Glucose homeostasis is regulated by insulin, which is produced in the β-cells of the pancreas. The synthesis of insulin is controlled by several transcription factors including PDX-1, USF1 and USF2. Both, PDX-1 and USF1 were identified as substrates for protein kinase CK2. Here, we have analysed the interplay of PDX-1, USF1 and CK2 in the regulation of PDX-1 gene transcription. We found that the PDX-1 promoter is dose-dependently transactivated by PDX-1 and transrepressed by USF1. With increasing glucose concentrations the transrepression of the PDX-1 promoter by USF1 is successively abrogated. PDX-1 binding to its own promoter was not influenced by glucose, whereas USF1 binding to the PDX-1 promoter was reduced. The same effect was observed after inhibition of the protein kinase activity by three different inhibitors or by using a phospho-mutant of USF1. Moreover, phosphorylation of USF1 by CK2 seems to strengthen the interaction between USF1 and PDX-1. Thus, CK2 is a negative regulator of the USF1-dependent PDX-1 transcription. Moreover, upon inhibition of CK2 in primary islets, insulin expression as well as insulin secretion were enhanced without affecting the viability of the cells. Therefore, inhibition of CK2 activity may be a promising approach to stimulate insulin production in pancreatic β-cells. Recently, the upstream stimulatory factor USF1 has been identified as a new substrate for CK27. Together with the second member of the USF family, namely USF2, both are involved in the transcriptional regulation of various genes whose gene products are implicated in the stress and immune response, cell cycle regulation, DNA repair and proliferation of cells and in lipid and carbohydrate metabolism12. Only USF1, but not USF2 was phosphorylated by CK2 and the major phosphorylation site was mapped to threonine 1007. Transactivation studies revealed that inhibition of the CK2 phosphorylation of USF1 stimulated the transactivation of some promoters such as the glucokinase promoter and the fatty acid synthetase promoter but not of the heme-oxygenase-1 promoter. Moreover, inhibition of the CK2 phosphorylation of USF1 led to an enhanced binding of USF1 to USF2. In another study it was shown that only a nuclear sub-population of CK2α and CK2β proteins bound to USFs13. One interpretation of these results might be that binding of CK2 to USFs facilitates phosphorylation of nuclear USF1. Another possibility might be that the USFs target CK2 to other substrates in the transcription factor complex in the nucleus. However, CK2 was not found within the transcription factor complex of USF1/USF2 at the DNA.Protein kinase CK2, which is composed of two catalytic α- or α′-subunits and two non-catalytic β-subunits, phosphorylates more than 400 different substrates of the human proteome. Among these substrates are a number of transcription factors whose transactivation factor activity was either enhanced or repressed upon phosphorylation by CK214. PDX-1 binds to its own promoter15 and regulates its expression in an auto-regulatory loop involving USFs bound to the E-box motif within the proximal PDX-1 promoter16. Expression of a dominant negative form of USF2 decreased the binding of USFs to the promoter, which resulted in a lower level of PDX-1 mRNA17. These various results prompted us to study the interplay of USF1 with protein kinase CK2 and within the regulation of the PDX-1 expression in the rat glucose-sensitive pancreatic β-cells (INS-1).Over the last couple of years CK2 was found to regulate another transcription factor, namely PDX-1 which is directly implicated in the regulation of the transcription of the insulin gene in pancreatic β-cellsWe found that PDX-1 and USF1 interact functionally at the PDX-1 promoter in INS-1 cells. The interaction of both proteins and the transcriptional activity are influenced by glucose and by the inhibition of CK2 activity. Both treatments abrogate the transrepressing effect of USF1 on the PDX-1 driven transcription of PDX-1. The measurable impact of CK2 inhibition in primary islets was an enhancement of insulin expression and secretion.7. We have now attempted to find out whether there is an influence of the CK2 phosphorylation on the functions of one or both transcription factors. For the experiments described here, we used the glucose-responsive pancreatic β-cell line INS-1 from rat18.PDX-1 and USF1 are transcription factors deeply involved in the regulation of glucose homeostasis. In addition, PDX-1 is the key transcription factor for the development of the pancreas and for maintaining the integrity of pancreatic β-cells. Both proteins have been described by us as substrates of protein kinase CK2et al. reported a functional interplay of USF1 and PDX-1 at the PDX-1 promoter16. To investigate whether this interaction also occurs in INS-1 cells under our experimental conditions, we first performed a transactivation assay using a luciferase construct under the control of the complete PDX-1 promoter (−6500/+68). In addition to the reporter construct we also transfected varying amounts of FLAG-USF1 and FLAG-PDX-1 or an empty vector as control (mock). When using more PDX-1 than USF1 the reporter assay showed a transactivation of the PDX-1 promoter construct   Fig. . In contF1) Fig. . When peF1) Fig. . Figure F1) Fig. . When us20. Thus, we were first interested in the cellular localization of the transcription factors USF1, USF2 and PDX-1 and protein kinase CK2  and cultivated the starved cells with glucose in concentrations ranging from 0–25 mM for 4 hours. The result of the transactivation experiment is shown in Fig. The transactivation potential of PDX-1 is an important functional determinant for glucose-induced transcription in pancreatic MIN6 β-cellsTo investigate the effect of USF1 on the PDX-1 transcription we simultaneously transfected the PDX-1 reporter along with a FLAG-tagged USF1 construct or the empty vector (mock) using different glucose concentrations. Figure 22 and detected bound nuclear proteins by Western blot  and quinalizarin (Q). We used those inhibitors in concentrations, which had previously been tested and found to inhibit the CK2 activity to similar levelsct. Fig. . CompareTo study the role of USF1 under these conditions we transfected INS-1 cells with the FLAG-USF1 construct along with the PDX-1 reporter construct and treated the cells either with the vehicle control DMSO or with CX-4945, TBB or Q for 24 h. Twenty-four hours after treatment we determined the luciferase activities and compared them with the mock-transfected, untreated control. Figure 24 and might also be affected. Therefore, we used a mutant USF1 where we had exchanged the CK2 phosphorylation site at threonine 100 by alanine. We transfected INS-1 cells with the PDX-1 promoter construct alone or together with the FLAG-tagged USF1 wild-type or USF1T100A phospho-mutant construct. We determined the luciferase activities 48 h after transfection and normalized them to equal amounts of FLAG-USF1 protein as shown in the Western blot analysis in Fig. Although, the inhibitors are quite selective for CK2 there is still the possibility that these inhibitors target other kinases or other molecules in the cell. Moreover, some components of the basal transcription machinery are known targets of protein kinase CK2We now wanted to know whether the reduction in the transrepression of USF1 might be due to altered DNA binding. Therefore, we performed pull-down assays using the PDX-1 probe after treatment of INS-1 with the different CK2 inhibitors. Nuclear extracts from cells grown in the presence of one of the CK2 inhibitors or with DMSO as a control for 24 h were used for DNA pull-down assays. DNA bound PDX-1 and USF1 were detected in a Western blot analysis. Protein amounts from several experiments were quantified by densitometry and presented as bar graph Fig. . The levT100A to 50% compared to wild-type USF1. The DNA binding of PDX-1 is also diminished, however not as strong as for the USF1 mutant  or CX-4945 to inhibit CK2 activity. After treatment, proteins were extracted and CK2 activity was determined with a kinase activity assay. As demonstrated in Fig. lot Fig.  as well lot Fig. . We founupstream stimulatory factors USFs were identified as transcriptional regulators of the adenovirus late promoter26. Two isoforms of USF were isolated as USF1 (43 kDa) and USF2 (44 kDa), which belong to the basic helix-loop-helix leucine zipper (b-HLH-LZ) family of transcription factors28. Like other b-HLH-LZ transcription factors USFs bind to E-boxes in the promoter region where both prefer CACGTGAC sequence elements. There are opposite results about a growth suppressing or pro-proliferating activity of the USFs29. USFs influence cellular proliferation by targeting hTERT, TGFβ2 or IGF2R, cell cycle regulating proteins like cdk4, cyclin B1 or cdk1 or growth suppressor genes like p53, BRCA1 or APC. USFs are stress responsive factors and are active in the tanning and immune response30. Besides these effects on cells, USFs are decisively involved in the regulation of glucose and lipid metabolism. Among the USF1-dependent targets of the glucose metabolism are targets of hormonal regulators such as the insulin or glucagon receptor32, or enzymes of glycolysis like glucokinase33 or L-type pyruvate kinase34. In addition to these targets the USFs were also found to bind to E-boxes within the promoter of the homeobox gene PDX-1, which plays a key role in the development of the pancreas as well as in the maintenance of pancreatic functions and integrity22. In the adult pancreas PDX-1 is responsible for the regulation of the insulin promoter, for GLUT2, as well as for glucokinase expression35. PDX-1 regulates its own transcription in an auto-regulatory loop16. In addition to the binding of the promoter sequences in the PDX-1 gene the homeobox domain of PDX-1 physically interacts with USFs thus, acting as a cofactor recruiting other cofactors, which are necessary for a successful transcription of the PDX-1 gene16. Another example for the co-ordinated action of both transcription factors is the regulation of Alx3 expression in pancreatic β-cells36. Alx3 is another homeodomain protein, which has been described to be an important transcription factor for the maintenance of glucose homeostasis37. Here, we have shown a functional interaction of USF1 and PDX-1 at the PDX-1 promoter in INS-1 cells. We confirmed the previous observation that PDX-1 acts in an auto-regulatory fashion and transactivates its own promoter. We observed, however, that USF1 had a concentration dependent transrepressing effect on the PDX-1 transcription. This observation seems to be opposite to findings of other groups who showed earlier that an adenovirus mediated overexpression of a dominant negative variant of USF2 (USF2ΔB) decreased the binding of both USF proteins to the E-box motif and this decrease led to a decrease in the PDX-1 gene promoter activity17. An explanation for the different result might be that we did not deplete functional USF dimers from INS-1 cells, but we used a controlled over-expression of USF1. Although in most cases USFs are considered as activating transcription factors, there are a few reports that USFs act as transrepressors. This was documented for the human intestinal monocarboxylate transporter 138 and for the catalytic subunit of the glutamate-cysteine ligase (GCLC)39. Sharma et al. also found that USFs were important components of the PDX-1 transactivating machinery22.About 30 years ago the 42. USF1 and USF2 bind to the glucose/carbohydrate response elements (GIRE/ChoRE) that are composed of E-box elements of the CACGTG type which seem to be necessary for the glucose response43. Those elements have been identified in the L-type pyruvate kinase promoter and as demonstrated with USF2−/− mice, binding of USF2 to the E-box elements is absolutely required for its glucose-dependent transcription34. Another glucose response element has been identified in the rat glucagon receptor gene and it was also demonstrated that USF1/2 are part of the glucose response complex32. However, the results concerning the modulation of the binding of USFs to those elements, their transactivation and the expression of USFs depending on the glucose concentration are contradicting and seem to be cell-type and species-specific. Kahn et al.43 postulated that the binding of USFs to the GRE/ChoRE is not modulated by nutritional conditions in liver. In rat mesangial cells USF1 and USF2 bind to the thrombospondin 1 promoter. Glucose stimulates nuclear USF2 accumulation, which results in an enhanced USF2 level and a higher DNA binding activity to the thrombospondin 1 promoter11. The increase in USF2 mRNA and protein expression