MOS14 encodes a nuclear protein with high similarity to previously characterized TRN-SR proteins in animals. Yeast two-hybrid assays showed that MOS14 interacts with AtRAN1 via its N-terminus and SR proteins via its C-terminus. In mos14-1, localization of several SR proteins to the nucleus was impaired, confirming that MOS14 functions as a TRN-SR. The mos14-1 mutation results in altered splicing patterns of SNC1 and another R gene RPS4 and compromised resistance mediated by snc1 and RPS4, suggesting that nuclear import of SR proteins by MOS14 is required for proper splicing of these two R genes and is important for their functions in plant immunity.Transportin-SR (TRN-SR) is a member of the importin-β super-family that functions as the nuclear import receptor for serine-arginine rich (SR) proteins, which play diverse roles in RNA metabolism. Here we report the identification and cloning of  Resistance (R) genes play essential roles in defense against pathogens. Multiple R genes are alternatively spliced. How plants regulate the splicing of these R genes is unclear. In this study, we identified MOS14 as an important regulator of two R genes, SNC1 and RPS4. Further analysis showed that MOS14 functions as the nuclear import receptor for serine-arginine rich (SR) proteins, which play diverse roles in RNA metabolism. Loss of the function of MOS14 results in altered splicing patterns of SNC1 and RPS4 and compromised resistance mediated by snc1 and RPS4, suggesting that nuclear import of SR proteins by MOS14 is required for proper splicing of these two R genes and is important for their functions in plant immunity.Plant immune receptors encoded by  MOS3, MOS6, MOS7 and MOS11 suppress the constitutive defense responses of snc1 , a gain-of-function mutant carrying a mutation in a Toll/interleukin-1 receptor-Nucleotide Binding-Leucine Rich Repeat (TIR-NB-LRR) R protein MOS3 encodes the nucleoporin Nup96 MOS11 encodes a putative RNA binding protein MOS6 encodes a putative importin-α MOS7 encodes another nucleoporin, Nup88, which is required for nuclear accumulation of snc1 and two general defense regulators, Enhanced Disease Susceptibility 1 (EDS1) and Nonexpresser of PR genes 1 (NPR1) In eukaryotes, the nuclear envelope forms a barrier between the cytoplasm and the nucleus. Trafficking of macromolecules across the nuclear envelope occurs through the nuclear pore complex (NPC) Nuclear import receptors play essential roles in transferring proteins from the cytoplasm to the nucleus. The largest group of nuclear import receptors belong to the importin-β super-family. Members of the importin-β super-family have rather low overall sequence similarity but they all have a conserved N-terminal RAN-binding domain The importin-β super-family can be divided into several sub-families according to the direction and the cargo type they transport SR proteins are a highly conserved family of nuclear proteins that play important roles in splicing R genes including the tobacco N gene Mla6SNC1RPS4RPS4RPS4-mediated resistance resulting from a lack of the TVs suggests that alternative splicing of RPS4 is required for its function R genes is controlled and why it is necessary for immunity. In this study, we report that Arabidopsis MOS14 encodes a TRN-SR that is required for proper splicing of SNC1 and RPS4, suggesting that SR proteins may play important roles in the control of the splicing of these two R genes.Several plant snc1 constitutively activates defense responses and displays enhanced resistance to pathogens. snc1 mutant plants exhibit dwarf morphology with curly leaves. Suppressor screens of snc1 have previously been carried out using fast neutron and T-DNA insertional mutagenesis snc1, we treated snc1 npr1 seeds with ethane methyl sulfonate (EMS) and screened the M2 plants for mutants that suppressed snc1 dwarfism. From this population, we identified mos14-1 snc1 npr1  Noco2. As shown in H. a. Noco2 is lost in mos14-1 snc1 npr1.In  mos14-1 . To testmos14-1 mutation, we crossed mos14-1 snc1 npr1 (in the Columbiaecotype background) with Landsberg erecta (Ler)-snc1mos14-1 is linked to the lower arm of chromosome 5  and C-terminus (282–958) of MOS14 were expressed in the bait vector and four selected Arabidopsis SR proteins  were expressed in the prey vector for yeast two-hybrid assays. As shown in mos14-1 mutation affects the nuclear import of Arabidopsis SR proteins, we made constructs expressing four SR genes AtRS2Z33, AtRSZ21, AtRS31 and AtSR34 with a C-terminal GFP tag. These constructs were transformed into protoplasts of wild type and mos14-1 plants to check for the localization of the SR-GFP proteins. A construct expressing the SARD1-GFP fusion protein was included as the control mos14-1 protoplasts, SARD1 was localized in the nucleus. Consistent with previous studies mos14-1 protoplasts, the SR-GFP proteins were mainly localized in the cytoplasm  DC3000 avrRps4 in mos14-1 is about ten-fold higher than that in wild type, suggesting RPS4-mediated immunity is compromised in mos14-1. We also tested whether MOS14 is required for basal resistance by challenging the mos14-1 plants with the virulent pathogen P.s.t. DC3000. As shown in mos14-1 compared to wild type, indicating that MOS14 is also required for basal resistance.Since snc1 suppressor mutants revealed that multiple components are involved in the regulation of plant immunity. In particular, pathways involved in mRNA export, protein import and protein export were found to contribute to immune responses. Here we report the identification of MOS14 as a novel component of nucleocytoplasmic trafficking required for plant immunity. Loss of MOS14 function suppresses the constitutive defense responses of snc1, compromises resistance mediated by RPS4 and impairs basal resistance against P.s.t. DC3000. These findings show that MOS14 plays a critical role in plant immunity.Previous studies on MOS14 encodes a nuclear protein with high sequence similarity to TRN-SR proteins in animals. TRN-SR proteins have been shown to function as nuclear import receptors for both phosphorylated SR proteins as well as the splicing repressor protein RSF1 which antagonizes SR proteins in the nucleus MOS14 is a single-copy gene, while the Arabidopsis genome has 18 genes belonging to six subfamilies of SR proteins, of which three are plant-specific RSF1 in Arabidopsis. Like the TRN-SR proteins in animals, the N-terminus of MOS14 interacts with AtRAN1 and the C-terminus interacts with SR proteins. In addition, localization of several SR proteins to the nucleus was impaired by mos14-1. These data indicate that the mechanism of nuclear import of SR proteins is conserved between plants and animals.C.elegans, RNAi of the MOS14 homolog Transporter of SR-1 (TSR-1) leads to embryonic lethality, suggesting TRN-SR proteins can be essential for viability mos14-1 mutation is not lethal, although it does cause multiple development phenotypes such as reduced stature and fertility. In addition to its functions in development, our genetic analysis of MOS14 revealed that it plays important roles in both R gene-mediated resistance as well as basal defense, suggesting that nuclear import of SR proteins is important for plant immunity. The reasons why mos14-1 leads to these pleiotropic defects and not lethality awaits further investigation.Very limited studies have been performed on the genetic characterization of TRN-SR proteins. In mos14-1 mutation affects the splicing of SNC1 and RPS4. Several R genes including SNC1, RPS4 and tobacco N gene are alternatively spliced, and alternative splicing of RPS4 and N gene are required for their function mos14-1, alternative splicing of both SNC1 and RPS4 are altered. This effect probably contributes to the suppression of snc1 mutant phenotypes by mos14-1 and compromised RPS4 function in the mos14-1 single mutant. In addition to the altered ratio of transcript variants, the expression levels of snc1 and RPS4 were also reduced. The reduced expression of snc1 and RPS4 is probably caused by splicing defects resulting from the reduced nuclear localization of SR proteins.SR proteins play important roles in general RNA splicing, alternative splicing, as well as other processes of RNA metabolism. Consistent with the function of MOS14 in the nuclear import of SR proteins, the mos14-1 snc1 npr1, the SNC1 TV-4 transcript level is only modestly reduced, suggesting that reduced accumulation of TV-4 may not be the only factor that contributes to the complete suppression of snc1 mutant phenotype. In addition to reduced accumulation of TV-4, levels of SNC1 TV-1, TV-2 and TV-3 are considerably increased in mos14-1 snc1 npr1. These transcripts are predicted to produce truncated snc1 proteins because of introduction of early stop codons. It is possible that these truncated proteins may interfere with the function of the full-length snc1. Because snc1 and RPS4 are not the only genes whose splicing are affected by mos14-1, altered splicing of one or more unknown positive regulators of plant defense could also contribute to the suppression of snc1 mutant phenotypes.In P.s.t. DC3000 