Our results support the hypothesis that Mss116p and Pet127p are involved in modulation of mtRNAP activity. Copyright © 2009 John Wiley & Sons, Ltd.The abundance of mitochondrial (mt) transcripts varies under different conditions, and is thought to depend upon rates of transcription initiation, transcription termination/attenuation and RNA processing/degradation. The requirement to maintain the balance between RNA synthesis and processing may involve coordination between these processes; however, little is known about factors that regulate the activity of mtRNA polymerase (mtRNAP). Recent attempts to identify mtRNAP–protein interactions in yeast by means of a generalized tandem affinity purification (TAP) protocol were not successful, most likely because they involved a C-terminal mtRNAP–TAP fusion (which is incompatible with mtRNAP function) and because of the use of whole-cell solubilization protocols that did not preserve the integrity of mt protein complexes. Based upon the structure of T7 RNAP (to which mtRNAPs show high sequence similarity), we identified positions in yeast mtRNAP that allow insertion of a small affinity tag, confirmed the mature N-terminus, constructed a functional N-terminal TAP–mtRNAP fusion, pulled down associated proteins, and identified them by LC–MS–MS. Among the proteins found in the pull-down were a DEAD-box protein (Mss116p) and an RNA-binding protein (Pet127p). Previous genetic experiments suggested a role for these proteins in linking transcription and RNA degradation, in that a defect in the mt degradadosome could be suppressed by overexpression of either of these proteins or, independently, by mutations in either mtRNAP or its initiation factor Mtf1p. Further, we found that Mss116p inhibits transcription by mtRNAP  They are responsible for production of most energy in eukaryotic cells through the process of oxidative phosphorylation  and, with the exception of the initiation factor Mtf1p  protocol to isolate highly pure protein complexes, while minimizing procedures that disrupt the association of their components, has become increasingly popular for such applications  was purchased from Open Biosystems Inc. Yeast cells were cultured on YEP media with 1.5% agar at 30 °C unless otherwise specified. E. coli XL10-Gold competent cells (Stratagene) were used for all subcloning and site-directed mutagenesis procedures. Yeast DNA was isolated from strain BY4743 as described ::pRS316-Rpo41p were carried out by standard methods  of a TAP tag from pMK33–NTAP vector  and submitted for DNA sequencing to Genewiz Inc.All reagents were purchased from Sigma-Aldrich unless otherwise specified. The heterozygotic diploid et al., 2PO4, 0.12% (NH4)2SO4, 0.05% CaCl2, 0.06% MgCl2, 0.0003% FeCl3, and 10 mg/l tetracycline. After vigorous agitation in 4 l baffled Erlenmeyer flasks with 10 drops of Antifoam Y-30, cells from mid-log 1.5 l culture (OD600nm = 5.0) were collected by centrifugation, and spheroplasts were obtained by rocking cells at 30 °C for 45 min with crude lyophilized lyticase from Arthrobacter luteus (∼600 units/g cells). Mitochondria were recovered from spheroplasts by osmotic lysis, with the addition of Protease Inhibitor Cocktail for use with fungal and yeast extracts (Sigma-Aldrich) and several steps of differential centrifugation, as described  were used immediately or flash-frozen in an acetone/dry ice mixture and stored at −70 °C for subsequent analysis.Isolation of a crude fraction of yeast mitochondria was performed as described , 7 mm β-mercaptoethanol). One µl nucleoid suspension was diluted into 10 µl transcription buffer  without addition of any external DNA template. Transcription reactions were carried out for 20 min at 30 °C, with or without addition of 10 mg/ml α-amanitin as indicated, and stopped by the addition of an equal volume of transcription stop buffer containing 90% formamide, 50 mm EDTA and 1 mg/ml bromophenol blue. After heating at 95 °C for 2 min, radioactively labelled RNA products were resolved by electrophoresis in polyacrylamide denaturing gels  for 1.5 h at 90 W. Transcription rate was quantified as amount of radioactivity incorporated into high molecular weight RNA products, using a PhosphorImager Typhoon 9200 system  and ImageQuant software.Mt nucleoids were isolated as originally described  and immediately resuspended in 3 ml ice-cold solubilization buffer . 