upon a high glucose stimulus is explained by a glucose-responsive element, which leads to an up-regulation of the CREB-dependent transactivation of the USF2 promoter44.As transcription factors are implicated in the maintenance of glucose homeostasis we expected a glucose dependent regulation of the activity and perhaps of the expression of PDX-1 and USFs. It is known for a long time that glucose influences PDX-1 activity, expression and sub-cellular localization, depending on the amount of glucose either positively or negatively46. Furthermore, elevated glucose concentration attenuated the transrepression of the PDX-1 promoter by USF1. Interestingly, an elevated glucose concentration did not influence the DNA binding activity of PDX-1 whereas binding of USF1 and USF2 to the PDX-1 promoter was reduced. These results are consistent with the role of PDX-1 in the regulation of the insulin production after a glucose stimulus. We have shown that this important function of PDX-1 is not counteracted by the transrepressing effect of USF1. Obviously, an elevated glucose concentration has the same effect as inhibition of the CK2 activity. In both cases the transcription of the PDX-1 gene is elevated, which is a prerequisite for an elevated insulin production.Here, we have shown that glucose increases the transactivation of the PDX-1 promoter by PDX-1 dose-dependently. The glucose-dependent increase in the transcription of PDX-1 was also observed with endogenous PDX-1. With the reporter assay, the strongest effect on transactivation was obtained with 11 mM glucose. With higher concentrations of glucose the effect decreased. This effect is in agreement with a loss of function of PDX-1 under diabetic conditions47 and USFs (for review see ref.29) are phosphoproteins and it is long known that different aspects of their functions can be regulated positively or negatively by phosphorylation. There are several examples that glucose regulates the expression of target genes via kinases or phosphatases. Glucose regulates the APOA5 gene via a dephosphorylation of PP1 and PP2A, which results in a stronger binding of USF1 and USF2 to their promoters48. PKG (cGMP dependent protein kinase) inhibits the glucose regulated transcription of the thrombospondin 1 (TSP1) gene via down-regulation of protein levels and DNA binding activity of the TSP1 promoter binding protein USF2; in contrast glucose leads to an enhanced nuclear accumulation of USF2 via PKC, p38 MAPK and ERK pathways11.It is well known that DNA binding and transcriptional activity of many transcription factors is regulated by phosphorylation/dephosphorylation. Also PDX-149, upstream binding factor UBF50, FoxM1c51, activating transcription factor 4 ATF41 and C/EBPδ6 whereas the transcriptional activity of CHOP is reduced52. The function of a transcription factor can switch from a repressing to an activating function by protein kinase CK2, as it was observed for the transcription factor CTCF53. CK2 plays a role in the hormonal regulation of carbohydrate metabolism and in modulating activities of enzymes involved in carbohydrate storage and metabolism (for review see ref.54). In a recent study we have identified PDX-1 as a CK2 substrate4. We have shown that phosphorylation of PDX-1 by CK2 results in a reduction of its transcription factor activity. By using pharmacological inhibition of CK2 activity in pancreatic β-cells, insulin transcription and release was enhanced14. Here, we have shown that inhibition of CK2 in pancreatic islets from mice led to an elevated production and elevated release of insulin into the culture medium. USF1 was also described as new CK2 substrate7. Inhibition of the CK2 kinase activity led to an enhancement of the transcriptional activity towards the fatty acid synthase and the glucokinase promoter7. CK2 was not found in the DNA bound USF complex, but an interaction between both proteins was observed in the nucleoplasm13. Here, we have demonstrated that inhibition of the CK2 phosphorylation resulted in a decreased transrepression of the PDX-1 promoter. This reduction in the transrepression went along with a reduction in the DNA binding activity of USF1. Pull-down analyses between USF1 and PDX-1 showed a much stronger interaction between PDX-1 and the phospho-mimicking USF1T100D mutant. Thus, it is tempting to speculate that CK2 phosphorylation of USF1 enhances its DNA binding to the PDX-1 promoter as well as its interaction with PDX-1, thus supporting the transrepression activity of USF1. CK2 is likely to act as a negative regulator of the auto-regulatory loop which finally leads to a down-regulation of insulin transcription and release. There are similar observations by Rossi et al.55 who found that CK2 phosphorylation of the G-protein coupled receptor M3R in pancreatic β-cells impairs its stimulatory effect upon insulin release.Over 400 substrates are known for protein kinase CK2, among them different transcription factors whose activity is regulated by CK2 phosphorylation. Phosphorylation by CK2 enhances the transcriptional activity of HIF1αOur results have shown that CK2 acts as a negative regulator of insulin transcription in pancreatic β-cells via the USF-dependent PDX-1 auto-regulatory loop.56, 4,5,6,7-tetrabromobenzotriazole 57 and quinalizarin 58 were dissolved in dimethyl sulfoxide (DMSO) to 10 mM stock solutions. The TRIzol® Reagent was obtained from Peqlab Biotechnology GmbH  and the QuantiTect® SYBR® Green RT-PCR kit was purchased from Qiagen GmbH .CX-4945 18 were cultured in RPMI1640 containing 10% foetal calf serum (FCS), 1% glutamine, 1 mM sodium pyruvate, and 50 µM β-mercaptoethanol. Cells were cultured at 37 °C and 5% CO2 in a humidified atmosphere in an incubator.INS-1 cellsPancreatic islets of C57B/6 mice (body weight of 25–28 g) were obtained from current animal experiments which are approved by the local governmental animal care committee  and conducted in accordance with the European legislation on protection of animals (Guide line 2010/63/EU) and the NIH Guidelines for the Care and Use of Laboratory Animals.et al.59. In brief, animals were anesthetized by intraperitoneal injection of ketamine  and xylazine . After laparotomy, the pancreatic duct was injected with collagenase  and the pancreas excised. To isolate the pancreatic islets, the dispersed pancreas was washed in PBS (10% FCS), islets were handpicked and transferred into a Petri dish containing DMEM .The isolation of pancreatic islets was performed as described in Gotoh 32P]- phosphate into the synthetic CK2 specific substrate peptide with the sequence RRRDDDSDDD [21]. Twenty µl kinase buffer  containing 20 µg proteins were mixed with 30 µl CK2 mix , 50 µM ATP, 0.19 mM substrate peptide) containing 10 µCi/500 µl [32P]γATP. The mixture was spotted onto a P81 ion exchange paper. The paper was washed with 85 mM H3PO4 for three times. After treatment with ethanol the paper was dried and the Čerenkov-radiation was determined in a scintillation counter.To determine the kinase activity of CK2, cells from pancreatic islets were lysed with RIPA buffer  and the extracts were used in a kinase filter assay. In this assay we measured the incorporation rate of [et al.60. Five islets from mouse pancreas were maintained in RPMI medium without glucose for 18 hours with 10 µM CX-4945 or an equal volume of DMSO as vehicle control. After washing two times with Krebs Ringer buffer KRB , islets were incubated 40 min with KRB supplemented with 1.1 mM glucose and either DMSO or 10 µM CX-4945. For another 20 min the glucose concentration was enhanced to 5.6 mM to induce the insulin secretion. After this treatment, insulin was determined in the medium with the rat/mouse insulin ELISA kit from Merck-Millipore  according to the recommendation of the provider.The determination of secreted insulin was essentially done as described by Kelly ® Site-Directed Mutagenesis Kit  to create the p3xFLAG-Myc-CMV24-USF1T100A. The mutation was verified by sequencing. For bacterial expression of GST fusion proteins of USF1 wild-type and mutants  we used the plasmids described in Lupp et al.7. For in vitro translation of USF1 (pcDNA-USF1) we cloned the cDNA of human USF1 into the EcoR1/Xho1 sites of the pcDNA3.1 vector (Invitrogen). For transient transfection assays p3xFLAG/CMV-7.1-PDX-1 constructs were produced by sub-cloning mouse PDX-1 cDNA into the BamH1 site of p3xFLAG/CMV-7.1 vector4. pGEX4T-1-PDX-1 plasmids are described by Meng et al.4. pRSET-PDX-1 was generated by cloning the cDNA of mouse PDX-1 in frame into the BamH1/HindIII sites of the pRSETA plasmid (Invitrogen).For eukaryotic expression, USF1 was cloned into the EcoR1 and Xba1 sites of p3xFLAG-Myc-CMV24 thus generating a fusion construct with an N-terminal FLAG-tag and a C-terminal myc-tag. A point mutation of USF1 was created with the p3xFLAG-Myc-CMV24-USF1 and the Q522 was a kind gift from Dr. Marc R. Montminy . The dominant-negative USF mutant CMV566 A-USF was a gift from Charles Vinson (Addgene plasmid # 33360).The PDX-1 promoter construct from −6500 to +6861. The mouse monoclonal antibody against CK2β  was purchased from Santa Cruz . Detection of CK2α was performed by using the mouse monoclonal antibody 1 A562. The mouse monoclonal antibody FLAG M2 (F1804), the β-actin and the α-tubulin (clone DM1A) antibody were from Sigma-Aldrich . Rabbit polyclonal serum #36 against nucleolin was prepared in our laboratory63. Insulin was detected by the monoclonal rabbit antibody AB181547 from abcam .The anti-USF1 antibody is a rabbit polyclonal IgG directed against the C-terminus of USF1  or a rabbit polyclonal IgG directed against the amino acids 75–160 of USF1 , whereas the anti-USF2 antibody is a rabbit polyclonal IgG directed against the C-terminus of USF2 . HA-tagged dominant negative USF mutant was detected with the HA specific mouse monoclonal antibody 12CA5 from Roche Diagnostics . PDX-1 was identified with a polyclonal antiserum generated by immunizing rabbits with recombinant mouse PDX-1TM Transfection Reagent  according to the manufacturer’s instructions.Transfection of cells was performed by using the ViafectTM Transfection Reagent. 24 h after transfection cells were starved overnight for glucose experiments and the next day treated with different glucose concentrations  over a period of 4 h. For glucose and/or parallel treatment with the CK2 inhibitors we used CX-4945 in a concentration of 10 µM, whereas TBB and quinalizarin were used in a concentration of 50 μM over a period of 4 h or 24 h.For the luciferase reporter assay, INS-1 cells were seeded into a 6-well plate in a total volume of 2 ml/well of cell culture medium and cultured overnight. Cells were then transfected with 2.5 µg plasmid DNA by using Viafect4 h or 24 h after treatment or 48 h after transfection cells were collected by lysing in 1x lysis buffer  and measured with the Luciferase Reporter Assay System (Promega) following the manufacturer’s recommendations. To normalize the experimental variations such as differences in transfection efficiencies we calculated the relative intensities of FLAG tagged protein amounts first in relation to α-tubulin and then in relation to the luciferase activity.For Western blot analysis, cell lysates were centrifuged at 13.000xg to remove cell debris. The protein content was determined with BioRad reagent dye . Protein extracts were immediately used for Western blot analysis or stored at −20 °C. Image acquisition of Western blots was performed with the ChemiDoc XRS+ imaging system from Bio‐Rad ; data were analyzed using the Quantity One 1‐D analysis software, version 4.6.2HPO4, 1.5 mM KH2PO4, pH 7.4), resuspended in buffer A  and incubated on ice for 20 min. After centrifugation, the supernatant containing cytoplasmic proteins was removed and the pellet was resolved in buffer C . Nuclear proteins were isolated by pressing the suspension through a 21 G and 23 G cannula and centrifugation . Two mg of nuclear proteins were pre-cleared twice with protein G sepharose beads  (control precipitate). The supernatant was incubated with rabbit polyclonal antibody USF1  for 2 h at 4 °C. Beads were washed four times with PBS , and bound proteins were analysed by Western blot with specific antibodies. Image acquisition of Western blots was performed with the ChemiDoc XRS+ imaging system from Bio‐Rad ; data were analyzed using the Quantity One 1‐D analysis software, version 4.6.For co-immunoprecipitation of USF1 and PDX-1, INS-1 cells were seeded on a 14.5 cm cell culture plate in a total volume of 12 ml/plate of culture medium. After a period of 24 h cells were starved overnight and the next day treated with glucose at different concentrations  over a period of 4 h. After 4 h nuclear proteins were