is also compromised in mos14-1. It remains to be determined how mos14-1 affects basal resistance. One possibility is that MOS14 is required for the splicing of one or more R genes that contribute to basal resistance against P.s.t. DC3000. Alternatively, mos14-1 may cause splicing defects in defense regulators required for basal resistance.In addition to the compromised resistance responses mediated by snc1 and RPS4, basal resistance against R gene splicing by SR proteins is critical for plant immunity. Future studies on individual SR proteins will help us better understand how SR proteins regulate the splicing of R genes.In summary, we have identified MOS14 as a nuclear transporter of SR proteins. Our data suggest that regulation of snc1, snc1 npr1 seeds were mutagenized with EMS. About 30,000 M2 plants representing about 1,500 M1 families were screened for suppression of the dwarf morphology of snc1 npr1-1. Mutants lacking the dwarf phenotype were further analyzed for suppression of the constitutive defense responses in snc1 npr1.All plants were grown at 23°C under 16 hr light/8 hr dark in plant growth rooms or chambers, if not specifically mentioned. To identify mutations that suppress the mutant phenotypes of ROC1. Real-time PCR was carried out using Takara SYBR® Premix Ex Taq™ II. The primers for real-time PCR analysis of PR1, PR2, SNC1ROC1  ROC1 is a housekeeping gene without introns. The sequences of primers used for SNC1 and RPS4 transcript variants analysis are shown in U1-70KAtSR30 and AtSR34About 0.1 g tissue was collected and RNA was extracted by Takara RNAiso reagent. The RNA was treated with Promega RQ1 RNase-Free DNase to remove contaminating genomic DNA. Reverse transcription was subsequently carried out using oligo-dT and the M-MLV RTase cDNA synthesis kit from Takara. About 200 ng of total RNA was included in each RT reaction. For semi-quantitative and real-time PCR , one fiftieth of the cDNA was used in each reaction. A total of 40 cycles were performed for semi-quantitative RCR except 28 cycles for H. a. Noco2, three-week-old soil-grown plants were sprayed with H. a. Noco2 at 5×104 spores/ml. The inoculated seedlings were subsequently kept in a growth chamber with high humidity (>80%) at 18°C under 12 hr light/12 hr dark cycle for seven days before growth of H. a. Noco2 was quantified, as previously described For infections with P.s.t. DC3000 or P.s.t. DC3000 avrRps4, five-week-old soil-grown plants were infiltrated with bacterial suspensions (OD600 = 0.001) in 10 mM MgCl2. Samples were taken at day 0 and day 3.For infections with To analyze the SA levels in the mutant plants, SA was extracted using a previously described procedure MOS14 is located were amplified from wild type genomic DNA. The primers used for amplification of F12R37, F14R38 and F23R19 are F12, R37, F14, R38, F23 and R19 respectively, and their sequences are provided in pMOS14:MOS14. The final construct containing MOS14 was transformed into mos14-1 snc1 npr1 through Agrobacterium-mediated transformation.For the transgenic complementation test, three PCR fragments, F12R37 (3.9K), F14R38 (3.8K) and F23R19 (2.9K) covering the 11 kb region where GFP to obtain pMOS14:MOS14-GFP.For the subcellular localization study of MOS14, PCR fragments F12R37 (3.9K), F14R38 (3.8K) and F23R20 (2.9K) were sequentially sub-cloned into pBluescript SK+. The primers used for amplification of F23R20 are F23 and R20 and their sequences are listed in the AtRS2Z33, AtRSZ21, AtRS31 and AtSR34 without the stop codons were amplified by PCR and cloned into the modified pUC19 vector pUC19-35S-cmGFP4 that expresses GFP under the 35S promoter.For transient expression of SR proteins in protoplasts, full-length cDNAs of snc1 cDNA, full-length snc1 cDNA was amplified from total cDNA of snc1 and cloned into a modified pGreen0229 vector. The cDNA clone was sequenced to make sure the sequence is correct and no intron was retained.To obtain transgenic plants expressing AtSR34 and AtRSZ21 without the stop codons were amplified by PCR and cloned into a modified pCambia1300 vector expressing C-terminal tagged GFP under 35S promoter. The constructs were transformed into Col-0 and mos14-1 through Agrobacterium-mediated transformation.To obtain transgenic plants expressing AtSR34-GFP and AtRSZ21-GFP, full-length cDNAs of AtRS2Z33, AtRSZ21, AtRS31 and AtSR34 were amplified by PCR and cloned into pGADT7a. cDNA expressing the N-terminal or C-terminal region of MOS14 were also cloned into pGADT7a. The plasmids expressing the MOS14 fragments were co-transformed with the vectors expressing AtRAN1 or one of the SR proteins into the yeast strain PJ694α for yeast two-hybrid analysis.To make constructs for the yeast two hybrid assays, an SfiI restriction site was first introduced to the multiple cloning site of pGBKT7 and pGADT7 to obtain pGBKT7a and pGADT7a, respectively. cDNA expressing the N-terminal or C-terminal region of MOS14 and AtRAN1 were amplified by PCR and cloned into pGBKT7a. Full-length cDNAs of 2O for at least three times. The concentration of PI used for staining the roots was 10 µg/ml, whereas the concentration of PI used for the leaves is 10 mg/ml. The stained sample was observed using a Zeiss Meta 510 confocal microscope. Excitation wavelengths for GFP and PI were 488 nm and 543 nm, respectively. For root samples, the emission filter used for PI was LP560 nm. For leaf samples, the emission filter used for PI was BP560 nm-615 nm. For both root and leaf samples, the emission filter for GFP was BP505 nm-530 nm.For confocal fluorescence microscopy analysis of MOS14-GFP, the roots or leaves of six-day-old seedling grown on MS plates were first stained with propidium iodide (PI) for 1 min and then washed in ddHmos14-1 protoplasts was performed as previously described Plasmids used for protoplast transfections were purified with Invitrogen PureLink™ HiPure Plasmid Filter Purification Kit. Transformation of wild type or Sequence data from this article can be found in the Arabidopsis Genome Initiative or GenBank/EMBL databases under the following accession numbers: At5g62600 (MOS14), At2g14610 (PR1), At3g57260 (PR2), At4g38470 (ROC1), At2g37620 (Actin1), At5g44340 (β-tubulin4 ), AAD38537 (hTRN-SR1), CAB42634 (hTRN-SR2), NP608708 (dTRN-SR), AF025464 (TSR1) and CAA99366 (MTR10a).Figure S1MOS14 wild type cDNA and the cDNA variants from mos14-1. Red asterisk indicates the site of the point mutation in mos14-1.Alignment of (PDF)Click here for additional data file.Figure S2mos14-1, and mos14-1 carrying the MOS14 transgene.Morphology of five-week-old soil-grown plants of Col-0 (WT), (PDF)Click here for additional data file.Figure S3Drosophila, TSR1 from C.elegans, MTR10a from S.cerevisiae and MOS14 were aligned by the CLUSTALW program  and the multiple sequence alignment result was further analyzed by the BOXSHADE software (http://www.ch.embnet.org/software/BOX_form.html). The importin β N-terminal domain is underlined.Alignment of MOS14 and transportin-SRs from other eukaryotes. Amino acid sequences of hTRN-SR1 and hTRN-SR2 from human, dTRN-SR from (PDF)Click here for additional data file.Figure S4mos14-1, and mos14-1 carrying the MOS14-GFP transgene.Morphology of five-week-old soil-grown plants of Col-0 (WT), (PDF)Click here for additional data file.Figure S5snc1, and three representative T1 transgenic plants expressing the snc1 cDNA under 35S promoter in Col-0 wild type background.Morphology of three-week-old Col-0 (WT), (PDF)Click here for additional data file.Figure S6SNC1 in wild type (WT), npr1-1, snc1 npr1 and mos14-1 snc1 npr1. (A) Gene structure of 5′ end of SNC1. Exons are indicated with boxes and introns are indicated with lines. Locations of the primers used to amplify the transcript variants (TV) are indicated. (B) Transcription patterns of SNC1 in wild type (WT), npr1-1, snc1 npr1 and mos14-1 snc1 npr1. PCR was performed on DNase I-treated total RNA. RNAs incubated in reverse transcription reaction without RTase M-MLV were used as the negative control to ensure that genomic DNA contamination did not occur. Primers F203 and R204 were used to detect transcripts with or without the second intron (upper panel). Primers F205 and R206 were used to detect transcripts with or without the third intron (lower panel). Primers are listed in Analysis of alternative transcripts of (PDF)Click here for additional data file.Figure S7Actin1, β-tubulin4, U1-70K, AtSR30, AtSR34 and ROC1 in wild type (WT) and mos14-1. Primers used to amplify Actin1 and β-tubulin4 are listed in Analysis of transcripts of (PDF)Click here for additional data file.Figure S8SNC1 and RPS4 splicing patterns in mos14-1 and mos14-1 snc1 npr1 by the MOS14 transgene. (A–B) SNC1 splicing patterns in the indicated genotypes. Primers used were F203 and R206. (C–D) RPS4 splicing patterns in the indicated genotypes. Primers used to amplified RPS4 are listed in MOS14g” stands for “mos14-1 snc1 npr1 containing the MOS14 transgene under its native promoter”.Reverse of (PDF)Click here for additional data file.Table S1Primers used in this work.(DOC)Click here for additional data file.