75 µl 20% NP-40 were added slowly, drop-by-drop, to the mt suspension to a final concentration of 0.5%. After 15 min of incubation on ice, each lysate was split into two 1.5 ml tubes and non-soluble components were removed by centrifugation at 20 000 × g for 10 min. After centrifugation was repeated, the cleared supernatant was transferred into 1.5 ml non-stick microtubes (Phenix Research Products Inc.), which contained 50 µl pre-washed Protein A Sepharose 4B FF beads (Sigma-Aldrich). After 1 h of incubation at slow rotation to prevent bead sedimentation, the supernatant was separated from the beads on a touch-spin table microfuge (1000 × g maximum), using Micro Bio-Spin columns (Bio-Rad), and transferred to a fresh non-stick tube with 25 µl pre-washed IgG Sepharose 6 FF beads . After 3 h rotation the supernatant was discarded and the beads were washed five times with 1.5 ml solubilization buffer containing 0.1% NP-40 and lacking EDTA (‘wash buffer’). Beads from two 1.5 ml tubes were combined in 0.6 ml non-stick microtubes (Phenix Research Products Inc.), resuspended in 500 µl wash buffer containing 10 µl AcTEV protease  and rotation was continued for 6 h. The eluate was separated from the Sepharose beads on the Micro Bio-Spin columns and transferred to a fresh 0.5 ml tube containing 25 µl Calmodulin–Sepharose 4B beads  that had been pre-washed with wash buffer containing 2 mm CaCl2. After 3 h rotation, the beads were settled, washed five times with 0.5 ml wash buffer with 2 mm CaCl2, and twice with CaCl2-free buffer. Protein was eluted from the beads with three consecutive washes, each using 50 µl wash buffer with 2 mm EGTA, on ice for 15 min. The washes were combined, polypeptides were precipitated with acetone/TCA at −20 °C for 3 h and separated by electrophoresis in 4–12% Bis–Tris NuPAGE Novex protein gels in an MES buffer system (Invitrogen). The gels were stained with Coomassie Brilliant Blue R-250 (Bio-Rad), and each lane containing visible protein bands was sliced into 18 equal gel samples, which were submitted for tryptic digestion and LC–MS–MS analysis, as described below.200 µl frozen mt pellet were defrosted at 30 °C, washed once with 5 ml pre-warmed (30 °C) respiration buffer (RB) containing 10 met al., Excised bands were subjected to in-gel reduction, alkylation and enzymatic digestion  in a HEPA-filtered hood to reduce the keratin background. LC–MS–MS analysis was performed on the in-gel digest extracts, using an Agilent 1100 binary pump  directly coupled to a mass spectrometer. 2–8 µl of sample was injected onto the column using a LC Packings FAMOS autosampler . Nanobore electrospray columns were constructed from a 360 µm o.d., 75 µm i.d. fused silica capillary with the column tip tapered to a 10 µm opening . The columns were packed with 200 Å 5 µm C18 beads , a reverse-phase packing material, to a length of 10 cm. The pre-column was used to achieve a flow rate of 320 nl/min. The mobile phase used for gradient elution consisted of (a) 0.3% acetic acid and 99.7% water, and (b) 0.3% acetic acid and 99.7% acetonitrile. Tandem mass spectra (LC–MS–MS) were acquired on a Thermo Finnigan LTQ ion trap mass spectrometer . Needle voltage was set to 3 kV, isolation width was 3 Da, relative collision energy was 30% and dynamic exclusion was used to exclude recurring ions. Ion signals above a predetermined threshold automatically triggered the instrument to switch from MS to MS–MS mode for generating fragmentation spectra. The MS–MS spectra were searched against the NCBI non-redundant protein sequence database, using the SEQUEST computer algorithm  to give pMS34. To express N-terminally 6His-tagged Rpo41p, the