway in which promoter two peaks according ChIP-seq analysis have been identified. (ZIP 499 kb)