F11), prekallikrein (KLKB1), and a cytochrome P450 family member (CYP4V2) genes are associated with deep venous thrombosis (DVT). These SNPs exert their effect on DVT by modifying the circulating levels of FXI. However, SNPs associated with DVT were not necessarily all in F11, but also in KLKB1 and CYP4V2. Here, we searched for evidence for common regulatory elements within the 4q35.2 locus, outside the F11 gene, that might control FXI plasma levels and/or DVT risk. To this end, we investigated the regulation of the orthologous mouse gene cluster under several metabolic conditions that impact mouse hepatic F11 transcription. In livers of mice in which HNF4α, a key transcription factor controlling F11, was ablated, or reduced by siRNA, a strong decrease in hepatic F11 transcript levels was observed that correlated with Cyp4v3 (mouse orthologue of CYP4V2), but not by Klkb1 levels. Estrogens induced hepatic F11 and Cyp4v3, but not Klkb1 transcript levels, whereas thyroid hormone strongly induced hepatic F11 transcript levels, and reduced Cyp4v3, leaving Klkb1 levels unaffected. Mice fed a high-fat diet also had elevated F11 transcription, markedly paralleled by an induction of Klkb1 and Cyp4v3 expression. We conclude that within the mouse F11, Klkb1, Cyp4v3 gene cluster, F11 and Cyp4v3 frequently display striking parallel transcriptional responses suggesting the presence of shared regulatory elements. Single nucleotide polymorphisms (SNPs) in a 4q35.2 locus that harbors the coagulation factor XI ( Blood coagulation serine protease factor XI (FXI) contributes to hemostasis by activating coagulation factor IX . AlthougF11) and prekallikrein (KLKB1) are located in tandem on the long arm of chromosome 4 (4q35.2) directly downstream from the cytochrome P450 family member (CYP4V2) gene and family with sequence similarity 149 A  were associated with DVT and plasma FXI antigen levels  [F11 gene (rs2036914 and rs2289252) [CYP4V2 was associated with DVT and FXI levels also after adjustment for rs2036914 and rs2289252 [F11 and CYP4V2 may also affect the expression of KLKB1, which is clearly also a strong candidate DVT risk gene. The above suggests that the DVT risk associated with variation in the 4q35.2 region may not necessarily be solely attributed to F11 gene variation and/or FXI protein levels. Furthermore, it opens the possibility that KLKB1 and CYP4V2 may be more than just neighbors of F11, but may also share regulatory elements.The genes encoding FXI  regulatory elements in their natural positions. Such analysis is limited by the size of the cluster (~100 kb). In mice, F11, Klkb1 and Cyp4v3 (the mouse orthologue of CYP4V2) are also located together , estrogen, and dietary status were varied. In contrast, Klkb1 only showed an F11-like response for the fat feeding condition.Experimental evidence for the possible regulatory interaction of the genes within 4q35.2 region should follow from a detailed together . Furthercluster ~0 kb. In Hnf4a-null mice with a liver-specific deletion of exons 4 and 5 of the Hnf4a gene  or control mice  were described previously [Livers from 45-day-old female liver-specific eviously . Liver feviously .C57BL/6J males and females were purchased from Charles River Laboratories  and housed under a 12-h light/dark cycle, with standard chow diet and drinking water provided ad libitum. For studying the impact of the natural estrogen 17ß-estradiol, 8-week-old female mice were bilaterally ovariectomized under isoflurane anesthesia, and after a 2 week recovery period, they were randomly assigned to either the experimental group or vehicle treatment group. 17ß-estradiol  stock was prepared in ethanol and diluted in arachid oil at a final concentration of 1% ethanol prior to injection. 17ß-estradiol was injected subcutaneously once or for 5 days, at a (daily) dose of 2 µg per mouse. For the vehicle treatment, mice were subcutaneously injected with 100 µL of arachid oil with an ethanol concentration of 1%.3) (Sigma Aldrich) stocks of 1 mg/ml were prepared in 4 mM sodium hydroxide and stored at 4 degrees Celsius. For injection, a T3 stock was diluted to 2.5 µg T3/ml in phosphate buffered saline supplemented with 0.02% bovine serum albumin with a final concentration of sodium hydroxide of 0.2 mM. Mice received a daily intraperitoneal injection of (0.5 µg) 200 µl thyroid hormone solution for 14 days.For study the effect of thyroid hormone, male C57BL/6J mice, 8 weeks of age, were fed a low iodine diet  and drinking water supplemented with 1% (wt/vol) potassium perchlorate (Sigma). 3,3′,5-triiodo-L-thyronine sodium salt  as a run-in for a period of 2 weeks. Subsequently, they were randomly assigned to either the experimental group fed a high-fat diet  or maintained on the control LFD for 1 or 7 days.GGU CCA UGG UGU UUA AUU-3 ´ and antisense: 5 ´-UUA AAC ACC AUG GAC CUC UUG-3 ´ . Control siNEG  and siHNF4α were complexed with Invivofectamine® 2.0  exactly according to the manufacturer’s protocol. Subsequently, C57BL/6J mice were intravenously injected via the tail vein with 200µl complexed siRNA, a dose of 7 mg of siRNA per kg body weight.For small interfering (si) RNA-mediated knockdown of HNF4α in 8-week-old female mice, a control siRNA  and a siRNA tested in mouse primary hepatocytes to be effective to reduce HNF4α transcript and protein levels by 90% at a concentration of 3 nM were used as described previously . The seq3-treated, fat-fed, or siHNF4α injected animals and the respective controls were anesthetized by a subcutaneous injection with a mixture of ketamine (100 mg/kg), xylazine (12.5 mg/kg) and atropine (125 µg/kg) after which the abdomen was opened by a midline incision and a blood sample on sodium citrate  was drawn from the inferior caval vein. Plasma was obtained by centrifugation and stored at -80°C until use [At the indicated time points, 17ß-estradiol-treated, Tketamine 0 mg/kg, t values of the individual samples were related to the mean ΔCt of the reference group (i.e ΔΔCt). Values are expressed as mean (2 POWER of mean ΔΔCt) with a lower range (2 POWER of mean ΔΔCt+SEM) and an upper range (2 POWER of mean ΔΔCt-SEM).RNA was isolated from mouse livers and subsequently analyzed for transcripts by quantitative real-time PCR as described previously . The genPlasma FXI activity levels were determined as described previously . Respectp-value <0.05 was considered to be significant.Data were analyzed with ‘GraphPad Instant’ software and statistical differences were assessed using the Student’s t-test  or the Mann-Whitney Rank sum test (transcript levels). The Pearson correlation coefficient (r) was used to evaluate whether hepatic transcript levels were correlated. A F11, Klkb1 and Cyp4v3 transcription in mice responds in a concerted manner to stimuli affecting F11 transcript levels i.e. estrogen hormone [F11 gene [F11 transcript levels and Cyp4v3 levels were strongly reduced or even absent when compared to their control littermates (FLOX)  RNA were also examined. Two days after intravenous injection of a double-stranded siRNA known to be effective for Hnf4a knockdown, a 61% reduction of hepatic Hnf4a transcript levels was observed that correlated with the reduction in F11 and Cyp4v3 transcript levels  for 10 days (1 µg/mouse/day) and subcutaneous injection of the natural estrogen 17ß-estradiol (E2) for 5 days (2 µg/mouse/day) to ovariectomized C57BL/6J mice induced moderate reductions of hepatic transcript for coagulation factor genes F2, F7, F10 and F12 as compared to the respective vehicle control treated animals . T3 results in a decrease in most hepatic coagulation gene transcript levels, with downregulatory effects of -35 and -27% observed for F2 and F10 respectively, while F7 levels remained unchanged . Increased hepatic F11 transcript levels at 14 days of T3 treatment coincided with a 45% decrease in Cyp4v3 transcript levels, while Klkb1 levels were not significantly affected. Consequently, under changing in T3 levels, significant correlations between hepatic F11, Klkb1 and Cyp4v3 transcript levels were not observed , without significantly altering the hepatic transcript levels of F2 (except at 1 day HFD), F7, F10 or F12  transcription also affected the mouse FXI protein activity at the level of the plasma. This resulted in statistically significant correlations between hepatic F11 transcript levels and plasma FXI activity, with the exception of synthetic and natural estrogen treatments  are present near the murine promoters and/or enhancers of the F11 and Cyp4v3 genes. Indeed, the mouse and human F11 loci are predicted to carry multiple functional HNF4α binding sites [Co-regulation of the transcription of genes in close proximity of one another, as the d Cyp4v3 , Table 1ng sites ,16. HencF11 gene. No binding sites for estrogen receptor α and thyroid hormone receptor were identified using these softwares. In addition, direct comparison of the human and mouse (enhancer and promoter) sequences did not show very convincing conservation of the putative binding sites. This exemplifies that a reliable identification of functional binding sites requires, perhaps unsurprisingly, functional data. These were beyond the scope of the present manuscript.Using Genomatix MatInspector and JASPAR programs, we identified two putative HNF4α binding sites in the enhancer (~5kb upstream of the start-site) and one in the promoter region (located just upstream of the transcription start site) of F11, Klkb1 and Cyp4v3 is highly responsive to changes in hormones and metabolism. Of note, these changes are much larger than, and in a different direction from the alterations in the expression of coagulation genes F2, F9, F10, F12, which were included as controls in the analysis. Both single/multiple estrogen doses were able to modulate F11 hepatic transcription. Studies indicated that there are ERα binding sites near the F11 gene [F11. Alternatively, prolonged thyroid hormone exposure was required to evoke a clear F11 transcriptional response. This could be explained by an indirect modulation involving an intermediate transcription factor (e.g. HNF4α) additional to thyroid hormone receptor. A small number of studies reported that estrogens, thyroid hormone and dietary fat may affect hepatic HNF4α [F11 and Cyp4v3. Alternatively, dietary fat can induce acute hepatic stress and inflammation [F11 gene. Whether stress-related FOXA1 or HNF4α contribute to possible concerted regulation in the human 4q35, 2 locus has not been studied.It was proposed that the human SNPs associated with thrombotic risk may modulate FXI expression in response to changing age and hormonal levels . Our mouF11 gene  which exic HNF4α -20. Althammation  that invammation  and ChIPammation  indicateF11 and CYP4V2 may impact the expression of KLKB1, and thereby increase risk for DVT [F11 and CYP4V2 on one hand and KLKB1 on the other hand, and make modulation of DVT risk through impact on KLKB1 by SNPs in F11 and CYP4V2 less likely.It was also suggested that human SNPs in  for DVT -20. The F11 gene and ChIP-seq analysis for HNF4α or FOXA1 binding sites, among others, does not provide any evidence for transcription factor binding sites in these regions. Also, the sequences around the SNPs are not strongly conserved in mammals. rs13146272 is a missense mutation in CYP4V2 and this position is relatively well conserved in other species and in other CYPs. It might be speculated that this SNP alters sensitivity or specificity of CYP4V2 and thereby affects plasma lipid levels, as some mutations in CYP4V2 have been shown to do [The mouse data encouraged us to investigate whether SNP rs2036914, rs2289252 and rs13146272 within the 4q35.2 locus are related to (common) regulatory sequences for this locus. SNPs rs2036914 and rs2289252 are located in introns of the wn to do . Thus, SF11 and Cyp4v3 transcript levels were produced when HNF4α and dietary status were varied. Such responses were also produced upon variation in estrogen, however minimal or not when using 17ß-estradiol (E2) . In contrast to 17ß-estradiol, ethinylestradiol is orally effective and thereby likely results in more effective exposure of the liver to the estrogenic compound as compared to a subcutaneous administration (17ß-estradiol). This is in line with our earlier observations using these compounds [F11 and Cyp4v3 transcript levels were affected in opposite direction. We have no explanation for these opposite effects, but these observations highlight that, although mouse F11 and Cyp4v3 may share regulatory elements, the transcription of these two genes is clearly not under control of one single shared regulatory region.Parallel responses in iol E2)  and 2. O  and 2. F11 transcript levels did not translate into increased FXI plasma protein levels, at least at the level of protein activity (F11 transcript levels truly were not translated into changes in plasma FXI (protein and activity) is at present subject to speculation. Although not determined, it would also be interesting to determine whether effects on hepatic Cyp4v3 transcription translate to effects on hepatic Cyp4v3 protein (activity) level. This would shine a light on whether parallel transcriptional responses of F11 and Cyp4v3 extends to protein level.Remarkably, only for the estrogen treatment conditions, changes in activity . This phF11, Klkb1, Cyp4v3 gene cluster, in particular F11 and Cyp4v3, frequently display a striking parallel transcriptional response suggesting the presence of regulatory elements. We speculate that, if present, SNPs within the human orthologues of these unidentified elements could causally influence DVT risk.In conclusion, our mouse data demonstrate that within the mouse Figure S1Hnf4a and F11 (A), Hnf4a and Klkb1 (B) and Hnf4a and Cyp4v3 (C) under siRNA-mediated HNF4α knockdown (●) and control siRNA (○) in mouse liver.Correlation between hepatic Data were statistically analyzed with Pearson correlation coefficient (r). p-values <0.05 were regarded as statistically significant. ‡p < 0.001.(TIF)Click here for additional data file.Figure S2Hepatic transcript and protein levels of HNF4α under metabolically challenged conditions in mice.(A) Hepatic transcript levels were determined by quantitative real-time PCR. Data are expressed as mean with error bars representing the difference between 2 POWER of upper and lower range of the mean ΔΔCt. β-actin was used as internal control for quantification and normalization. The ΔCt values of the individual samples were related to the mean ΔCt of the reference group. On the x-axis the metabolic conditions are depicted. Data were statistically analyzed using Mann Whitney Rank Sum Test. P-values less than 0.05 were regarded as statistically significant. *p<0.05. (B) Immunoblotting for HNF4α was perfromed for liver homogenates that were prepared for three randomly selected mice per condition. 15µg total protein lysate was loaded in each lane and HNF4α was detected using anti-HNF4α antibody . β-actin was used as a protein loading control. 3; 3,3′,5-Triiodo-L-thyronine.siNEG/siHNF4α mice; mice injected with control (negative) or HNF4α siRNA respectively, EE; ethinylestradiol, E2; 17-β estradiol, T(TIF)Click here for additional data file.Table S1(DOC)Click here for additional data file.Table S2(DOC)Click here for additional data file.