extracted. For CK2 inhibition, cells were treated with 10 µM CX-4945 or 50 µM quinalizarin (Q) over a period of 24 h and then nuclear proteins were extracted. For extraction, cells were washed two times with PBS .For ′-CAGGACAGGAGAGATCAGCC-3′ and the biotinylated reverse primer 5′-biotin/GCAGCTCACGGACTCTCA-3′ for PCR amplification to prepare a biotinylated PDX-1 promoter probe from −221 to +22.Several biotinylated primers were obtained from Integrated DNA Technologies . We used the forward primer 5in vitro pull-down assays with the PDX-1 promoter, INS-1 cells were seeded on a 14.5 cm cell culture plate in a total volume of 12 ml/plate of culture medium. After a period of 24 hours, cells were starved overnight and the next day treated with different glucose concentrations  over a period of 4 h. For treatment with the CK2 inhibitors we used CX-4945 in a concentration of 10 µM, whereas TBB and quinalizarin were used in a concentration of 50 μM over a period of 24 h.For TM-column. The column was washed 15 times with buffer C (150 mM NaCl) and the proteins were eluted by the addition of 50 µl pre-heated SDS sample buffer and after SDS polyacrylamide gel electrophoresis analysed by Western blot with specific antibodies. Image acquisition of Western blots was performed with the ChemiDoc XRS+ imaging system from Bio‐Rad ; data were analyzed using the Quantity One 1‐D analysis software, version 4.6.Nuclear extracts were prepared as described above and 1 mg was incubated with salmon sperm DNA (100 µg/ml) for 20 minutes at 4 °C. One µg of biotinylated PDX-1 DNA probe was added and the extract was again incubated for 30 minutes at 4 °C. 50 µl of streptavidin-agarose beads were added for 12 minutes at room temperature and the DNA-protein complex was passed through an equilibrated μMacset al.64. Purified GST- or GST-tagged proteins (10 μg) were immobilized on GSH-sepharose and equilibrated with PBS-T binding buffer . Immobilized proteins were incubated for 2 h at 4 °C with 7.5 µl of PDX-1 product from an in vitro translation reaction. The translation from a pRSET-PDX-1 plasmid was done as recommended by the manufacturer (Promega). After washing with cold PBS-T, bound proteins were eluted with SDS sample buffer  and analysed by SDS polyacrylamide gel electrophoresis, followed by protein staining with Coomassie blue and autoradiography.The pull-down assay was essentially done as described by Sun 65 employing the LightCycler 2.0 instrument  using the QuantiTect® SYBR® Green RT-PCR kit . The following primers were used for real-time RT-PCR: PDX-1_for: 5′-AGCGAGATGCTGGCAGA-3′ and PDX-1_rev: 5′-TCAGTTGGGAGCCTGATTCT-3′. 18 S rRNA was used as an endogenous control employing the primers 18S_for: 5′-GTAACCCGTTGAACCCCATT-3′ and 18S_rev: 5′-CCATCCAATCGGTAGTAGCGA-3′.Total RNA and protein were isolated using TRIzol® Reagent  following the manufacturer’s protocol. One-step real-time RT-PCR was performed as described previouslyMicrosoft Excel 2007 and the software “Quantity one 1-D Analysis” from Bio-Rad Laboratories Inc. were used to analyse the data. Results from the luciferase assay were expressed as mean ± standard deviation (SD) of at least three or four independent experiments. Statistical analysis of the data was performed using the student’s t-test , statistical significant differences were shown as follows: ***p < 0.001, **p < 0.01 or *p < 0.05.Supplementary figure S1


SGI1 is mobilized by the IncA/C family plasmids. The island exploits not only the conjugation apparatus of the plasmid, but also utilizes the plasmid-encoded master regulator AcaCD to induce the excision and formation of its transfer-competent form, which is a key step in the horizontal transfer of SGI1. Triggering of SGI1 excision occurs via the AcaCD-dependent activation of xis gene expression. AcaCD binds in Pxis to an unusually long recognition sequence. Beside the Pxis promoter, upstream regions of four additional SGI1 genes, S004, S005, S012 and S018, also contain putative AcaCD-binding sites. Furthermore, SGI1 also encodes an AcaCD-related activator, FlhDCSGI1, which has no known function. Here, we have analysed the functionality of the putative AcaCD-dependent promoter regions and proved their activation by either AcaCD or FlhDCSGI1. Moreover, we provide evidence that both activators act on the same binding site in Pxis and that FlhDCSGI1 is able to complement the acaCD deletion of the IncA/C family plasmid R16a. We determined the transcription start sites for the AcaCD-responsive promoters and showed that orf S004 is expressed probably from a different start codon than predicted earlier. Additionally, expression of S003 from promoter PS004 was ruled out. Pxis and the four SGI1 promoters examined here also lack obvious -35 promoter box and their promoter profile is consistent with the class II-type activation pathway. Although the role of the four additionally analysed AcaCD/FlhDCSGI1-controlled genes in transfer and/or maintenance of SGI1 is not yet clear, the conservation of the whole region suggests the existence of some selection for their functionality.The integrative genomic island SGI1 and its variants confer multidrug resistance in numerous S Salmonella and other Gram-negative pathogenic bacteria. Salmonellae, particularly several serovars such as Typhimurium and Enteritidis, are among the most prevalent zoonotic pathogens worldwide. The MDR clone of S. Typhimurium DT104  emerged in the early 1980s in the UK and became globally distributed in the 1990s among humans and livestock . Acc. Accxis d SGI1-C . Interesxis four additional potential target sites have been predicted on SGI1 [S004, S005, S012 and S018  and included the whole predicted AcaCD-binding site with 20 to 75 bp upstream flanking regions. As orf S003 was previously suggested to be expressed from the putative promoter PS004 [S003 from the start codon of S003 to the stop codon of S005, including the entire S004, was also created (pMSZ956). On the other hand, the predicted AcaCD-binding site in the upstream region of S004 is located only 3 bp from the start codon of the annotated S004 orf, which rules out the possibility of expression of the annotated S004 protein (designated here as S004L) from this putative AcaCD-dependent promoter. Comparing the spacing between the AcaCD-binding site and the start codon in AcaCD-driven promoters [S004, if it is expressed at all, starts from the in-frame ATG codon located 66 bp downstream of the AcaCD-binding site (3474 bp in SGI1 sequence AF261825) and produces a shorter protein (designated as S004S). This assumption was further supported by the presence of a potential SD-box preceding this ATG codon. Accordingly, a tester plasmid, pMSZ965, containing the upstream region of the in-frame ATG located in S004 to the stop codon of S005, was also constructed  independently of the presence of activators, which suggested that S003 (repA) is not controlled by AcaCD or FlhDC SGI1 and expressed from a weak promoter probably located in S004. These results confirmed that all the predicted AcaCD-binding sites on SGI1 are parts of functional promoters that can be induced by either AcaCD or FlhDCSGI1, and indicate that both activators can potentially participate in the regulation of the genes located downstream of these promoters in vivo.Induction of PSGI1-dependent activation of PS004, PS005, PS012 and PS018 promoters, the transcription start sites (TSS) were determined using the appropriate tester plasmids in primer extension assays. In case of all four promoters, extension reaction was observed only when the activator AcaCD was expressed from a compatible plasmid   also showed unambiguous TSS. The strongest signals showed that the transcript begins 22 or 25 bp upstream from the start codon of S005, however, weaker signals could be seen at 24 and 26 bp positions, as well. Majority of the S012 transcripts begins 26 bp upstream of the start codon, but a small fraction starts at 27 bp. The longest 5’ UTR was observed in S018 transcript where TSS was detected at 135 bp from the ATG. Although a relatively high basal activity was measured in the β-galactosidase assay for PS018, the TSS of this constitutively expressed transcript was not detectable  . The TSS in Pxis .S004 starts at a different position than was predicted by the annotation of SGI1 orfs (GenBank: AF261825), we examined whether the expression of S004 has any detectable effects on the growth of host bacteria. According to the original annotation, orf S004 encodes 96 amino acids, while our results suggested that it might be expressed from an in-frame ATG as a shorter (75 aa) polypeptide . The two orfs beginning with the different ATGs were placed under the control of Ptac  and their effect on bacterial growth was monitored by determination of growth curves. For comparison, an analogous plasmid expressing S003 and the empty vector as negative control were also applied , S004S (8.8 kDa) and S003 (35.3 kDa) was tested by PAGE of total protein samples obtained from plasmid-bearing strains, which showed that S004L appears to be expressed in lower level compared to S004S and S003  related to HTH of MerR regulatory proteins and shows 38–42% identity and 59–64% similarity (mostly in this region) to AlpA phage regulatory proteins. Although the HTH motif is present in both possible variants of S004, the growth rates of strains expressing them support that S004S may have more significant biological effects and S004L, if expressed at all, is probably inactive and/or degraded rapidly.The proper expression of proteins S004and S003 . The exp 5 hours . While eSGI1 master activators. Similarly to Pxis, the four additional SGI1 promoters analysed in this work also lack obvious -35 promoter box and their -10 box is located 22–24 bp downstream of the AcaCD binding site. In general, their promoter profile appears to be consistent with promoters of the class II-type activation pathway [We have demonstrated that AcaCD binding sites in SGI1 predicted previously  are loca pathway .SGI1 is able to act similarly on the AcaCD-dependent SGI1 promoters, and can also substitute for AcaCD in the regulation of the whole conjugation apparatus of the IncA/C plasmids. This compatibility of FlhDCSGI1 and AcaCD may raise the possibility of complex interactions between SGI1 and IncA/C helper plasmids when they occur in the same cell. However, the functions of FlhDCSGI1 and the AcaCD-responsive SGI1 genes have not yet been established in detail with the exception of xis. Pxis appears to be a sensor of the helper plasmid entry as the plasmid-borne activator induces xis expression through binding to Pxis, which leads to triggering SGI1 excision [S005 to S012 has no significant effect on the SGI1 conjugation frequency, indicating that neither FlhDCSGI1 (encoded by orfs S007-S006) nor the AcaCD-responsive S004 and the putative conjugation-related genes S005 (traN), S011 (traG) and S012 (traH), are required for efficient mobilization of SGI1. Similarly, deletion of the S013-S018 region containing short orfs without known functions has no effect on the transfer frequency under laboratory circumstances (G. M. unpublished data). A possible explanation can be that flhDCSGI1 and its target promoters are remnants from the earlier evolution of the island. Accordingly, the ancient SGI1 could possess the whole conjugation apparatus and its regulatory functions, like SXT elements or the related IncA/C plasmids [xis, might have contributed to the evolution of the recent form of SGI1 that has lost the ability for self-transfer but can efficiently hijack IncA/C plasmids. Although few deletions can be found in AcaCD-controlled genes , the whole region is highly conserved at the DNA sequence level (85–100% identity) even in distant relatives of SGI1, which might imply some kind of selection for the integrity of these functions.The two activators are close relatives as they share 79 and 46% identity in the amino acid sequence of C and D subunits, respectively . Despiteexcision . We haveplasmids . In thisS1 FigS003 and S005 are not shown as full length orfs). The elements of the AcaCD-responsive promoter are indicated by grey boxes . The promoter regions inserted into pMSZ956 and pMSZ965 tester plasmids are shown below the graph.Orfs are represented by purple arrows ((TIF)Click here for additional data file.S2 FigTwo parallel colonies harboring the expression vectors were used in the assays. Lanes—and + show the total proteins obtained from non-induced and induced cultures, respectively.(TIF)Click here for additional data file.S1 Table(DOC)Click here for additional data file.S2 Table(DOC)Click here for additional data file.S3 Table(DOC)Click here for additional data file.S1 Text(DOC)Click here for additional data file.