expression plasmid pMS11 was created as previously described  was constructed following a previously reported approach (Schubot E. coli BL21(DE3) codon plus RIPL cells (Stratagene) carrying the pMS34 plasmid were vigorously aerated at 20 °C in 0.5 l LB Miller broth supplemented with 0.1 g/l ampicillin until the culture reached an optical density of 0.6 at 600 nm. After addition of 0.1 mm IPTG followed by a further 8 h of aeration, cells were collected by centrifugation (6000 × g for 10 min), resuspended in 25 ml Mtf1p lysis buffer , and disrupted on a Sonic Dismembrator 500 (Fisher Scientific), using 30 s pulses alternated with 30 s pauses over a total disintegration time of 20 min. The cell lysate was clarified by centrifugation (25 000 × g for 15 min) at 4 °C and applied to a 0.2 ml HisPur™ Cobalt Spin Column (Pierce) at room temperature (RT). The column was washed with 10 ml lysis buffer, and Mtf1p was eluted with 3 ml lysis buffer containing 150 mm imidazole. 1 ml fractions of eluate were collected, and the fraction containing the largest amount of protein was dialysed overnight at 4 °C against 500 ml dialysis buffer . The dialysed protein was further purified using 1 ml HiTrap™ Heparin HP column  and a 10 ml gradient of 0.3–1.0 m NaCl in dialysis buffer on an AKTA HPLC system . The peak 1 ml fraction(s) corresponding to 0.42 m NaCl were pooled, diluted two-fold with glycerol and stored at −20 °C. Typically, the resulting solution contained ∼1 mg His-tagged Mtf1p at a concentration of 0.5 mg/ml, as determined by UV spectroscopy (OD of 1.8 at 280 nm corresponded to 1 mg/ml concentration of Mtf1p).To purify His-tagged Mtf1p, E. coli BL21 codon plus RIL cells (Stratagene) containing pMS11 were grown at 25 °C in 0.5 l LB Miller broth supplemented with 0.1 g/l ampicillin until the culture reached an optical density of 0.5 at 600 nm. After the addition of 0.6 mm IPTG followed by a further 4 h of vigorous aeration, cells were collected by centrifugation, resuspended in 25 ml Rpo41p lysis buffer  and disrupted on a Sonic Dismembrator 500 (Fisher Scientific), as described for Mtf1p. The cell lysate was clarified by centrifugation as described, and NaCl was added to a final concentration of 0.8 m. 0.7 ml 10% poly(ethyleneimine) solution was added slowly, drop-by-drop, to the stirred lysate, and the precipitate was removed by centrifugation (10 000 × g for 5 min) at 4 °C following a 10 min still incubation on ice. The supernatant was diluted two-fold with a saturated solution of (NH4)2SO4 and incubated for 30 min on ice. The precipitate was collected by centrifugation (10 000 × g for 5 min), dissolved in 10 ml lysis buffer at 4 °C and filtered through a Poly-Prep Chromatography Column (Bio-Rad) packed with 0.5 ml Ni-NTA agarose (Qiagen) at RT. The column was washed with 5 ml lysis buffer followed by 5 ml lysis buffer containing 15 mm imidazole. The protein was eluted with 3 ml elution buffer . 1 ml fractions of eluate were collected, the fraction containing the largest amount of protein was loaded directly on a 1 ml HiTrap™ Heparin HP column , and the protein was eluted from the column with a 10 ml gradient of 0.2–1.0 m NaCl in buffer containing 40 mm Tris–HCl, pH 7.9, 5 mM β-mercaptoethanol, 5% glycerol. The peak 1 ml fraction(s) corresponding to 0.6 m NaCl were combined, distributed into aliquots and stored frozen at −70 °C. The typical yield of purified His-tagged Rpo41p was 3 mg at a concentration of 1.5 mg/ml, as determined by UV spectroscopy (1 mg/ml His-tagged Rpo41p corresponded to OD of 0.99 at 280 nm).To purify His-tagged Rpo41p, et al., m Tris–HCl, pH 7.5, 500 mm KCl, 1 mm EDTA, 1 mm DTT, 50% glycerol) at 5 µm concentration.Mss116p was expressed and purified as previously described  phosphine , 7 nm double-stranded DNA template , 20 nm Rpo41p, 20 nm Mtf1p, and Mss116p as indicated (in 0.8 µl Mss116p storage buffer) at 30 °C for various times, and stopped with an equal volume of transcription stop buffer as described above. Labelled RNA products were resolved by electrophoresis