Genome-wide distribution of the majority of H2A and H3 variants  was simultaneously investigated in mouse embryonic stem cells by chromatin immunoprecipitation sequencing. Around the transcription start site, histone variant distribution differed between genes possessing promoters of high and low CpG density, regardless of their expression levels. In the intergenic regions, regulatory elements were enriched in H2A.Z and H3.3, whereas repeat elements were abundant in H2A and macroH2A, and H3.1, respectively. Analysis of H2A and H3 variant combinations composing nucleosomes revealed that the H2A.Z and H3.3 combinations were present at a higher frequency throughout the genome than the other combinations, suggesting that H2A.Z and H3.3 associate preferentially with each other to comprise the nucleosomes independently of genome region. Finally, we found that chromatin was unstable only in regions where it was enriched in both H2A.Z and H3.3, but strongly quantified stable in regions in which only H3.3 was abundant. Therefore, histone variant composition is an important determinant of chromatin structure, which is associated with specific genomic functions. These genomic components play roles in various genomic functions, such as gene expression, DNA replication and maintenance of genome stability. Emerging evidence from epigenetic studies revealed that various genome regions contain specific modifications on their nuclear core histones, which are involved in functional regulation The eukaryotic genome consists of genes and intergenic regions, which include enhancers, insulators, repeat elements, Of the H2A variants, canonical H2A is the most abundant in chromatin i.e., H3K4me3, H3K36me3) and acetylation of various N-terminal lysines Arabidopsis thaliana showed that H3.2 enrichment in genes is negatively correlated with their expression levels, and H3.2 was deposited abundantly in regions with methylated CpG and H3K9me2, as well as H3K27me3 Arabidopsis thaliana does not possess H3.1, the difference in the function of H3.1 and H3.2 remains to be elucidated.Of the H3 variants, H3.3 is the evolutionarily conserved replacement H3 Although several studies have assessed the genome-wide distributions of histone H2A and H3 variants, most examined only a single variant In the present study, we examined the distribution of the majority of genomic H2A and H3 variants in mES cells using chromatin immunoprecipitation (ChIP) followed by high-throughput sequencing. We found that the enrichment of histone variants in gene regions (promoter and gene body) differed depending on gene expression level. Further, the enrichment was markedly different around the transcription start sites (TSSs) of genes with high- and low-CpG promoters. Furthermore, specific H2A and H3 variant compositions were observed in various regulatory and repeat elements. Also, H2A.Z and H3.3 associated preferentially with each other throughout the genome. Taken together, our results suggest that specific H2A and H3 variant compositions regulate the functions of various genome regions.Feeder-free ZHBTc4 ES cells Pac) gene obtained by SalI digestion of pCre-Pac was inserted into the SalI site of the vector. The vector coding a Flag-histone variant was transfected into ZHBTc4 cells by lipofection, and cells were selected in ES medium using 100 μg/mL of puromycin. A single colony of cells for each variant was picked up, cultured and subjected to ChIP-sequencing analysis. The difference in the genome distribution of a Flag-histone variant among the cell lines was assessed using the Flag-H2A.X cell lines. ChIP-quantitative polymerase chain reaction (qPCR) analysis of several genes showed no significant difference among the four cell lines examined . A DNA fragment encoding a histone variant tagged with Flag at its N-terminal region was inserted into the EcoRI site of the pCAGGS vector. The puromycin N-acetyl-transferase , following the manufacturer's protocols, and high-throughput sequencing was carried out. Sequences (35 bp) were aligned against the cDNA sets from Ensemble GRC38 using Bowtie. The number of mapped reads per kilobase of exon per million reads (RPKM) for each gene was calculated using the following formula: RPKM  =  ) ×107 cells per plate). One plate was used per ChIP aliquot. Formaldehyde solution  was directly added to culture media at a final concentration of 1% for fixation. After 10-min incubation at room temperature, a glycine solution was added to the plates at a final concentration of 125 mM, and the plates were incubated at room temperature for 10 min to stop fixation. Cells were transferred to 15 mL tubes and washed twice with cold phosphate buffered saline (PBS). All buffers used in the following procedures were supplemented with protease inhibitor solution  before use. Cells were suspended in lithium borate (LB) buffer , and sonicated (30 s pulse +1 min break) seven times on ice using a SONIFER 250 (Branson) to obtain 150–300-bp chromatin fragments. Triton X-100 was added into the tubes to a final concentration of 1%, and then the tubes were centrifuged  to obtain the supernatant used for immunoprecipitation. For the input sequencing, DNA was purified—without being immunoprecipitated—from cells expressing Flag-mH2A. To bind the antibodies to beads, 50 μL of Dynabeads Protein G (Invitrogen) were washed twice with PBS, 5 μg of the antibody in blocking solution  was added and gently flipped overnight at 4°C. Anti-FLAG M2 , anti-H3K4me3 , anti-H3K27me3 , and anti-H3K36me3  antibodies were used for immunoprecipitation. After three PBS washes, the antibody-bound beads were added to the supernatant containing the chromatin fragments, and gently flipped overnight at 4°C. The beads were washed once with a low salt buffer , twice with a high salt buffer , five times with RIPA buffer , and once with TE (pH 8.0) supplemented with 50 mM NaCl. To elute the chromatin, the beads were suspended in elution buffer , and incubated at 65°C for 15 min. After centrifugation , the supernatant was collected and incubated overnight at 65°C to de-crosslink the chromatin. At this time, the samples for input sequencing were added to the elution buffer and then subjected to the same treatments as those for the immunoprecipitated samples in the following procedures. After de-crosslinking, RNase A  was added to all samples, which were incubated at 37°C for 2 h, followed by the addition of proteinase K  and incubation at 55°C for 2 h. Finally, DNA was isolated by the phenol-chloroform method followed by ethanol precipitation; isolated DNA was dissolved in Milli-Q water. Adapters were ligated to both ends of the DNA following the Illumina protocol for “Preparing Samples for ChIP Sequencing of DNA.” The ligated DNA was subjected to electrophoresis, and 250–400-bp DNA fragments were excised from the gel and purified. The purified DNA was subjected to PCR amplification following the Illumina protocol and then sequenced using the Illumina Genome Analyzer (Illumina).ES cells were cultured in 10-cm plates to ∼80% confluence (1.2×10DNA fragments obtained from ChIP were applied to the deep sequencer following the manufacturer's protocol. Sequences (36 bp) obtained by high-throughput sequencing were aligned against the mouse reference genome (mm9) using Bowtie. The total number of uniquely mapped reads in each sample is shown in Uniquely mapped 36-bp sequences in each ChIP sample were extended to 150–300 bp, which is the length appropriate for sequencing without adapters. At each mapped nucleotide position, the number of reads mapped there ×1,000,000/total number of reads was calculated. For input samples, the calculated value at each position was added to the minimum calculated value, except for zero, to avoid a value of zero at any position. The calculated values of ChIP samples were divided by those of input samples to determine the enrichment at each nucleotide position in the genome.The whole genome was compartmentalized into 150-bp bins . The ratTo determine major isoform among transcripts from a gene, we deterimined the transcript with the highest RPKM value in transcripts from a gene registered in Ensemble (GRC38) as a major isoform of the gene.et al.Genes registered in Ensemble (GRC38) were classified into HCP, ICP, and LCP groups as described by Mikkelsen http://genome.ucsc.edu/), and used for the determination of CpG islands.The CpG island annotation file was downloaded from the UCSC Genome Bioinformatics site  and CTCF data were obtained from Chen http://www.girinst.org/repbase/). The number of the reads from ChIPseq data mapped on each repeat element was determined and corrected for the length of the repeat element and the number of reads for the input sample.Repeat element sequences were obtained from Repbase (http://trace.ddbj.nig.ac.jp/dra/index.html) under accession number DRP001103.Gene expression and sequencing data have been deposited in the DDBJ Sequence Read Archive (DRA)  that expressed Flag-tagged H3.1, H3.2 and H3.3. We also