LCYb1 in citrus with abundant carotenoid accumulation are still unclear. To understand the molecular basis of CsLCYb1 expression, we isolated and functionally characterized the 5′ upstream sequences of CsLCYb1 from citrus. The full-length CsLCYb1 promoter and a series of its 5′ deletions were fused to the β-glucuronidase (GUS) reporter gene and transferred into different plants  to test the promoter activities. The results of all transgenic species showed that the 1584 bp upstream region from the translational start site displayed maximal promoter activity, and the minimal promoter containing 746 bp upstream sequences was sufficient for strong basal promoter activity. Furthermore, the CsLCYb1 promoter activity was developmentally and tissue-specially regulated in transgenic Arabidopsis, and it was affected by multiple hormones and environmental cues in transgenic citrus callus under various treatments. Finer deletion analysis identified an enhancer element existing as a tandem repeat in the promoter region between -574 to -513 bp and conferring strong promoter activity. The copy numbers of the enhancer element differed among various citrus species, leading to the development of a derived simple sequence repeat marker to distinguish different species. In conclusion, this study elucidates the expression characteristics of the LCYb1 promoter from citrus and further identifies a novel enhancer element required for the promoter activity. The characterized promoter fragment would be an ideal candidate for genetic engineering and seeking of upstream trans-acting elements.Lycopene β-cyclases are key enzymes located at the branch point of the carotenoid biosynthesis pathway. However, the transcriptional regulatory mechanisms of  Carotenoids are a class of 40-carbon terpenoid molecules present in most tissues of higher plants. They are one type of the most important secondary metabolites, and play a variety of roles in many biological processes in plants, including flowering and fruit coloration , light hCarotenoid metabolism is a complicated pathway involving the expression of many genes, which are regulated by various factors, such as developmental cues and environmental conditions . AlthougArabidopsis phytoene synthase (PSY) promoter identified a G-box-like element involved in light induction and discrimination between different light qualities, and also identified a novel cis-acting element ATCTA which contributes to strong basal promoter activity ((Pds) promoter developmentally drives high GUS expression in the organs where chromoplasts are formed, and promotes gene transcription in green tissues in response to end-product regulation (Arabidopsis carotenoid cleavage dioxygenases 7 (AtCCD7) promoter exhibits a vascular-specific expression pattern in transgenic plants (AtBCH) promoter shows strong constitutive expression in dicot plants (Gentiana lutea zeaxanthin epoxidase (GlZEP) promoter in transgenic tomato plants showed that GlZEP-GUS expression is closely associated with fruit development and chromoplast differentiation, suggesting an evolutionarily conserved link between ZEP and the differentiation of organelles that store carotenoid pigments (Chrysanthemum morifolium (CmCCD4a-5) and assessed its petal-specific promoter activity.To date, some promoters of the genes in the carotenoid biosynthetic pathway have been analyzed and some progresses have been achieved. The analysis of the activity . The tomgulation . The Arac plants , while tt plants . The funpigments . Imai etLCYb1 and LCYb2, respectively) in citrus. LCYb1 is predominantly expressed in leaf tissues, while LCYb2 is mainly expressed in fruit tissues and shows a marked induction during fruit development  results in lycopene accumulation in red grapefruit (Citrus paradisi) (C. sinensis) revealed that the down-regulation of both LCYb (LCYb1) and capsanthin capsorubin synthase (CCS) (LCYb2) may be responsible for the abnormal lycopene accumulation in the mutant  . Howevere mutant . Zhang en tomato  and waten tomato , little CsLCYb1 promoter from sweet orange (C. sinensis) as well as to analyze the LCYb1 promoters from different citrus species. This study will contribute to understanding the expression characteristics of LCYb1 promoters and is expected to help future transcriptional regulation studies of LCYb1 expression in citrus.The objectives of the present study were to isolate and functionally characterize the C. grandis; White-flesh Guanxi pummelo, Red-flesh Guanxi pummelo, Huanong red pummelo, HB pummelo), three genotypes of grapefruit , four genotypes of sweet orange , and three genotypes of mandarin . Leaves of all these citrus varieties were obtained from the National Center of Citrus Breeding, Huazhong Agricultural University, Wuhan, China. The tissues were frozen in liquid nitrogen and stored at -80°C until use. Tomato (Lycopersicon esculentum cv Ailsa Craig) and Arabidopsis  plants were grown under standard greenhouse conditions. Embryogenic callus used in this study was derived from Marsh grapefruit and subcultured on solid MT (Murashige and Tucker) basal medium containing 50 g L-1 sucrose under normal conditions (16 h light/8 h dark cycles at 25°C).The materials included four genotypes of pummelo (CsLCYb1 cDNA sequence (orange1.1t00772) was used as a query to search the C. sinensis genomic database1 (CsLCYb1 mRNA was set as the transcription start site (TSS). Promoter regions and plant regulatory motifs were searched using the Softberry TSSP and Nsite-PL program2. A search for putative cis-elements in the promoter sequence was performed by using the PLACE3  and its five deletions  were amplified by PCR from the pMD18-T basic vector containing the 5′ full-length flanking sequence. Specific primers with EcoRI and NcoI restriction sites were designed (Supplementary Table CaMV35S promoter (the cauliflower mosaic virus 35S promoter). The six vectors were designated as LP (-1584), LP1 (-1255), LP2 (-1045), LP3 (-746), LP4 (-406), and LP5 (-247), respectively. The CaMV35S::GUS fusion in pCAMBIA1301 was used as a positive expression control (designated as 35S).The entire n CGTGAAGGAGGAAAAGTGAGAAGAAAAAAAATTATATATTTTTTAATT), was synthesized to yield two fragments denoted as W1 (n = 1) and W2 (n = 2), respectively. Then, two pairs of primers  were used for PCR amplification of the full-length CsLCYb1 promoter sequences to obtain two fragments denoted as LMa and LMb, respectively. Next, overlapping PCR was performed with three fragments  simultaneously as templates and with two oligonucleotides (LPF and LPR containing EcoRI and NcoI sites at their 5′-ends) as primers. The obtained fragments were double digested and subcloned into the correspondingly enzymatic sites of the pCAMBIA1301 plasmid to yield two internal deletion vectors WP1 and WP2. The promoter-GUS vectors are schematically represented in Figures 2 and 6. All constructs were verified by sequencing and then transformed into the Agrobacterium tumefaciens stain GV3101 by the freeze-thaw method. The generated constructs were subsequently transformed into plants to test promoter activities.Internal deletion vector construction: Double stranded DNA, (GTGACTGAAATCATCAACCCTTGATGAACATCCTTTGCTATTGGGCATGAATGGAGAAGGAAGAAAATGAG (ATTGAAGGAAGAAAAATGAG)Agrobacterium cultures (0.5 mL) from individual colony were grown at 28°C for 24 h in LB liquid medium supplemented with kanamycin (100 mg L-1) and rifampicin (25 mg L-1), then transferred to 50 mL induction medium  containing corresponding antibiotics and grown again. In the following day, the bacterial cells were sedimented by centrifugation and re-suspended in infiltration medium  to an OD600 of approximately 1.0, and then incubated at room temperature with gentle agitation (20 rpm) for about 2 h. Cultures were collected with a syringe, and then injected into detached tomato fruits (L. esculentum cv Ailsa Craig) at a total volume of 600 μl. Three days later, the injected fruits were cut into slices for histochemical GUS staining.Tomato transient transformation was performed according to the method described by Arabidopsis transformation was done using the floral dip method established by -1 of hygromycin, then were transferred to soil, and finally were grown in the greenhouse at 22°C under a 16 h light/8 h dark photoperiod. The positive transformations were further confirmed through PCR amplification of genomic DNA by using the primer sets, respectively  were collected during five developmental stages for developmental expression assay. Seedlings of other transgenic lines were only collected on day 24 after seed germination to compare the promoter activities among different truncated fragments.-1 of hygromycin and 250 mg L-1 of cefotaxim. PCR amplification was used to further confirm the positive lines. Each independent line was propagated on solid MT basal medium containing 50 g L-1 sucrose under normal conditions (16 h light/8 h dark cycles at 25°C). Twenty-day-old callus was harvested for GUS assays or various stimuli treatments.Embryogenic callus transformation was performed with the method described by In various stimuli treatments, transgenic callus was cultured on solid MT medium for about 20 days at 25°C, followed by culturing for 4 days in liquid MT medium with shaking. The stable cell suspension cultures in a good state were immersed in MT liquid medium supplemented with 100 μM ABA, 100 μM auxin (IAA), 100 μM gibberellin (GA), 100 μM salicylic acid (SA), 100 μM Methyl Jasmonate (JA), 100 μM Kinetin (KT), 10% (w/v) sucrose (Suc), 10% (w/v) glucose (Glu), 200 mM NaCl for 12 h under light. Callus cells in MT liquid medium without any supplement were used as negative control. All parallel samples were grown under the same conditions. After treatment, all samples were frozen in liquid nitrogen and used for subsequent GUS assays. Three biological replicates were performed for each set of treatments.Histochemical staining and fluorometric assays were performed according to the method proposed by Various tissues were submerged in X-gluc buffer  overnight at 37°C. After staining, the tissues were kept in 70% ethanol until the chlorophyll was removed, and then photographed with a digital camera or under a stereomicroscope (Leica MZFL III). All the experiments were repeated ten times for each construct.TM M200 plate reader at 365 nm excitation and 455 nm emission. GUS enzyme activity was expressed as umoles of 4-MU per min per mg protein. Three replicates were performed for each sample.Quantitative GUS assays were performed as follows. The samples were frozen in liquid nitrogen and ground into powder. Protein was extracted with GUS extraction buffer . After centrifugation, the supernatant of various extraction liquids was used for the subsequent protein quantification and fluorometric assays. Protein concentrations were determined using the BCA Protein Assay Kit . Fluorometric assays were performed in microtiter plates at 37°C in the presence of 1 mM 4-methylumbelliferyl glucuronide . The appearance of 4-methylumbelliferone (MU) was monitored using a Tecan InfiniteLCYb1 promoter sequences  were designed according to above isolated P-value <0.05 (∗/Lowercase letters) or <0.01(∗∗/Uppercase letters) was considered as significant.The data were presented as mean ± SD of three independent experiments. Statistical analyses were