on denaturing 15% polyacrylamide denaturing gels for 2 h, as described above.Steady-state transcription reactions were carried out in a volume of 10 µl containing 20 me Figure A, 0.5 mmet al., et al., et al., et al., et al., et al., et al., Previous studies have shown that whereas alterations in the C-terminal region of T7-like RNAPs Figure C result et al., et al., et al., et al., in vivo, as even the smallest deletion resulted in a ts-petite phenotype and thus appeared to disrupt Rpo41p function  would abolish enzyme activity and result in loss of functional mitochondria, while an N-terminal tag might block delivery of Rpo41p to mitochondria or would be cleaved off with the targeting peptide. To circumvent this problem, we sought to identify internal locations in Rpo41p that would tolerate small affinity tags, e.g. an eight aa histidine (His) tag, without disrupting function, and would be solvent-exposed.Structural analysis of T7 RNAP reveals many surface exposed loops, and a number of these have been shown to tolerate substantial changes without disrupting function. Some of these regions are conserved in Rpo41p, suggesting that substitutions in these sites would also be tolerated by the mtRNAP Figure .et al., The resulting mutant mtRNAPs were substituted for the wild-type protein by a plasmid-shuffling technique. With the exception of mutant EI  salt conditions, which are commonly used. To rule out the possibility that the observed salt-resistant transcription activity might be due to contaminating nuclear RNAPs, control reactions were carried out in the presence of α-amanitin, a potent inhibitor of nuclear RNAPs . Experiments to determine the mechanism of action of Mss116p on mtRNAP activity are under way.One of the most abundant proteins identified in the mtRNAP–TAP pull-down was a DEAD-box protein, Mss116p, which has previously been shown to be involved in mtRNA processing . More importantly, our data identified a limited set of proteins that were not previously thought to be involved in interaction with the mt transcription complex, even though they had been implicated in mt gene expression by biochemical and genetic experiments  structural rearrangements in all organisms . To explore this further, it will be necessary to purify Yml6p and MrpL4p and examine their effects in a yeast mt transcription system.Regarding the observed association of ribosomal proteins Yml6p and MrpL4p with yeast mtRNAP, it has recently been shown that human ribosomal protein L12 interacts with human mtRNAP and stimulates mt transcription Some of the proteins that we identified in the TAP–mtRNAP pull-downs may have been recovered due to their association with the mt nucleoids, rather than directly with Rpo41p. For example, Hsp-like proteins have previously been shown to be a major component of human mt nucleoids  and to act as a negative regulator of Ire1p in the ER stress response  or oxygen-less conditions (e.g. argon-saturated buffers) in future experiments may enhance detection of proteins that are particularly sensitive to oxidation, although the oxidation of peptides during electrospray MS is difficult to avoid.et al., in organello  that do not affect TAP pull-down (Cojocaru et al., Only a small number of peptides were found in an informed search for Mtf1p. This may reflect the nature of the association of this protein with the transcription complex, which occurs only during initiation (Mangus In conclusion, we have demonstrated the potential of using a TAP pull-down procedure that has been optimized for mitochondria for the characterization of mt transcription complexes. This approach, and additional modifications that we have suggested, may prove useful in the analysis of other mt complexes. In addition, scanning insertion mutagenesis of the mtRNAP has identified a number of sites that appear to tolerate substitutions and modifications. This information may prove useful in the subsequent manipulation and engineering of yeast mtRNAP, as well as mtRNAPs from other organisms.