prepared ES cells expressing Flag-tagged H2A variants to conduct ChIP with identical specificity and efficiency for H2A and H3 variants. Thus, we established seven cell lines that constitutively expressed Flag-tagged H2A, H2A.X, H2A.Z, mH2A, H3.1, H3.2 or H3.3.We first investigated the distribution of H2A and H3 variants both in genes and in their vicinities. Genes were classified as active or inactive based on RNA sequencing data . We founTo further assess the correlation between the expression level and the enrichment of histone variants in genes, genes were classified into six groups according to their expression levels . In thesi.e., HCP, ICP and LCP, respectively) according to Mikkelsen's criteria Chromatin structure and the regulation of transcription are known to be differ markedly between genes with promoters of high and low CpG densities A detailed analysis indicated that all H2A and H3 variants—with the exception of H2A.Z—were depleted from the nucleosome at the TSS of genes with HCP promoters, regardless of their expression levels . H2A.Z wSmad1, suggesting that the nucleosomes at enhancer elements comprise mainly H2A.Z and H3.3  and H3.3 .see Materials and Methods) and compared the enrichment of histone variants at sites with the highest and lowest quartile of bound CTCF. All variants were absent from the CTCF binding sites in both the highest and lowest quartiles, although the extent of the removal was greater in the highest quartile , which contributes to higher-order chromatin architecture for long-range interactions quartile , suggesti.e., promoters, gene bodies, and intergenic regions), suggesting that their enrichment at CGIs is not regulated by a particular epigenetic environment but is dependent only on the presence of a CG-rich DNA sequence.Many genes have CpG islands (CGIs) in their upstream regulatory regions; moreover, a number of CGIs are present in gene bodies and intergenic regions Repeat elements are generally enriched in H3K9me3 and H4K20me3, and are constitutively heterochromatic To investigate the existence of a preferential combination of H2A and H3 variants comprising nucleosomes in various genome regions, we compartmentalized the genome into 150-bp bins, and identified those in which the particular histone H2A or H3 variant was enriched . Then, wBoth H2A.Z and H3.3 are associated with H3K4me3 i.e., H2A, H2A.Z, mH2A, and H2A.Bbd) was published e.g., insects and fungi). Therefore, H2A and H2A.X must be investigated simultaneously to understand their roles in vertebrate genomic function. In addition, the amino acid sequence of H3.1 is similar to that of H3.2, and is present only in mammals. Because no antibody that can distinguish these proteins is available, the difference in their genome distribution and function has not been determined. Here we report that the genome distributions of H2A and H2A.X, and H3.1 and H3.2 differ, suggesting that each of these variants has a specific function that is different from their counterparts.Several reports of the genome-wide distribution of various histone variants have been published We found that H3.1 but not H3.2 was enriched in repeat elements other than telomeres . These eArabidopsis thalianaet al. (2010), who reported that H3.2 enrichment was not correlated with gene expression in mouse ES cells. Therefore, the role of H3.2 in gene expression seems to differ between mice and plants.Histone variants play important roles in the regulation of transcription. Previous reports showed that H2A.Z and H3.3 enrichment in promoters and gene bodies was well correlated with the expression levels of the genes Previous studies have shown that nucleosomes are depleted at the TSSs of actively expressed genes Nucleosomes containing H2A.Z and H3.3 are preferentially subjected to the H3K4me3 modification. Previous studies showed that regions of H2A.Z enrichment overlapped substantially with those of H3K4me3 in promoter and enhancer regions i.e., enhancers, insulators, and CGIs; H2A.Z and H3.3 were enriched in regulatory elements  i.e., the promoters of active HCP genes, insulators, enhancers and CGIs; Previous studies suggested that nucleosomes containing both H2A.Z and H3.3 compose unstable chromatin. Jin and Felsenfeld e.g., gene expression and heterochromatin formation) and histone modifications.In summary, we clarified the histone variant composition of various genome regions by genome-wide analysis of the majority of H2A and H3 variants expressed in mouse ES cells. We found that in HCP, but not LCP, genes, regardless of their expression levels, most of the variants were depleted at the TSS but that H2A.Z and H3.3 were enriched in the vicinity of the TSS. H2A.Z and H3.3 were also enriched in enhancers, CpG islands and insulators. We found that H2A and H3.1 were enriched in repeat elements, which is the first knowledge of the differential distribution between H2A and H2A.X, and between H3.1 and H3.2 on specific genome regions. The analysis of the combinations of H2A and H3 variants that comprise nucleosomes revealed that the frequency of the H2A.Z and H3.3 combination was high throughout the genome when compared to other combinations, and that nucleosomes containing this combination were preferentially trimethylated at the lysine 4 residue. None of these results could have been obtained without simultaneous analysis of the majority of histone variants. Taken together, these data suggest that specific combinations of histone variants influence the regulation of various genomic functions  was examined in four cell lines expressing Flag-H2A.X by ChIP-qPCR.(TIF)Click here for additional data file.Figure S2Morphology of ES cells expressing Flag-tagged histone variants. ZHBTc4 cells expressing Flag-tagged H2A, H2A.X, H2A.Z, macroH2A (mH2A), H3.1, H3.2 or H3.3, and wild-type (WT) cells, were observed before (0 h) and after (72 h) induction of differentiation.(TIF)Click here for additional data file.Figure S3Clustering analysis of gene expression profiles in ES cells expressing Flag-histone variants and wild-type cells before and after the induction of differentiation. cDNA microarray data sets of wild-type ZHBTc4 cells before (WT_0 h) and 24, 48, 72, and 96 h after induction of differentiation  and cells expressing H2A, H2A.X, H2A.Z, macroH2A (mH2A), H3.1, H3.2 or H3.3 without differentiation induction were subjected to hierarchical clustering. Results are shown as a tree view.(TIF)Click here for additional data file.Figure S4Identification of the regions in which a particular histone H2A or H3 variant is enriched. As an example, the identification of the bin in which H2A.Z is enriched is shown.(TIF)Click here for additional data file.Figure S5Histogram of the expression levels of protein-coding genes in mouse ES cells. The expression levels of coding genes were obtained from the RNA sequence data. The histogram of gene expression levels was smoothed by kernel density estimation, which is depicted by bimodal peaks. The genes in the first and second peaks are defined as inactive and active, respectively.(TIF)Click here for additional data file.Figure S6Classification of genes according to expression level. The expression level of coding genes was obtained from the RNA sequence data. The histogram of gene expression levels was smoothed by kernel density estimation. The genes were classified into six groups according to their expression levels. Groups 1, 2, 3, 4, 5 and 6 contained 6728, 447, 1161, 2526, 2760 and 1009 genes, respectively.(TIF)Click here for additional data file.Figure S7Correlation between the expression level and enrichment of histone variants in gene promoters. Genes were classified into six groups according to their expression levels as shown in (TIF)Click here for additional data file.Figure S8Correlation between the expression level and enrichment of histone variants in gene bodies. Genes were classified into six groups according to their expression levels as shown in (TIF)Click here for additional data file.Figure S9Enrichment of histone variants in the promoters of HCP and LCP genes. Genes were classified into HCP & active, HCP & inactive, LCP & active, and LCP & inactive on the basis of CpG density at the promoter and gene expression levels. Enrichment of histone variants at promoters (TSS±2 kb) in each group is shown as a box plot.(TIF)Click here for additional data file.Figure S10Enrichment of histone variants in the bodies of HCP and LCP genes. Genes were classified into HCP & active, HCP & inactive, LCP & active, and LCP & inactive on the basis of CpG density at the promoter and gene expression levels. Enrichment of histone variants at gene bodies (TSS+2 kb∼TES) in each group is shown as a box plot.(TIF)Click here for additional data file.Table S1Classification of the genes by their expression levels and the CpG density at promoter.(TIF)Click here for additional data file.Table S2The number of reads uniquely mapped to the genome for all ChIPseq and Inputseq data sets.(TIF)Click here for additional data file.