done using the One-way ANOVA test on the Microsoft Excel program . The difference with a CsLCYb1 genomic DNA sequence and its 5′ flanking region were downloaded from the genomic database of sweet orange with the full length CsLCYb1 cDNA (orange1.1t00772) as a query sequence. The 1584 bp fragment located in the upstream from the ATG start codon was obtained through PCR-based method and tentatively designated as the full-length promoter of CsLCYb1 . The TSS was located at -263 bp upstream from the ATG (the position of the ATG start codon was designated as 0). Bioinformatics analysis revealed that the CsLCYb1 promoter was a typical eukaryotic promoter containing a potential TATA box at -292 bp, a CAAT-box at -356 bp, and many TA-rich enhancer elements. A large number of hormone-responsive elements were predicted in the promoter, such as the ATCTA-motif in response to ethylene, the CGTCA-motif to Jasmonate, the GARE motif to gibberellin, the TCA motif to SA and the TGA motif to auxin. We also discovered some stress-responsive elements, such as the ARE-motif involved in anaerobic induction, the CATGTG-motif in dehydration response, the E-box in defense signaling, and the MYB-binding sites in drought inducibility. In addition, the CsLCYb1 promoter carried numerous light-responsive elements, such as the Box 4, Box II, CATT-motif, GA-motif, GAG-motif, SP1 and TCCC-motif. Among these motifs, the GA-motif characterized by the AGATT sequence existed as a tandem repeat in the promoter region between -588 and -522 bp. We also compared the cis-elements in the CsLCYb1 promoter with those in the previously isolated CsPSY promoter .Firstly, we applied a transient expression method to identify whether the cloned CsLCYb1 promoter, we examined stable Arabidopsis transgenic plants. Homozygous single-insertion T2 lines of each construct were used for histochemical staining and quantitative GUS assays. Different tissues  from each construct were subjected to histochemical GUS staining . GUS staining was observed in all tested tissues of the CaMV35S construct, but not in the wild type control. GUS expression of LP, LP1, LP2, and LP3 constructs was apparently detectable in leaves. No significant difference in GUS staining intensity was found among LP, LP1 and LP3, while the staining intensity of LP2 was relatively lower than that of the above-mentioned three constructs. Little or no GUS staining was found in the leaves of LP4 and LP5. In addition, several tissues of some constructs were slightly stained, such as the root of LP, flower of LP4, stem ends of LP1 and fruit ends of LP, LP1, LP3, and LP4. Epidermal hairs in the stems of LP, LP1, LP2, LP3, and LP4 were also stained.To elucidate the spatial and temporal expression patterns of the Figure 4). The results showed that the promoter activities increased along with the seedling development, reached the maximum on day 24, and subsequently decreased on day 28. Then, GUS expression of different promoter constructs was compared on day 24. In accordance with the results of GUS staining assay, the LP construct carrying the full-length sequence of the CsLCYb1 promoter produced the highest level of GUS expression in leaf tissues. With deletions of the 5′ fragments, the promoter activity gradually decreased. However, no significant difference in GUS activity was found among LP, LP1, LP2, and LP3. By comparison, the GUS activities of LP4 and LP5 were remarkably reduced. The GUS activity of LP4 was about fourfold lower than that of LP, and that of LP5 was too low to be detected.Glucuronidase enzyme activities were quantified by fluorometric 4-MUG assay at different developmental stages of seedling in the full length promoter transgenic lines . In order to eliminate the possible effect of heterogeneous expression on promoter activity, we stably transformed the promoter constructs into citrus callus, respectively. The expression level of each construct in transgenic citrus callus was also evaluated by histochemical GUS staining . The results showed that GUS staining was obviously visible in callus transformed with constructs LP, LP1, LP2, and LP3, suggesting that these fragments could functionally drive GUS expression in callus. The callus transformed with construct LP4 was slightly stained, indicating possible promoter activity of this fragment in callus. Pale blue was observed only in a few cells of transgenic callus with LP5.Previously we detected the transcripts of CsLCYb1 promoter function in detail by GUS expression assay under various stimuli treatments. Based on the GUS staining results, we speculated that the sequence region from LP1 (-1255) to LP4 (-406) was likely to significantly contribute to the regulation of the CsLCYb1 promoter activity. Thereby, we tested the expression levels of constructs LP1, LP2, and LP3 under various stimuli including hormones, sugar and salt stress to examine the responses of different 5′ sequence regions. GUS expression level driven by LP5 promoter was also tested under all conditions. As shown in Figure 5, LP1 promoter activity was significantly induced under ABA and JA treatments. In contrast, the promoter activities of LP2 and LP3 were not strongly affected by both ABA and JA. IAA treatment induced the activities of LP1 and LP2 promoter , and SA treatment also induced their activities . However, the LP3 construct did not show any significant differences in promoter activity under IAA and SA treatments. When callus cells were incubated with GA, we observed significant induction of GUS expression. Compared with under normal conditions, the promoter activities of LP1, LP2, and LP3 were increased to 2.1-, 3.3-, and 2.2-fold, respectively. Under KT treatment, GUS expression driven by either LP1 or LP2 was increased to about 1.6-fold. Notably, a deletion from LP2 to LP3 resulted in a sharp increase of GUS activity to 1.1 μmol MU min-1 mg-1 protein (2.6-fold). Both sucrose and glucose treatments significantly enhanced the promoter activities of LP1 and LP2. However, GUS expression of LP3 was only promoted by glucose, not by sucrose. Additionally, we analyzed the promoter activities under NaCl treatment to determine whether the promoter activity responded to salt stress. The results showed that the promoter activity of LP1 was significantly increased, while the activities of LP2 and LP3 were decreased to some extent, even though no significant differences were observed.Although the roles of phytohormones, glucose (sucrose or mannitol) and various other stimuli in carotenoid accumulation have been studied, little has been known about the molecular mechanisms that regulate carotenoid metabolism and gene expression. To fully reveal the regulation, we analyzed cis-elements in the 20 bp sequence identified five reported cis-elements: Inr-element (YTCANTYY), CAAT-box (CAAT), GT1-motif (GAAAAA), GT-element (GRWAAW), and pollen-specific element (AGAAA). In order to verify whether the 20 bp fragment was essential for promoter function, we performed finer deletion analysis. Additional vectors with the deletion of one or two copies of the 20 bp fragment were constructed and transformed into citrus callus to test the promoter activities. Compared with the complete CsLCYb1 promoter, the deletion of one copy caused the promoter activity to dramatically decrease to 55%, while the promoter activity with the deletion of two copies dropped to approximately 23% . Taken together, these data clearly indicated that the 20 bp fragment acted as a positive cis-acting regulatory element to affect promoter activity.Since a deletion from LP3 to LP4 resulted in a significant reduction in promoter activity, we speculated that an enhancer (or enhancers) may be located in this region. Further sequence analysis revealed the existence of a 20 bp fragment (ATTGAAGGAAGAAAAATGAG) in the region as a tandem repeat (between -574 and -513 bp upstream from the ATG). A search of the PLACE database for the potential LCYb1 promoter, we isolated promoters of LCYb1 alleles from other citrus species. Due to the high heterozygosity in citrus genome, most of the gene loci have two different alleles termed as a and b, respectively. CsLCYb1 in sweet orange has two different coding sequences (data not shown), while we got only one promoter sequence named as pCsLCYb1. We tried our best but failed to get the other one. However, in pummelo and grapefruit, we obtained two different promoter sequences named as pCgLCYb1a and pCgLCYb1b, pCpLCYb1a and pCpLCYb1b, respectively  from the C. clementina genome databases and found that a large fragment was inserted in the enhancer region of the LCYb1 promoter , while no band was found for mandarin. These results indicated that the SSR markers based on the 20 bp enhancer element could be used to distinguish different citrus species.To further confirm the association between the copy numbers of the 20 bp enhancer element and genetic evolution of citrus species, a pair of primers was designed to develop a derived SSR (simple sequence polymorphism) DNA molecular marker (Supplementary Table trans-lycopene to produce the cyclic α- and β-carotenes in the carotenoid biosynthetic pathway (LCYb1 and LCYb2) in citrus. Functional analysis showed that both enzymes participate in the formation of β-carotene, and play important roles in fruit ripening and plant development . Due to the difficulty of transformation, long life cycle (at least 1 year from sowing to fruit ripening), and the difficulty of GUS measurement of citrus species, we investigated the promoter activities by transient expression assay in tomato fruit and stable transformation method in Arabidopsis plants. These two methods had been successfully used for studying the promoter function of carotenogenic genes, such as the tomato phytoene desaturase , suggesting that the three analyzed species shared some transcription factors binding to the promoter. A similar finding was reported based on the functional analysis of a dahlia mosaic virus subgenomic transcript (DaMVSgt) promoter in transient protoplasts, transgenic tobacco and Arabidopsis plants  exhibited very weak promoter activity . However, the shortest fragment LP5, which did not contain any core promoter element, also drove very little GUS expression in transgenic callus . One explanation may be that the actual positions of core elements in the promoter regions are not consistent with the bioinformatics predictions, which needs to be verified by more experiments. Another reason could be that the promoter sequences containing no core elements such as TATA-box still have promoter activity as demonstrated previously .Promoter deletion analyses performed, respectively, in tomato, s plants . The 158eviously . In the CsLCYb1 transcripts accumulate predominantly in leaf and fruit flavedo which contain high proportions of chloroplasts . A similar result was observed in transgenic Arabidopsis expressing the citrus PSY promoter-GUS construct . Some results are consistent with the change of CsLCYb1 gene expression under the same treatments (PSY (the key rate-limiting enzyme in the carotenoid biosynthetic pathway) was affected by various stimuli. Additionally, we found that the GUS expression levels driven by different CsLCYb1 promoter deletions were influenced by NaCl treatment , suggesting that the promoter activity of CsLCYb1 could respond to salt