Alteration of bacterial DNA structure and/or associated functions in vivo by [Cu(hsm)H2O]Cl·3H2O was demonstrated by the induction of a recA-lacZ fusion integrated at the amyE locus of a recombinant Bacillus subtilis strain. Results from circular dichroism and denaturation of calf thymus DNA (CT-DNA) suggested that increased amounts of copper complex were able to stabilize the double helix of DNA in vitro mainly by formation of hydrogen bonds between chn and the sugars of DNA minor groove. In vivo and in vitro biological activities of the chn complex were compared with the chemical nuclease [Cu(phen)H2O]NO3 · 3H2O (cpn) where phen is phenanthroline.A mixed copper complex with deprotonated nalidixic acid  and histamine (hsm) was synthesized and characterized by FTIR, UV-Vis, elemental analysis, and conductivity. The crystal structure of [Cu(hsm)H Since Lescher and col. prepared the nalidixic acid in 1962 . LevofloFourth generation quinolones include the trovofloxacin with an effectiveness against anaerobic organism . Quinolo2 · 2H2O (J. T. Baker products) were used as provided. Far-IR spectra were taken on a Bruker IFS-55 spectrophotometer as polyethylene pellets. Medium-IR spectra were carried out on Perkin-Elmer spectrophotometer model. FT 1600 as KBr pellets. Electronic spectra were recorded on an Agilent 8453 UV-Vis spectrophotometer using water as solvent. The molar conductivity measurements were made with a Conductronic PC18 conductivity bridge with a nickel-platinezed electrodes cell using water solutions prepared immediately before used. Elemental analyses were performed by Desert Analysis, Organic Microanalysis, Tuczon AZ, USA. The histamine dihydrochloride, nalidixic acid (Sigma-Aldrich products), and CuCl2O]Cl · 3H2O: Nalidixic acid  was dissolved with NaOH  in 70 mL of water. This solution was added into a previously prepared equimolar mixture of histamine dihydrochloride  and CuCl2 · 2H2O  dissolved in a total volume of 15 mL of water. The final solution was brought to pH 7.5 and left for slow crystallization in a refrigerator (4°C). Purple crystals suitable for X-ray analysis were isolated. Anal. Calc. for C17H31CuN5O8Cl: C, 38.35; H, 5.87; N, 13.15. Found: C, 39.82; H, 5.50; N, 13.65%. IR : 1634(s), 1607(m), 1522(m), 1501(m), 539 (s), 503 (m). UV-Vis : λmax  212 (ε212 = 25674 L mol−1 cm−1), 257 (ε257 = 25596 L mol−1 cm−1), 323 (ε323 = 11138.15 L mol−1 cm−1), 625 (ε625 = 63.47 L mol−1 cm−1). Conductivity: 105 Ω−1 cm2 mol−1 in water 10−3 M.[Cu(hsm)H2O]NO3 · 3H2O was obtained as previously reference indicates , R1 = 0.0455, wR2 = 0.1053, R indices , R1 = 0.0655, wR2 = 0.1161. Selected bonds lengths and bond angles are presented in A crystal specimen of 0.35 × 0.20 × 0.15 mmB. subtilis strain YB-3001 carrying pCCR202 (a recA-lacZ fusion integrated in the amyE locus) was kindly provided by Dr. Ronald E. Yasbin . To propagate this strain, Luria Bertani (LB) medium was used. Chloramphenicol was added to the culture medium at a final concentration of 3 μg/mL.B. subtilis YB-3001 was grown to an optical density of 0.3 at 600 nm, and at this point, the culture was split into 6 subcultures. One of the cultures was used as control, and the other five were supplemented with CuCl2, nalidixic acid, [Cu(hsm)]2+, hsm, and [Cu(hsm)H2O]Cl · 3H2O complex to a final concentration of 300 μM. Samples of the culture were collected before and after 5 hours of addition of the compounds and they were processed for β-galactosidase activity as previously described Cl · 3H2O or [Cu(phen)H2O]NO3 · 3H2O complexes, 100 μM, and Mercaptopropionic acid (MPA), 7 mM. The reaction mixture was incubated for 50 minutes at 37°C, diluted with loading buffer, which contained 50 mM Tris-acetate buffer, pH 7, glycerol 50% (v/v), and Bromophenol blue as a tracking dye, and loaded on a 2% agarose gel containing 50 mM Tris-acetate buffer, pH 7, and 1 mg/mL ethidium bromide. The gel was run in the same buffer at 90 V. The DNA on the gel was observed and photographed using an Eagle Eye gel documentation system. A typical reaction mixture contained in a final volume of 25 280/Abs260 = 2 was used as an indication of a DNA free of proteins. The concentration of the CT-DNA stock solution was 6.22x10−3 M, determined at 260 nm and applying the molar extinction coefficient of 6600 M−1 cm−1 given in literature Cl · 3H2O /CT-DNA ratio . The DNA concentration was kept equal to 2.07 × 10−4 M in all experiments. After 1 hour of incubation at 37°C, the spectrum of each sample was recorded on a Jasco—715 spectropolarimeter at room temperature under constant nitrogen flush. The wavelength range between 200 and 300 nm was monitored. CD spectra were recorded at increasing [Cu(hsm)H2O]NO3 · 3H2O complex. The sodium cacodylate buffer was employed as background.All CD spectra were carried out in 5 mM sodium cacodylate, 0.6 M NaCl, pH = 7.1 buffer, using a quartz cell. Similar