Saccharomyces cerevisiae has a variety of highly conserved factors necessary for these reconstructions. Chromatin remodelers, histone modifiers and histone chaperones directly associate to promoters and open reading frames of exposed genes and facilitate activation and repression of transcription. We compare two distinct patterns of induced transcription: Sustained transcribed genes switch to an activated state where they remain as long as the induction signal is present. In contrast, single pulsed transcribed genes show a quick and strong induction pulse resulting in high transcript levels followed by adaptation and repression to basal levels. We discuss intensively studied promoters and coding regions from both groups for their co-factor requirements during transcription. Interplay between chromatin restructuring factors and dynamic transcription is highly variable and locus dependent.Regulation of transcription involves dynamic rearrangements of chromatin structure. The budding yeast  Fin. FinGAL1l levels . In this2.Induction of gene transcription is triggered by the binding of transcriptional activators to specific promoter elements (upstream activation sequences) followed by recruitment of co-activators such as mediator and chromatin restructuring factors . Thereafter, components of the general transcription machinery including TATA binding protein (TBP), general transcription factors (GTFs) and RNA Pol II assemble into the pre-initiation complex (PIC). Some loci contain preformed PICs which are paused for transcription and require additional factors for release into productive elongation . HoweverS. cerevisiae contain this histone variant which is encoded by the HTZ1 gene . Dis. Dis8]. the gene .INO1 promoter where it removes H4 K5 acetylation under non-induced conditions [As already mentioned, the histone deacetylase Rpd3 is a subunit of two complexes: Rpd3L and Rpd3S. Both have been associated with transcriptional repression. The Rpd3L large histone deacetylase complex (HDAC) is recruited to the nditions . Rpd3S rnditions ,53. Thesnditions ,48.GAL1/10 system Asf1 is necessary for histone eviction and positioning and thus is involved in activation and repression [Histone chaperones also function in the repression of induced genes. They are predominantly associated with elongating RNA Pol II and restore canonical chromatin structure. Asf1 and Spt6 have been intensively studied for their activities during transcriptional regulation. Asf1 interacts with H3–H4 dimers and is associated to promoters and coding regions of active genes. In case of the inducible pression . In casepression .CHA1 locus to the delocalization of the +1 nucleosome [The histone chaperone Spt6 interacts directly with phosphorylated Serine2 of RNA Pol II CTD . Spt6 hacleosome . Spt6 iscleosome . In addicleosome . The unicleosome . This mecleosome .Basically there are two different activities of INO80 described which could contribute to the observed transcriptional effects. INO80 is able to slide nucleosomes along DNA without dissociation and able to reduce the distance between nucleosomes on an artificial DNA construct . In addi4.In summary, activation and repression of induced genes are influenced by chromatin restructuring factors. The factors involved as well as the importance of these factors for transcriptional regulation varies for different loci. Some are essential for PIC formation and thus for activation whereas others are required for fine-tuning of transcript levels. Multiple studies have shown that transcriptional activation and repression are regulated at the level of initiation and elongation. Although functions of these factors have been extensively characterized, many of the signals which direct them to distinct loci remain unclear. It is speculated that characteristic induction kinetics may also be a signal to attract certain chromatin restructuring factors.


Yersinia. RovA levels are kept in check by a sophisticated layering of distinct transcriptional and post-transcriptional regulatory mechanisms. In the enteropathogen Y. pseudotuberculosis, we have previously reported that the extracytoplasmic stress sensing CpxA-CpxR two-component regulatory system modulates rovA expression.RovA is a global transcriptional regulator of gene expression in pathogenic in vitro, and determined that phosphorylation was necessary for CpxR to efficiently bind to the PCR-amplified upstream regulatory region of rovA. The precise CpxR∼P binding site was mapped by a nuclease protection assay and directed mutagenesis confirmed that in vivo binding to the rovA promoter inhibits transcription. Reduced RovA production was most pronounced following CpxR∼P accumulation in the Yersinia cytoplasm during chronic Cpx pathway activation and by the indiscriminate phosphodonor action of acetyl phosphate.In this study, we characterized CpxR phosphorylation (CpxR∼P) Yersinia envelope, to include genes involved in environmental survival and pathogenicity.Cpx pathway activation restricts levels of the RovA global regulator. The regulatory influence of CpxR∼P must therefore extend well beyond periplasmic quality control in the  Extracytoplasmic stress (ECS) has a deleterious effect on bacterial fitness, since it impacts on cell envelope integrity, protein folding and function. Bacteria have consequently evolved multiple signaling pathways to counter these situations E. coli – autokinase, CpxR kinase and CpxR phosphatase activity E. coli, CpxR∼P is then capable of up- or down-regulating the transcription of many genes that function to alleviate the effects of ECS E. coli, as would result from a full length cpxA deletion mutant or so-called gain-of-function mutants (designated cpxA*), would be to accumulate CpxR∼P Spanning the inner membrane, CpxA is a histidine kinase possessing three distinct activities in Yersinia pseudotuberculosisY. pseudotuberculosis virulence factors. The fact that we could demonstrate in vitro CpxR∼P binding to inv and rovA promoters supported this view, although typical consensus CpxR∼P binding sites in these regions were never located Y. pseudotuberculosis lacking CpxA phosphatase activity must accumulate CpxR∼P, serving as the fundamental basis for transcriptional repression of virulence gene expression in this mutant background Homologues to components of the Cpx pathway exist in a number of clinically important bacterial pathogens. We have recently studied the function of the Cpx TCRS in the enteropathogen Yersinia because it was required for transcription of the inv gene encoding for invasin YersiniaYersinia. Thus, this study therefore seeks to substantiate the contribution of the Cpx TCRS to control of RovA-dependent virulence gene regulation in Yersinia.RovA was first identified in Y. pseudotuberculosis YPIII/pIB102 (serotype III) as the parental strain. pIB102 is a virulence plasmid encoding for the Ysc-Yop T3SS and is a variant of cryptic pIB1. It differs only by a kanamycin resistance cartridge inserted into the yadA gene, which does not reduce the pathogenicity of YPIII/pIB102 in mouse models phoP located on the chromosome. This is predicted to generate an inactive truncated variant of the PhoP response regulator that impairs bacteria growth and survival inside macrophages Y. pseudotuberculosis) or 37°C (E. coli) with aeration. When examining transcription and translation from rovA and inv, bacteria were grown at both 26°C and 37°C in LB broth to late stationary phase. These conditions were selected on the basis that reports from other laboratories have clearly established the peak rovA and inv expression occurs in bacteria grown at low temperature to stationary phase Bacterial strains and plasmids can be viewed in E. coli S17-1λpir into Y. pseudotuberculosis. Mutated alleles were initially introduced into the recipient genome by a single cross-over event. A second single cross-over event followed to secure the complete allelic exchange, which was initially screened for on the basis of sacB-dependent sucrose sensitivity Y. pseudotuberculosis genome was then confirmed by diagnostic PCR and sequence analysis of the amplified regions flanking the mutation.To construct individual in-frame deletions, site-directed amino acid substitutions and nucleotide ‘shuffle’ mutations, we applied the standard overlap PCR technique using the relevant primer combinations listed in Protein derived from lysates of the bacterial pellet was fractionated by 12% (for invasin) or 15% (for RovA) SDS-PAGE. Protein was then transferred to Schleicher and Schuell Protran® nitrocellulose  using a Hoefer semi-dry transfer assembly. Proteins of interest were bound with specific rabbit polyclonal antibodies that were a gift from Petra Dersch (anti-RovA), Hans Wolf-Watz (anti-invasin), Thomas Silhavy (anti-MBP-CpxR) or Shu-ichi Nakayama (anti-CpxR). These were then detected with an anti-rabbit monoclonal antibody conjugated with horse radish peroxidase  and a homemade chemiluminescent solution.Y. pseudotuberculosis nlpE is listed in EcoRI/XbaI restriction to generate pJF027. This placed nlpE expression under arabinose control. The nlpE allele from E. coli cloned under arabinose control in pBAD18 (termed pND18) was a gift from Thomas Silhavy. These two constructs along with the vector control were electroporated into Yersinia. To induce nlpE expression, overnight bacterial cultures were used to seed fresh growth medium supplemented with 0.2% (w/v) L-arabinose and appropriate antibiotic selection.The primer combination for PCR amplification of D51A and CpxRM199A variants were amplified with gene specific primers (Y. pseudotuberculosis or the respective mutants and cloned with NdeI and XhoI in front of the IPTG inducible promoter of pET22b(+). These expression plasmids were maintained in E. coli BL21(DE3) plysS allowing for the IPTG-inducible expression of individual CpxR variants fused to a C-terminal His(6) tag. Recombinant protein was purified as previously described Alleles encoding CpxR wild type and the CpxR primers  using te32P end-labeled forward primers listed in cpxR/P, rovA, ppiA, ail, ail-like, inv, psaA and psaE. The amplified DNA fragments were purified by agarose gel electrophoresis. Purified CpxR was mixed with the DNA fragments (∼2 to 5 nM) in a 20 µL reaction volume containing 20 mM Tris-HCl pH 7.0, 30 mM Acetyl phosphate, 125 mM KCl, 10 mM MgCl2, 1 mM EDTA, 1 mM dithiothreitol, 0.25 mg per ml BSA and 5 µg per ml sonicated salmon sperm DNA. BSA corresponding to the highest CpxR concentration (1.5 µM) was used as a negative control, and