stress. Very weak GUS staining was observed at the mechanical cut ends of some transgenic Arabidopsis tissues , indicating that the promoter activity of CsLCYb1 may be induced by oxidative stress resulting from mechanical wound. Similar results were observed by CCS  gene was dramatically activated by various ROS progenitors under different oxidative stress conditions. The stress-related and hormone-responsive cis-elements predicted in the promoter sequences . A similar finding was previously reported, which suggested that four tandem repeats of a 20 bp sequence in the promoter of the melon cucumisin gene are sufficient to confer fruit-specific gene expression pattern to the minimal promoter, and that the 20 bp sequence contains a regulatory enhancer . Previous studies have reported that Inr-elements and GT-elements are present in the promoter of many light-regulated genes, and the GT-1 motifs are present in the promoter of stress-induced genes (G. lutea lycopene β-cyclase gene (JQ417648), suggesting a common regulatory mechanism. Additionally, the deletion of the 20 bp fragment may disrupt adjacent cis-elements, such as the ARR (Arabidopsis response regulator) transcription factor binding site (NGATT) existing in the enhancer region as four copies. The ARR proteins belong to the GARP superfamily, two members of which have recently been reported to be related to carotenogenesis. One member is the GOLDEN2-LIKE (GLK) gene, which controls the dominant Uniform ripening (U) locus of tomato fruit. Tomato carrying the u mutation produced fruit with defective chloroplasts and low levels of sugar and lycopene (ARABIDOPSIS PSEUDO RESPONSR REGULATOR2-like (APRR2-like) gene, which affects plastid number and size in tomato fruit, and enhances the levels of chlorophyll in immature fruit and carotenoids in red ripe fruit (Arabidopsis and further discovered that the transcription factor RAP2.2 (a member of the APETALA2 (AP2)/ethylene-responsive element-binding protein) interacting with the SINAT2  bound to the ATCTA element to coordinately regulate AtPSY expression. These analyses indicate that the enhancer element identified in this study could be used as a good candidate to seek upstream trans-acting factors.ed genes . These rlycopene . The othpe fruit . Welsch pe fruit , 2007 idODORANT1 (ODO1) gene, a key regulator in the volatile benzenoid pathway in petals, identified an enhancer region which distinguished a fragrant from a non-fragrant petunias cultivar . These differences may affect the expression level of LCYb1 gene, thus resulting in carotenoid diversity among different citrus species. However, this speculation needs to be confirmed by more experiments. Sweet orange is a natural hybrid of pummelo and mandarin , which provides an ideal molecular marker to study the genetic relationship between different species.Previous studies of the cultivar . This stmandarin . In sweehe world . Previouhe world . This sthe world . The seqLCYb1 promoter from citrus, thus facilitating the understanding of the complex regulatory mechanisms of LCYb1 expression in higher plants. The identified novel enhancer element is required for promoter activity; besides, it also can be used as a marker to distinguish different citrus species. These data give a clue to further study of the differences in gene expression among species. The promoter cloned and functionally validated in this study would be an ideal candidate for genetic engineering and seeking of upstream trans-acting elements.This study elucidates the expression characteristics of the SL performed the major experiments and wrote the manuscript. YZ and KZ, assisted in the experiments. XZ give suggestions for writing improvement. QX designed the experiment. XD proposed and supervised the research, and provided valuable comments on the manuscript. All authors read and approved the manuscript.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.


Drosophila Maleless (MLE) protein, it is an NTP-dependent helicase consisting of a conserved helicase core domain, two double-stranded RNA-binding domains at the N-terminus, and a nuclear transport domain and a single-stranded DNA-binding RGG-box at the C-terminus. With an ability to unwind DNA and RNA duplexes, as well as more complex nucleic acid structures, DHX9 appears to play a central role in many cellular processes. Its functions include regulation of DNA replication, transcription, translation, microRNA biogenesis, RNA processing and transport, and maintenance of genomic stability. Because of its central role in gene regulation and RNA metabolism, there are growing implications for DHX9 in human diseases and their treatment. This review will provide an overview of the structure, biochemistry, and biology of DHX9, its role in cancer and other human diseases, and the possibility of targeting DHX9 in chemotherapy.DHX9 is member of the DExD/H-box family of helicases with a “DEIH” sequence at its eponymous DExH-box motif. Initially purified from human and bovine cells and identified as a homologue of the  Drosophila, C. elegans, and Arabidopsis, although the majority of the research has focused on human DHX9. Many of the biological processes in which DHX9 participates are deregulated during oncogenesis, or are hijacked by viruses to promote their own replication. A wealth of recent studies have implicated DHX9 in human diseases such as various cancers and viral infections, and there is evidence supporting the targeting of DHX9 in disease intervention. This review will discuss the structural, biochemical and biological properties of DHX9 as well as its implications in disease.DHX9  and RNA Helicase A (RHA)) is an NTP-dependent helicase protein capable of unwinding both RNA and DNA , as wellvia Mg2+ . Other helicases may act as RNA clamps or chaperones to aid in RNA folding. These functions have made them important players in nucleic acid unwinding and remodeling during replication, transcription, translation, DNA repair, RNA splicing and editing, ribosome biogenesis, RNA transport, and RNA decay , 8. TheyDrosophila melanogaster protein Maleless (MLE) [Drosophila as a gene which when mutated caused lethality in male zygotes, and was found to play a role in X-chromosome dosage compensation in males [C. elegans [Mammalian DHX9 was first purified from the nuclear fraction of calf thymus and designated Nuclear DNA Helicase II (NDHII) . The humss (MLE) , 18. MLEin males , 20. Subin males , C. eleg elegans , and Ara elegans .The human DHX9 gene maps to the prostate cancer susceptibility locus on chromosome 1q25, with a pseudogene, DHX9P, mapping to chromosome 13q22 . The actD. melanogaster, C. elegans, and Arabidopsis homologues respectively  in DExD helicases, is not present in DHX9 and that DHX9 lacks base-selective contacts, thereby enabling it to utilize all four NTPS  for its energy requirements. The region immediately preceding motifs I-III consists of 3 α-helices arranged perpendicular to each other on the surface of the α/β core, while the MTAD region consists of 2 short β-strands which lie in a hydrophobic groove on the surface of the helicase core [The structure of DHX9 has been partially solved. X-ray crystallography was used to determine the structure of domain 1 of human DHX9's helicase core in the presence of ADP and MnThe structure of the two dsRBD domains has also been elucidated. Both dsRBDI and dsRBDII are arranged into a core α-β-β-β-α fold, with the two α-helices lying on one surface of the three-stranded antiparallel β-sheets, and both domains were able to co-crystallize with dsRNA. Interaction with dsRNA is mediated primarily by surface-exposed residues which tend to be highly conserved amongst dsRBDs of various proteins . DespiteIn vitro studies using human, bovine, and Drosophila DHX9 demonstrated that all three homologues are able to bind both DNA and RNA [M values, in concordance with the lack of NTP selectivity (due to the absent Q-motif) demonstrated by structural data [In vitro experiments have shown that human DHX9 also unwinds DNA or RNA forks composed of either partially complementary DNA duplexes or DNA/RNA hybrids respectively [via Hoogsteen base pairing [ and RNA , 17, 34. and RNA . It exhiral data , 34, 36.ectively   is responsible for X-chromosome dosage compensation, a process initiated in early embryogenesis which is critical to male development [DHX9 is an abundant protein and is ubiquitously expressed. Expression is high in the skin, small and large intestines, stomach, pancreas, kidney, breast, skeletal muscle, bone marrow, and reproductive organs. The liver, spleen, lung, heart, smooth muscle, adipose tissues, and lymph nodes show a moderate level of expression –76. DHX9 meiosis . In Droselopment . Homozygelopment . In humaelopment . DHX9 alelopment . Hence, DHX9 has a large number of interacting partners, a reflection of the many biological processes it participates in. Table in vitro experiments illustrating DHX9's ability to unwind complex nucleic acid structures suggest a role in DNA replication and maintenance of genomic stability, since these are transient intermediates that form during replication, transcription, or recombination and need to be resolved. This is supported by several lines of evidence. First, DHX9 associates with numerous proteins involved in DNA replication and/or the DNA damage response. These include the breast cancer specific tumour suppressor protein BRCA1, which remodels chromatin, facilitates orderly homologous recombination, and ensures DNA replication fidelity [The fidelity . The repfidelity –89. DHX9fidelity , 91. DHXfidelity . This deIn vitro characterization of DHX9). These two helicases were found to interact, with the interaction sites mapping to the dsRBDII and RGG domains of DHX9 and the N-terminal exonuclease domain of WRN [in vitro results suggest that DHX9 may be loaded onto these sites, aiding WRN to remove the primer RNA-containing Okazaki fragments and promoting lagging strand maturation. Holliday junction-like intermediates are formed during replication fork stalling [Mechanistic insight into DHX9's role in DNA replication is provided by studies investigating its relationship with WRN, a RecQ ATP-dependent helicase containing both 3′to 5′ helicase and 3′to 5′ exonuclease activity. WRN is involved in DNA replication, recombination, and repair, and defects in this helicase are a cause of Werner's syndrome, a rare autosomal recessive genetic disorder characterized by premature aging, increased genomic instability, and increased cancer susceptibility. In terms of substrate specificities, DHX9 shares many similarities with WRN /p300 and RNA PolII. During CBP/p300-mediated transcriptional activation, phosphorylation of the cAMP response element-binding protein (CREB) results in complex formation with co-activator CBP/p300 and binding to a cAMP responsive element (CRE) in the promoter of specific genes . DHX9 bithe MTAD .via its C-terminal RGG region, and its catalytic activity is not required [Other instances of human DHX9 serving as a bridging factor between PolII and transcriptional co-factors have been uncovered. DHX9 links PolII to BRCA1, which in addition to its roles in DNA replication and DNA repair, also functions in transcriptional regulation . Bindingrequired .DHX9 participates in nuclear factor-κB (NF-κB)-mediated transcriptional