experiments were made with Cl · 3H2O complex of  τ. Tau-descriptor (τ) for five coordination complexes expressed as the difference between the angles of the bonds O(2)–Cu(1)–N(3) and O(3)–Cu(1)–N(8) divided by 60 gives a value of 0.13, being the ideal values of 1 for a trigonal bipyramid and 0 for a square pyramid Cl · 3H2O compound are quite complex due to the presence of numerous functional groups in the molecules; selected signals are listed in −1 can be assigned to the O–H stretching vibrations of lattice water molecules. Also between 3261 and 2661 cm−1 some medium bands appeared corresponding to amine and amide vibrations forms. A broad band between 1634 and 1501 cm−1 which is splitted and includes the vibrations vα (COO) and vs (CO) is present, showing the interaction of these groups (the 4-oxo and to the 3-carboxylate groups) with the metallic ion. Similar results are observed for far infrared spectrum. At 538 and 503 cm−1 two bands assigned to Cu–NH2 combination modes appeared, as it had reported previously [π − π transitions of the ligands as showed in similar hsm complex previously reported [2O]Cl · 3H2O compound with [Cu(phen)H2O]NO3 · 3H2O complex NO3 · 3H2O metal complex to interact with DNA in vivo and its capacity to promote the complete degradation of plasmid and chromosomal DNA under reductive conditions has been previously reported [2O]Cl · 3H2O complex has similar biological activity. For this purpose, we investigated here whether chn complex behaves as a chemical nuclease by inducing the degradation of double stranded DNA under both, reductive, and nonreductive conditions. As a positive control we used cpn complex at the same concentration. The results shown on chn complex does not posses chemical nuclease activity even in the presence of Mercaptopropionic acid (MPA) Hreported . Therefotrations , Lane 6 chn with DNA we took advantage of the bacterial SOB response of the gram-positive microorganism Bacillus subtilis. This response, similar in several aspects to the SOS response of E. coli, is triggered by physicochemical agents which promote alterations in the structure of DNA and when B. subtilis is grown under conditions which induce genetic competence Cl · 3H2O complex and with each of its components, ]2+). As shown in β -galactosidase induced by the [Cu(hsm)H2O]Cl · 3H2O complex were 20% lower than those induced by the analogous [Cu(phen)H2O]NO3 · 3H2O complex [2O]Cl · 3H2O complex were able to induce the β-galactosidase activity from the recA-lacZ fusion to levels comparable to those observed for the full complex, neither its activity can be interpreted as an additive effect of the independent components. To investigate the interaction of mpetence –29. Thus complex . HoweverTherefore, we can conclude that the complex acts as a whole, being able to cross the cell membranes and interact with the DNA directly. 2O]Cl · 3H2O DNA interaction, the in vitro interaction of chn and cpn with CT-DNA was analyzed by melting temperature and circular dichroism (CD). In 2O]Cl · 3H2O. To investigate the way of [Cu(hsm)H2O]NO3 · 3H2O. It is important to mention that both complexes are CD inactive. The observed changes in the CD spectra suggest that both complexes interact with DNA through the sugar-phosphate bone, in the minor groove. This result is in agreement with the increment in the melting point observed when the complex concentration increases, Hses, see  and suggf CT-DNA . HoweverThis difference between both complexes should be related with the type of coordinated ammine. Being histamine able to form stronger hydrogen bonds, it is reasonable to speculate that when complex concentration increases, more hydrogen bonds will be formed and therefore more stable the double helix will be. On the other hand, when phenantroline is the diammine at low concentration, it is possible that the main interaction between the complex and DNA is dominated by hydrogen bonds with the antibiotic, but when concentration increases, it is possible to have two types of interactions between the complex and DNA, one via hydrogen bonds as stated before and a second one weaker due to the intercalation of the phenanthroline rings, which will be reflected in a destabilization of the double helix. In conclusion, from this study is possible to say that quinolones behave very similar as ligands; also it is observed that their coordination mode is relatively independent of the type of coordinated diammine. However the activity as chemical nuclease is strongly dependent of the diammine. Our work suggests that complexes of the quinolone family drugs with copper coordinated to other ammines may be active, even if the nuclease activity is not present as in those complexes with phenantroline. It seems that DNA-complex interaction is dominated by hydrogen-bond type, and therefore the design of new potential copper-based drugs should consider this.