about 7 fold excess of unlabeled PCR amplified DNA was used in a competition reaction. After incubation at 30°C for 1 h and addition of the DNA dye solution , the mixture was loaded directly onto a pre-run 5% polyacrylamide gel. Gel electrophoresis was performed in 1× TBE at 100 V for 2 h at 4°C. After gel drying, signals were detected by autoradiography with a Storm 860 PhosphorImager (Molecular Dynamics).32P using γ32P-ATP (Perkin Elmer) and T4 polynucleotide kinase (Fermentas). The labeled pE-rovAfor3, pE-ppiAfor and pcpxRb were paired with unlabeled pE-rovArev3, pE-ppiArev and pcpxPb respectively and used to PCR amplify promoter regions of rovA (314 bp), ppiA (276 bp) and the divergent cpxR-cpxP (245 bp). For a control, the labeled pcpxRfor was paired with unlabeled pcpxRrev for the PCR amplification of an internal region of cpxR (389 bp). Subsequently, 1.5 nM of the amplified DNA fragments and 0, 50, 100, 200 and 400 nM acetyl∼P phosphorylated CpxRwt::His6 were mixed in a 40 µL reaction containing 25 mM Hepes (pH 8), 100 mM potassium glutamate, 0.5 mg/mL BSA. After incubation of the reaction mixture at room temperature for 10 minutes, 4 µl DNase 1  was added and the reaction was left for 80 seconds at 37°C. The DNase 1 digestion was stopped by addition of phenol/chloroform and 200 µL DNase 1 STOP (0.4 M sodium acetate (pH 5), 2.5 mM EDTA, 10 ug/mL herring sperm DNA), and then the DNA was precipitated using ethanol. Samples were analyzed on a 7% denaturing polyacrylamide gel and visualized with a Storm 860 PhosphorImager (Molecular Dynamics).The primers pE-rovAfor3, pE-ppiAfor, pcpxRb and pcpxRfor were radioactively labeled with in vitro and in vivo visualization of TCRS response regulator aspartate phosphorylation Purified CpxR variants labeled with acetyl∼P were mixed with 2× loading buffer (0.02% (w/v) Bromophenol blue, 20% (v/v) Glycerol). For analysis, samples were either fractionated on a SDS-12%-PAGE or a Manganese(II)-Phos-tag™ (33 µM) 12.5% acrylamide gel. Purified CpxR∼P was detected by the Pro-Q® Diamond phosphoprotein gel staining method as described by the manufacturer (Invitrogen). To confirm equal loading, subsequent detection of total protein present on the same gel was revealed by SYPRO® Ruby staining (Invitrogen). In both cases, fluorescent output was recorded using a Fluor-S™ MultiImager (BioRad) and band intensity quantified with Quantity One® quantitation software version 4.2.3 (BioRad). The presence of recombinant purified CpxR∼P or that which is produced endogenously and present in bacterial lysates was also assessed by coupling Manganese(II)-Phos-tag™ acrylamide gel fractionation to immunoblotting with monospecific polyclonal anti-CpxR antiserum. The Manganese(II)-Phos-tag™ approach is an affinity based system for the recognition and separation of anionic substrates, such as phosphorylated proteins, during polyacrylamide electrophoresis 18 micro columns and dried by vacuum centrifugation The reduction of CpxR∼P using sodium borohydride was performed as described previously 18 columns Intact mass determination of CpxR and CpxR∼P utilized purified recombinant CpxR, and this CpxR phosphorylated with acetyl∼P. Duplicate samples of both phosphorylated and non-phosphorylated CpxR were then desalted on homemade C18 micro columns and dried by vacuum centrifugation 18 material  and dried by vacuum centrifugation. In addition, enrichment and purification of phosphorylated peptides was performed by affinity chromatography on titanium dioxides as described Peptides of CpxR and CpxR-P were prepared using trypsin and pepsin. Protein was desalted using CMALDI-MS of peptides were acquired using a Voyager DE-STR mass spectrometer  in the reflector mode and 2,5 dihydroxybenzoic acid (DHB) solution containing 0.5% (v/v) phosphoric acid as a matrix  from Agilent Technologies, Dalco Chromtech AB, Sollentuna, Sweden. LC-MS/MS combined with ESI-TRAP and ETD-TRAP was performed using a HCT ultra ETD II mass spectrometer from Bruker linked to Easy nano LC from Proxeon. Spectra were acquired using the enhanced scanning mode covering a mass range from m/z 200 to m/z 1300.Protocols detailing the isolation of total RNA, the reverse transcription of mRNA into cDNA and its use as template for subsequent PCR amplification with the gene specific primers listed in Yersinia virulence, we first wanted to characterize CpxR phosphorylation in vitro using a high energy phosphate donor, acetyl∼P. We initially determined the minimal concentration of acetyl∼P needed to sufficiently in vitro phosphorylate CpxR. Purified CpxRwt::His6 was incubated with increasing concentrations of acetyl∼P. Aliquots were then fractionated on Manganese(II)-Phos-tag™ 12.5% acrylamide gels to monitor the extent of CpxR∼P. As little as 6.25 mM acetyl∼P was sufficient to phosphorylate the majority of CpxR  is the predicted phosphorylation site of CpxR cpxR mutation in which Asp51 was replaced with alanine. As a control, another mutant was constructed in which methionine at position 199 (Met199) was replaced with alanine. Based on the helix-turn-helix DNA-binding motif prediction algorithm of Dodd and Egan 6-tagged fusions of CpxRwt and the two CpxRD51A and CpxRM199A variants were then purified by affinity chromatography from E. coli. Acetyl∼P was used to investigate which of these purified proteins could still be phosphorylated. The Manganese(II)-Phos-tag™ acrylamide system was used to visualize phosphorylated protein. Only CpxRwt::His6 and CpxRM199A::His6 were deemed to be phosphorylated in vitro by acetyl∼P -Phos-tag™ acrylamide gel, we also confirmed the absence of phosphorylation using conventional SDS-12%-PAGE followed by visualization with the independent Pro-Q® Diamond phosphoprotein stain  and Ail-like (YPTB2113), as well as the confirmed adhesins pH6 antigen (psaA) and invasin (inv), are all influenced by loss of CpxA wt and CpxRD51A His6-tagged variants derived from Yersinia CpxR and performed an EMSA to examine if CpxR∼P bound to radiolabeled PCR- amplified DNA control regions upstream of these Yersinia virulence genes. All regions of amplified target DNA included a minimum of ∼300 bp upstream and 40 bp downstream of the translational start codon. Our experiments were controlled by incorporating DNA encompassing the cpxR/cpxP divergent promoter and the ppiA promoter, both of which are known to bind CpxR∼P either from Y. pseudotuberculosisE. colirovA gene encoding the transcriptional activator of invasin psaE gene, which encodes a regulator for pH6 antigen expression in vitro phosphorylated CpxRwt bound the promoters of inv and rovA, as well as those known CpxR∼P regulon members ppiA and cpxR/cpxP  template DNA could compete for CpxR∼P binding. Although the degree of successful competition varied for each template, the addition of cold DNA did reduce the amount of radiolabeled (‘hot’) cpxR/P, ppiA, rovA, inv, psaA and psaE template DNA being retarded in migration during electrophoresis (ail (YPTB2867) promoter also represents a target of CpxR∼P considering that the higher CpxR∼P concentration (1.5 µM) bound to all available free DNA resulting in the complete retardation of the hot template . This is distinct from the reduced amount of ail-like (YPTB2113) promoter template shifted by equivalent amounts of CpxR∼P. In this case, significant signal representing free unbound ail-like hot template still exists at the bottom of the gel  virulence gene promoters. On the other hand, modulation of expression of the Ail-like homologue (YPTB2113) by CpxR∼P is apparently not via direct binding, but must presumably involve at least one other unknown regulatory intermediate.Having established that CpxR∼P carries only one phospho group that is likely to be at AsppxR/cpxP . Interesphoresis . Quite p the gel . In factal start . Finallytemplate . Taken tYersinia gene expression, being responsible for fine-tuning expression of a multitude of genes involved in general housekeeping, environmental survival and pathogenicity rovA transcription, we performed a nuclease protection (footprinting) analysis to map the DNA binding site of CpxR∼P in the rovA promoter. A protected sequence of the sense strand estimated to be 5′-gcgtgctaacgataatgacaaaaattgacaaatcta-3′ was identified within the P2 promoter region of rovA (ppiA promoter (5′-ttctgttacgtaaatatccgtaaatgggtgg-3′) and the divergent cpxR-cpxP promoter (5′-gtcagggcatgtaaagctga-3′) rovA promoter, while more obviously conserved consensus CpxR∼P binding motifs were observed in the protected sequence upstream of ppiA and between cpxP/cpxR  . As expepxP/cpxR .ppiA, cpxR (and cpxP) and rovA (rovA might overlap with the −35 box of the P2 promoter 5ACAAA-3′5′-ACAAA(N) overlapping with the −35 box of the P2 promoter and roughly located 360 nucleotides upstream of the rovA ATG start codon contributes to CpxR∼P binding. In addition, the 5GTAAA-3′5′-GTAAA(N) sequences situated ∼80 and ∼55 nucleotides upstream of ppiA and cpxR respectively are also bona fide CpxR∼P binding sites.Based on the analysis of these ‘footprints’, we used sited-directed mutagenesis to shuffle the order of the nucleotides predicted to compose the CpxR∼P binding sites upstream of and rovA . Since trovA promoter restricts transcription. Therefore, elevating available CpxR∼P in the Yersinia cytoplasm in turn should reduce detectable RovA levels. To test this, we took advantage of work performed in E. coli, whereby phosphatase deficient CpxAT253P encoded by the allelic variant designated cpxA101* generally lead to a constitutively active Cpx pathway Yersinia, we constructed an in cis cpxA101* mutation in Y. pseudotuberculosis. We then utilized the Manganese(II)-Phos-tag™ acrylamide gel system to measure in vivo levels of CpxR∼P. Formic acid-lysed bacteria grown in LB broth to early stationary phase (OD600 in the range of 0.85 to 0.95) at 26°C were fractionated on a Manganese(II)-Phos-tag™ 12.5% acrylamide gel and blotted with affinity purified anti-CpxR antiserum. Impressively high levels of both CpxR and CpxR∼P was detected in the cytoplasm of the Yersinia mutant completely lacking the cpxA allele  lipoprotein Yersinia with pBAD18 derivatives encoding E. coli nlpE (termed pND18) or Y. pseudotuberculosis nlpE (NlpEY.p – pJF027) under the control of an arabinose inducible promoter. Since NlpEY.p shares less than 50% identity at the amino acid level with NlpEE.c  used to identify CpxR∼P binding in vitro   double mutant indicating that the ‘Mt 2’ mutation made the rovA promoter non responsive to Cpx pathway activation. Similarly, RovA levels were also