activation, where members of the NF-κB family including p65, RelB and c-Rel upregulate transcription in response to exposure to a variety of inducers, such as interleukin-1 and tumour necrosis factor. DHX9 directly binds p65 and enhances NF-κB-dependent transcriptional activation, an event dependent on functional NTPase/helicase activity . Since pcis-acting element in the MDR1 promoter [via an AT-rich sequence (ATRS) in the promoter of target genes [DHX9 also binds directly to promoters in a sequence specific manner. It enhances transcription of the tumour suppressor p16INK4A by binding specifically to the sequence 5′CGGACCGCGTGCGC3′ within its promoter . Anotherpromoter . As wellet genes . EGFR-reet genes .via its dsRBDII to stimulate HIV-1 transcription, a process that is dependent on DHX9's ATPase and PolII-binding activities [DHX9 can interact with RNA to regulate transcription. It has been found to bind both the insulin-like growth factor 2 (IGF2) mRNA and miR-483p, a microRNA that enhances IGF2 transcription, promoting the miR-483-5p-mediated induction of IGF2 mRNA . In an etivities , 134.C. elegans homologue, RHA-1, enables normal germline proliferation and development by silencing transcription. A temperature-sensitive mutant, RHA-1(tm329), caused loss of lysine 9 histone H3 methylation  and resulted in transcriptional desilencing. This resulted in defects in germ cell mitosis, meiosis, and gametogenesis, leading to a sterile phenotype [In addition to activating transcription, it appears that in some situations DHX9 can also repress it. Association with the transcriptional activator TonE (tonicity-responsive enhancer)-binding protein (TonEBP) inhibits TonEBP activity . Althoughenotype . Again, via conserved lysine residues in the distal α-helices of the two dsRBDs and facilitates translation of said mRNAs by stimulating polyribosome incorporation [via interaction with the poly(A)-binding protein (PABP), although this has not been verified [Beyond its role in transcription, DHX9 also participates in regulation of gene expression at the translational level. Many mRNAs contain highly structured 5′UTRs, a feature that represents an impediment to translational initiation, the rate-limiting step of cap-dependent translation. The eIF4A (DDX2) subunit of the eIF4F complex is responsible for unwinding structures to facilitate ribosome recruitment to the mRNA template –138. Howporation , 142. Thporation . Associaporation . It has verified . Since Jvia both its N- and C- terminal regions. This interaction promotes DHX9 association with polysomes and stimulates translation of Oct4 mRNA [DHX9 also facilitates translation of type I collagen, another mRNA with a unique 5′UTR structural element. Type I collagen is a heterotrimer composed of two α1(I) polypeptides and one α2(I) polypeptide. The mRNAs encoding both polypeptides contain a unique 5′ stem-loop structure (5′sL) in their 5′UTR. La ribonucleoprotein domain family member 6 (LARP6) is known to bind with high affinity to the 5′sL . DHX9 doct4 mRNA , 151. Itct4 mRNA .Recent studies indicate that DHX9 also helps regulate IRES-mediated translation. Exposure to DNA damaging agents causes stabilization of the p53 tumour suppressor protein. However, recent evidence has shown that p53 translation is also increased upon DNA damage –154. It Involvement in microRNA (miRNA) biogenesis and processing is another means by which DHX9 regulates post-transcriptional gene expression. It associates with pri-miRNA, and along with its binding partner, BRCA1, forms part of the DROSHA microprocessor complex, which also contains DGCR8, DDX5, and DDX17. The concerted action of DHX9 and BRCA1 enhances processing of the pri-miRNA into their mature forms . FurtherDrosophila. In a process distinct from its role in dosage compensation, MLE links editing and splicing of the para sodium channel pre-mRNA. A mutation near the NTP-binding site of MLE results in aberrant splicing and exon skipping, again suggesting that the Drosophila DHX9 homologue may act to resolve secondary structures concealing splice sites [As described above, human DHX9, while normally excluded from the nucleolus, can shuttle into said compartment under conditions of transcriptional inhibition or cellular stress , 71, 72.ce sites , 168.A role in RNA transport is suggested by the observation that DHX9 binds directly to both filamentous actin (F-actin) and hnRNP C1 in the nucleus, mediating their association . F-actinThe role played by DHX9 in a multitude of cellular processes, including multiple levels of gene regulation, and its association with a large number of key regulatory binding partners, makes it an important protein in maintaining normal cellular homeostasis. However, this also means that defects in DHX9 may have serious effects on cell growth or viability, and usurpation of its functions may lead to a variety of human diseases.The human DHX9 gene maps to the major susceptibility locus for prostate cancer at chromosome band 1q25 , and DHXAs discussed previously, DHX9 interacts with EGFR to activate transcription of EGFR-responsive genes. EGFR is an oncogene overexpressed in several human cancers and drugs targeting EGFR are in clinical use  –187. An DHX9 is also implicated in osteosarcoma and Wilms' tumour. Gene expression profiling of osteosarcoma cell lines showed overexpression of DHX9 in cells with high metastatic ability compared to those with low metastatic ability. Several other genes in the NF-κB pathway were upregulated as well; since DHX9 is a NF-kB binding partner, this supports a role for NF-κB signaling in osteosarcoma metastasis . DHX9 coAlthough much research implicates DHX9 as a promoter of tumourigenesis, there are also indications that it has tumour-suppressive properties. Its ability to unwind aberrant polynucleotide structures and to aid WRN in ensuring the fidelity and efficiency of DNA replication shows that it plays a role in maintaining genomic stability. It also activates transcription of p16INK4A, a tumour suppressor . Its coo−/−ArfEμ-myc/Bcl-2 mouse lymphoma cells, which overexpress MYC and exogenous BCL-2 and are resistant to ABT-737, a shRNA screen performed in search of sensitizers to ABT-737 uncovered DHX9 as a synthetic lethal hit [−/−ArfEμ-myc/Bcl-2 model recapitulates several clinically relevant features of non-Hodgkin's lymphomas; DHX9 may therefore show promise as a candidate target to suppress in combination with ABT-737 or its derivatives. Knockdown of DHX9 also synergizes with the glucocorticoid dexamethasone in human multiple myeloma cell lines [Recent research has supported the notion of targeting DHX9 as a chemotherapeutic approach. DHX9 was identified as a modifier of ABT-737 resistance in a mouse lymphoma model . ABT-737thal hit . DHX9 suthal hit . The Arfll lines , 194, suex vivo and in vivo and extended survival of mice harbouring these tumours [Aside from the targeting of DHX9 in a combinatory approach, it has also effectively been targeted as a single agent. In a representative panel of human cell lines derived from different types of cancer, including multiple myeloma, osteosarcoma, breast, lung, and cervical cancers, suppression of DHX9 was lethal in the majority of these lines . This wa tumours .Ewing's sarcoma family tumours (ESFTs) is a pediatric cancer driven by a t chromosomal translocation which fuses the 5′ transactivation domain of EWS with the 3′ ETS domain of the transcription factor FLI1. The resulting fusion protein, EWS-FLI1, is oncogenic and acts as a potent aberrant transcription factor . DHX9 inversus normal tissues may be due to a higher dependency of the former on DHX9. Replication, transcription, translation, and many other biological processes are often deregulated in cancer, and since DHX9 is an important player in all of these processes, cancer cells may become “addicted” to DHX9. It should be noted that the shRNA mouse model used in the aforementioned study represents partial inhibition of DHX9. Because of the importance of DHX9 in many regulatory processes, it is quite possible that total loss of DHX9  may be detrimental to the adult organism. A straight knock-out mouse model showed that DHX9 is embryonic lethal, but does not inform on the consequences of acute DHX9 loss in the adult [In evaluating the feasibility of targeting DHX9 in as a neoplastic approach, two important considerations must be taken into account: (a) The availability of DHX9 inhibitors and (b) an achievable therapeutic index. At the moment, a specific inhibitor of DHX9 activity has not been identified - the approaches for curtailing DHX9 expression in the scenarios discussed above used shRNAs. However, attempts have been made to remedy this. A primary screening assay was recently developed to uncover inhibitors of DHX9 activity, where it was found that aurintricarboxylic acid prevents DHX9-mediated ATP hydrolysis . Althoughe adult . If DHX9Viruses can hijack various aspects of the host cell machinery for their own purposes. This includes using host proteins to facilitate the replication, transcription, translation, or transport of their own nucleic acids or proteins. DHX9 interacts with the viral TAR RNA to stimulate HIV-1 transcription , 134. AsSurprisingly, DHX9 has also been implicated in antiviral immune responses. For example, it is recruited to PML bodies in response to IFN-β stimulation and is phosphorylated by the dsRNA-binding kinase PKR , 133. ItDHX9 knockdown induces a pronounced p53-dependent growth arrest and premature senescence in primary human fibroblasts . This isSystemic lupus erythematosus (SLE) is an autoimmune disease in which the body launches an immune response against healthy tissues. It is characterized by the generation of antibodies against the body's own proteins, termed autoantigens, most of which are nuclear proteins. This leads to inflammation and complications such as skin rashes, photosensitivity, and atherosclerosis . DHX9 waDHX9's interaction with an extensive and varied array of nuclear and cytoplasmic protein and nucleic acid partners indicates that it holds a privileged position as a central regulator of gene expression. Although much has been uncovered about DHX9 in the past three decades, there is still a lot of information to be gained with respect to its structure, substrates, binding partners, and functions. Future work should focus on obtaining greater mechanistic insight on DHX9 - for example, elucidating the processivity of its helicase activity, why it prefers certain substrates to others, and how it is able to recognize and bind to specific sequences or structures on DNA or RNA. As well, further characterization of non-human homologues would be of interest to determine if many of the functions and binding partners observed in humans are conserved across different species, or if there may be yet-undiscovered species-specific activities. Finally, the potential of using DHX9 as an anti-neoplastic or anti-viral target is still in its infancy. Additional studies characterizing the mechanistic consequences of DHX9 overexpression or mutation in various cancers, as well as studies in animal models of disease, would be beneficial in understanding the role of DHX9 in tumourigenesis, and the identification of DHX9 inhibitors would contribute to the feasibility of targeting DHX9 in chemotherapy.