restored in the cpxA101*, cpxR/P(Mt) double mutant (cpxR transcription is not induced limiting the accumulation of CpxR∼P (data not shown). To confirm these data, we performed semi-quantitative RT-PCR analysis to gain some insight into rovA transcription levels. Compared to the single cpxA101* mutant, rovA transcription was elevated in both cpxA101*, rovA(Mt 2) and cpxA101*, cpxR/P(Mt) double mutants  mutant was still low, which explains the absence of RovA detection by western blotting  . Signifie mutant . In this mutants . Interesblotting . This inblotting  predictaY. pseudotuberculosis is two-fold; not only does CpxR∼P serve to directly up-regulate the expression of genes encoding for periplasmic quality control factors, but it also down-regulates genes encoding virulence associated determinants – especially the global regulator RovA. In Y. pseudotuberculosis, the CpxA-CpxR TCRS therefore serves to coordinate the maintenance of bacterial envelope integrity with a reduction in virulence gene expression to ensure that vital energy resources are used to maximize the chances of survival in the prevailing environment.Pathogenic bacteria experience diverse environmental conditions both inside and outside of a host. Multiple regulatory pathways are required to incorporate these diverse signals that then converge to fine tune spatial and temporal virulence gene expression. Herein, we report that the effect of CpxA-CpxR pathway activation in the enteropathogen Yersinia? A review of studies on other OmpR/PhoB family members provides some insight into the many assorted possibilities. CpxR∼P binding to target DNA may physically recruit the RNA polymerase. This principle is well-established for the phosphate starvation regulon, whereby PhoB∼P facilitates specific interactions between RNA polymerase and the pho promoters ompF porin gene induced by changes in osmolarity ompC and ompF by OmpR∼P Yersinia gene expression.How can CpxR∼P both induce and repress the expression of target genes in pathogenic Streptococcus spp. can also bind DNA as a non-phosphorylated monomer, although binding is enhanced by phosphorylation-dependent dimerization Salmonella enterica that senses environmental Mg2+, dimerizes and binds DNA independent of phosphorylation D51A variant was not able to bind DNA under the assay conditions used here. However, our previous results suggested the possibility of weak binding when nonphosphorylated CpxR was used in EMSAs with rovA and invResponse regulator activation is thought to first require phosphorylation for the triggering of dimerization that is necessary for target DNA binding rovA transcriptional regulation are complex  promoter, the cornerstone of invasin control is positive regulation by RovA. We currently have no explanation for why CpxR∼P-dependent invasin regulation requires two mechanisms – one direct via binding to the inv promoter and the second indirect through binding to the rovA promoter. These two mechanisms may be a way to unite multiple overlapping signals to establish exquisite spatial and temporal control of inv expression – an important Yersinia virulence determinant. With respect to the control of other Yersinia surface adhesins, our DNA binding data suggests CpxR∼P might repress ail expression directly. Additionally, the controlled production of the pH 6 antigen appears to involve a complex mechanism requiring direct binding of CpxR∼P to the promoter of the tip adhesin gene psaA as well as to the promoter of psaE, encoding a membrane-associated protein required for transcriptional activation of psaApsaA transcription is currently unknown. Intriguingly, CpxR activation must also influence pH 6 antigen levels through an effect on RovA, since RovA binds to both psaA and psaE promoter regions cpxA101*-encoding CpxAT253P variant duplicates the effects of completely removing cpxA. In this context, it is curious that the cpxA101* and ΔcpxA full length mutants lacking ackA and pta both similarly failed to accumulate detectable CpxR∼P  or Y. pseudotuberculosis (NlpEY.p) to stimulate an intact Cpx pathway. NlpEY.p and NlpEE.c are about 49% identical at the amino acid level. Even though both proteins activated the Cpx pathway, NlpEY.p did so to a greater extent. Exactly how the Cpx pathway is induced by NlpE is unknown, but it likely involves sensing of bacterial adhesion to surfaces E. coli, only the over-expression of NlpE and one other – the inner membrane located YafY of unknown function – caused Cpx pathway activation Y.p and NlpEE.c could potentially provide important insight into the activating mechanism of the Cpx pathway.We made use of NlpE lipoprotein over-production, either from 51, peptides were generated by fast enzymatic digestion. Commonly used proteases such as trypsin, chymotrypsin, and the endoproteinases AspN and GluC, were not suitable to generate peptides for a study of Asp51. All produced theoretically predicted Asp51-containing peptides with masses above 4000, which is relatively high for successful structural analyses by tandem mass spectrometry (MS/MS). Nevertheless, a protocol for fast digestion with trypsin and pepsin was developed. While MALDI-MS and ion trap MS combined with ESI and ETD ionization 51 or any other phosphorylation sites were detected (data not shown). This was even the case after enrichment of phosphopeptides using titanium dioxide In an effort to provide experimental evidence for a phosphorylation of CpxR on AsprovA expression in Yersinia. RovA is a global regulator targeting in excess of 60 genes in each of Y. enterocolitica and Y. pestisYersinia pathogenicity can be widespread. We see CpxR∼P as an ‘over-ride mechanism’ capable of a quick response to repress virulence gene expression in over-committed, fully-induced bacteria that suddenly find themselves in an unfavorable environment exposed to ECS where virulence factors are no longer useful for their survival – to continue to express them would waste precious energy resources.In this study we have described how CpxR∼P accumulation has a direct negative effect on Table S1Bacterial strains and plasmids used in this study.(RTF)Click here for additional data file.Table S2Oligonucleotides used in this study.(RTF)Click here for additional data file.Figure S1Mass determination of intact CpxR and CpxR∼P by MALDI-MS. The respective CpxR and CpxR∼P peaks are indicated along with the experimental mass .(TIF)Click here for additional data file.Figure S2Y. pseudotuberculosis bacteria. Overnight cultures of parental and mutant bacteria were sub-cultured into fresh LB broth (time point 0 hours) and their growth during aerobic culturing with agitation at 26°C was monitored over a period of 9 hours by optical density measurement at 600 nm (A and B). Parent, YPIII/pIB102; YPIII07, ΔcpxA; YPIII08, ΔcpxR; YPIII46, mutated cpxR encoding CpxRM199A; YPIII49, ΔcpxA, ΔackA, pta null mutant; YPIII51, cpxA101* encoding CpxAT253P; YPIII52, mutated cpxR encoding CpxRD51A; YPIII69, ΔackA, pta null mutant; YPIII74, cpxA101*, ackA, pta null mutant; YPIII112, cpxR/cpxP(Mt) placed in the parent background; YPIII113, rovA(Mt 1) placed in the parent background; YPIII114, rovA(Mt 2) placed in the parent background; YPIII115, cpxR/cpxP(Mt) placed in the cpxA101* background; YPIII120, rovA(Mt 1) placed in the cpxA101* background; YPIII121, rovA(Mt 2) placed in the cpxA101* background. The asterisk (*) signifies modest to severe growth restriction in those bacteria with defects in the Pta-AckA biosynthetic pathway cpxA101* background, strain YPIII115/pIB102 grows quite well (B). Presumably, this is because CpxR∼P does not accumulate in this strain consistent with poor cpxR expression (data not shown) resulting from a shuffle mutation that makes the divergent promoter of cpxR/cpxP unable to bind CpxR∼P.Growth curves of various (TIF)Click here for additional data file.Figure S3rovA transcription in vivo. For transcription analysis, crude semi-quantitative RT-PCR was performed on mRNA isolated from Y. pseudotuberculosis. Given the limitations of semi-quantitative RT-PCR, we attempted to increase the robustness of our assay by reverse transcribing mRNA isolated from bacteria grown at 26°C in LB broth to two different growth phases as measured by optical density at 600 nm; the first being an OD600 value between 0.45 to 0.55 (mid-logarithmic phase) and the second being an OD600 measurement between 0.85 to 0.95 (early-stationary phase). This was necessary to control for the altered growth rate of those bacteria expected to accumulate toxic levels of CpxR∼P (rovA (plus RT). As an mRNA loading control, we analyzed the transcription of rpoA encoding for the α-subunit of RNA polymerase, which remained the same regardless of genetic background or phase of growth. To confirm the purity of the RNA isolated, PCRs with rpoA and rovA primer pairs was performed on template derived from RT reactions in which the enzyme was intentionally excluded (minus RT). PCR analysis on these samples indicated that the RNA isolation was essentially free of genomic DNA contamination. All images were acquired with a Fluor-S MultiImager (Bio-Rad) and analyzed with Quantity One software version 4.2.3 (Bio-Rad). DNA fragment sizes in base pairs are given in parentheses. Strains: parent, YPIII/pIB102; cpxA101* encoding CpxAT253P, YPIII51/pIB102; cpxR/cpxP(Mt)  placed in cpxA101*, YPIII120/pIB102; rovA(Mt 2) placed in cpxA101*, YPIII121/pIB102.CpxR∼P DNA binding is required for repression of f CpxR∼P . Samples(TIF)Click here for additional data file.Figure S4rovA and inv expression. The Cpx pathway is a sensor of extracytoplasmic stress (ECS) Y. pseudotuberculosis pathogenicity is also becoming apparent (this study) rovA and inv promoters repressing transcriptional output (red line). This influence may also be augmented indirectly through as yet unknown (indicated by a dashed line and a ‘?’ symbol) regulatory links to other positive (green) and negative (red) factors controlling rovA and/or inv expression in vitro electrophoretic mobility shift analysis did not reveal any CpxR∼P bound to the hns or ymoA promoters . Not shown in this diagram is the influence of CpxR∼P on lon expression Established and potential mechanisms of Cpx-dependent modulation of (TIF)Click here for additional data file.Figure S5rovA. Y. pseudotuberculosis rovA transcription is initiated from two sites (P1 and P2) upstream of the translational start codon (TTG – green) rovA DNA-binding regulators.Regulator binding sites in the upstream flanking sequence of (TIF)Click here for additional data file.