Bioessays published by WILEY Periodicals, Inc.The third annual meeting of the EpiGeneSys network brought together epigenetics and systems biologists to report on collaborative projects that apply quantitative approaches to understanding complex epigenetic processes. The figure shown represents one meeting highlight, which was the unexpected emergence of genotype versus epigenotype in control of cell state. The European Commission network of excellence “EpiGeneSys” has been in operation since 2010 and the third annual meeting took place at the Babraham Institute in Cambridge from 4th to 6th December 2013. EpiGeneSys is an ambitious project studying epigenetics with an emphasis on systems biology. The marriage of these two disciplines is desirable because systems biology approaches are essentially designed to make sense of biological systems that are highly dynamic and involve a plethora of components, both of which are features of epigenetics.In addition to the 22 permanent members of EpiGeneSys, there are 20 RISE1 (research integrating epigenetics and systems biology) members, who are early career stage researchers, and 97 associates. The meeting in Babraham gave this illustrious group of scientists and their lab members the opportunity to come together for a few days of excellent talks and discussions to catch up on the scientific highlights of the network, as well as the network's other functions such as training, career development and public engagement.The research of EpiGeneSys is divided into four main areas covering dynamics of epigenetic regulators, the relationship between genotype and epigenotype, the influences of environment and nutrition on the epigenome and finally, building an integrated computational epigenetics framework. At the Babraham meeting there were highlights from all of these areas, and in this report we present a selection to illustrate key issues under discussion and to indicate the future trajectory of EpiGeneSys research.A central theme of the meeting was the extent to which genotype can influence epigenotype. Adrian Bird  suggested that we should consider CpG islands in the context of “CG signaling” rather than focusing on CpG methylation. Bird challenged the audience to think about a reader of CG signalling, CFP1, as a sequence‐specific DNA binding protein, even though CG is clearly a very short sequence, that is able to influence epigenetic modifications by recruiting histone‐modifying machinery. Examination of artificial DNA insertions showed that altering GC‐content and CpG density of an ectopic locus is sufficient to establish different epigenetic states Hmr) from Drosophila melanogaster and lethal hybrid rescue (Lhr) from Drosophila simulans, causes hybrid incompatibility due to mislocalisation of an HMR/LHR complex throughout the genome, altered regulation of transposable elements and impaired cell proliferation. Levels of HMR and LHR are different between the two Drosophila species, which may have arisen as a response to species‐specific changes in the abundance of transposable elements that occurred during divergent evolution.Evolutionary diversity can also be used effectively to explore the relationship between DNA sequence and epigenetic landscape. Axel Imhof  presented work about the molecular basis for species divergence The relationship between genetics and epigenetics is not all one‐way however; Daniel Rico  described how the timing of DNA replication can define the rate at which copy number variants arise. He demonstrated that replication timing reflects the evolutionary age of duplicated genes, and that the late replicating regions of genomes tend to be newer. Replication timing, an almost completely epigenetic property, allows fast and slow evolving regions to be defined. Consistently, primate‐ and rodent‐specific genes tend to be in late replicating regions, suggesting that this conserved epigenetic property controls the rate of genetic change and evolutionary innovation Drosophila cells. New data presented at the meeting showed that the same five chromatin types are also observed in human K562 cells, revealing that many common features at the genome‐wide level are conserved between Drosophila and human cells. Filion extended these studies by performing the same analysis for human embryonic stem cells (ESC), and although broadly similar, several interesting differences were observed. “Black” chromatin, for example, which is characterised by the absence of DNA binding factors, is detected with a similar abundance between K562 and ESC, yet shows relatively poor overlap in its location within the genome. These findings remind us that chromatin state must be defined by more than DNA sequence alone.Connections between underlying genomic features and the epigenome are also revealed by genome‐wide analyses of chromatin states. Guillaume Filion  has previously used computational classification strategies to interrogate combinations of proteins and epigenetic marks, and reported that five major types of chromatin can be distinguished in There are situations where chromatin state, however, can be affected by genetic changes depending on genome position. Gael Yvert  described experiments comparing histone acetylation patterns in three different yeast strains. Nucleosome positions and acetylation patterns varied in complex ways; sites with conserved nucleosome positioning and conserved acetylation were observed, but sites showing conservation of neither or only one of these properties were also readily detected. To elucidate genetic and epigenetic contributions to this heterogeneity, histone acetylation was stripped by drug treatment and then allowed to re‐establish over a number of generations. Quantitative differences in levels of histone modifications were found to be either “labile” or “persistent” depending on whether they were re‐established or not after drug treatment. Remarkably, “persistent” variations correlated well with those linked to genetic determinants whereas “labile” variations did not, showing clear contributions of both genetic and epigenetic information to intra‐species variation of the histone modifications landscape FXN gene. The expanded triplet repeat tract seeds an aberrant region of heterochromatin, which spreads bidirectionally, leading to transcriptional silencing of the FXN gene. Importantly, heterochromatinisation could be inhibited using the histone deacetylase inhibitor nicotinamide. Administration of nicotinamide to a cohort of Friedreich's Ataxia patients in a Phase II clinical trial led to a significant increase in FXN mRNA and protein levels, proving the concept that drugs targeting epigenetics can have clinical applications The influence of the genome on the epigenome is of more than theoretical importance. Cihangir Yandim  reported the results of a study on the autosomal recessive disorder Friedreich's Ataxia, which is caused by an expansion of GAA triplet repeats in an intron of the Tsix transcription, thereby limiting Xist expression. In contrast, NSL positively regulates pluripotency factors, including Oct4 and Sox2, in turn leading to suppression of Xist expression. These findings reinforce the concept that multiple mechanisms are used to ensure correct dosage regulation during development.Dosage compensation in various species has long provided a paradigm for epigenetic regulation of gene expression. Asifa Akhtar  showed that two related MOF‐containing complexes, MSL and NSL, can control dosage compensation in mouse ESC, but achieve it through two distinct pathways Arabidopsis thaliana. But the important question now addressed by the Dean group is how this process is reset every generation? They screened for mutants impaired in the epigenetic reprogramming of FLC. In one hypomorphic mutant, FLC is switched on, but fails to reach wild‐type levels. The mutated gene was identified as Elf6, encoding a jumonji C domain protein likely to be an H3K27me2/3 demethylase. H3K27me3 levels remain elevated in mutant seeds, therefore demethylase activity is required to erase H3K27me3 in the resetting between generations Caroline Dean  addressed how environmental signals can influence gene expression and phenotype. An interesting epigenetic system allows environmental signals perceived at one stage to be “remembered” until later in development. Vernalisation, the promotion of flowering by cold, involves gradual PcG‐mediated epigenetic silencing of floral repressor FLC in Numerous studies have shown that epigenetic marks can be deposited by non‐protein coding RNAs (ncRNAs), particularly those involved in transcriptional repression and heterochromatin formation. This has been well studied at fission yeast centromeres, where ncRNAs from the outer centromeric repeats are processed by the RNA interference (RNAi) machinery to form siRNAs. These siRNAs direct the formation of centromeric heterochromatin, but the connection between the RNAi machinery and the chromatin modifying enzymes only became clear with the recent discovery of the connector protein Stc1. Elizabeth Bayne  reported a structural analysis of Stc1, showing that the zinc finger domain interacts directly with Argonaute while the C‐terminal interacts with the CLRC silencing complex, providing the molecular basis of the connection between RNAi and heterochromatin formation How the RNAi machinery selects targets for processing into siRNA and hence defines heterochromatin domains has remained somewhat mysterious, as many genomic regions produce sense and antisense RNAs that could technically form RNAi substrates. Using an RNAi system reconstituted in budding yeast, Jon Houseley  demonstrated that transcripts from high‐copy genomic regions are inherently better at forming double‐stranded RNA than those from single copy regions. This unexpected influence of genome sequence on an apparently epigenetic process provides a simple answer to an old problem, as selective silencing of high‐copy regions has long been thought to require a mechanism by which the cell can count DNA copies. To be effective however, this RNAi‐mediated silencing mechanism would require the whole genome to be transcribed, providing a potential function for recently discovered pervasive transcription of eukaryotic genomes XIST, but human ESC express an apparently antagonistic ncRNA, XACT, which coats the active X‐chromosome. X‐inactivation is variable in human ESC compared to mouse, potentially reflecting a balance between XIST and XACT expression, and here it was shown that XACT expression precedes XIST loss and X‐reactivation, and that XACT is present on both X‐chromosomes in embryos when both Xs are active.Mammalian cells also use ncRNAs to control transcriptional silencing. In this area, Claire Rougeulle  described recent work from her laboratory regarding the control of X‐inactivation in human ESC. X‐chromosome inactivation in mammalian cells is orchestrated by the ncRNA Single cell sequencing approaches have the potential to revolutionise epigenetics. Heather Lee (Babraham Institute) revealed that even apparently homogenous ESC cultures in fact show distinct epigenetic heterogeneity. However, some cell populations are indeed highly homogenous; Sebastien Smallwood (Babraham Institute) presented the first genome‐wide methylomes from single cells, in this case from oocytes, which showed very little sample‐to‐sample variability providing an important validation of single cell methodologies Platynereis, combined with in situ profiling for selected marker genes, allowed reconstruction of the expression profile of the brain with single cell resolution. Expanding such techniques to more complex organisms is highly challenging. However, Andreas Ladurner (Ludwig Maximilians University of Munich) presented surprisingly effective approaches for profiling selected neuronal cell populations in Drosophila. By expressing tagged ribosomes, RNA polymerases or histones using specific GAL4 driver lines, it was possible to measure gene expression and chromatin structure changes in specific sets of neurons, glia and adipocytes within the fly head. This was used to demonstrate how gene expression changes when a fly is fed normally or fasted.John Marioni  described how single cell expression profiling of brain cells from the marine annelid It was exciting to see the emergence of an unexpected theme at this meeting: the influence of DNA sequence on the epigenome. This concept unites many of the core research areas of EpiGeneSys and was a common discussion point during the coffee breaks. Overall, a model emerges whereby epigenetic processes coordinate nuclear function and transcriptional states, but on one level these states can be defined by DNA sequence elements and their associated binding proteins. Of course, external signalling events require changes in the transcriptional output of the genome and trigger changes in epigenetic profiles, however, these profiles are constrained by underlying sequence considerations Fig. . Hence,