Geomyces destructans and is responsible for the deaths of >1,000,000 bats since 2006. This disease and fungus had been restricted to the northeastern United States. We detected this fungus in a bat in France and assessed the implications of this finding.White-nose syndrome is caused by the fungus  Biologists are struggling to understand a recent emerging infectious disease, white-nose syndrome (WNS) (Geomyces destructans)  found on March 12, 2009, near Périgueux , showed a powdery, white fungal growth on its nose , and narrowed at each end, one of which was broadly truncate, often showing an annular frill , panel CG. destructans, which was recently isolated from WNS-positive bats in the northeastern United States  following the manufacturer’s instructions with slight modifications . The internal transcribed spacer (ITS) regions  and the small subunit (SSU) of the rRNA gene were amplified separately.2, 0.4 μmol/L of each primer, 0.2 mmol/L dNTP, 1× PCR buffer, and 1 U of Platinum Taq DNA Polymerase High Fidelity . Identical PCR cycling conditions were used for both fragments: an initial step at 95°C for 15 s; 10 cycles at 95°C for 30 s, 60°C (reduction of 2°C every 2 cycles) for 1 min and 45 s, and 72°C for 1 min; 30 cycles at 95°C for 30 s, 50°C for 1 min and 45 s, and 72°C for 1 min; and a final step at 72°C for 10 min. PCR products were purified and sequenced in both directions by using primers listed in the www.codoncode.com/aligner/download.htm).PCRs were performed in 25-μL volumes containing 1 μL of DNA (10–75 ng/μL), 1.5 mmol/L MgClG. destructans fungus  and SSU (GQ489025). Sequences obtained for the 2 genetic markers showed a 100% sequence identity with the described M. myotis bats may migrate up to 436 km to reach their summer roosts  in Bat, France


Killer cell lectin-like receptor G1 (KLRG1) is a type II transmembrane glycoprotein inhibitory receptor belonging to the C type lectin-like superfamily. KLRG1 exists both as a monomer and as a disulfide-linked homodimer. This well-conserved receptor is found on the most mature and recently activated NK cells as well as on a subset of effector/memory T cells.2+-dependent cell-cell adhesion molecules comprises of an extracellular domain containing five cadherin repeats responsible for cell-cell interactions, a transmembrane domain and a cytoplasmic domain that is linked to the actin cytoskeleton. Using KLRG1 tetramer as well as other methods, E-, N-, and R-cadherins  were identified as KLRG1 ligands. These CaGeneration of the KLRG1 tetramer was essential to the identification of the KLRG1 ligands. KLRG1 tetramer is also a unique tool to elucidate the roles cadherin and KLRG1 play in regulating the immune response and tissue integrity. Inoculate 4 x 500 ml flasks of TB each containing 50 μg/ml of carbenicillin and chloramphenicol with an overnight culture of bacteria expressing the KLRG1 plasmid construct.  When the OD reaches 0.6 Å at 600 nm, induce with the addition of 0.4 M IPTG and incubate the culture for an additional 4 hours shaking at 37°C. Resuspend the harvested cells in resuspension buffer pH=8.0  sucrose, 1 mM EDTA, 10 mM DTT).  Note: pellets can be frozen at this stage.Pool no more than 60 ml of bacterial resuspensions in a polypropylene beaker. If necessary adjust to 60 ml with TE buffer pH 8.0 and stir mixture at half speed.To stirring mixture add drop wise: lysozyme , MgCl2 , 2 mg DNAse I in 50% glycerol contained 75 mM NaCl, Triton-X100 , DTT .Decant the supernatant.Add 15 ml of wash buffer pH=8.0 .On ice, sonicate solution for 1.5 min until the pellet is completely resuspended.Centrifuge the samples at 5,000 RPM for 10 min at 4°C.Repeat the wash 5 times.Sonicate the solution on ice for 1.5 min and centrifuge the lysates at 5,000 RPM for 10 min at 4°C.  Repeat 5b with wash buffer without Triton X-100, centrifuge and resuspend in 4 ml of TE Buffer.Take the wet weight inclusion bodies slurry and store in TE buffer at a concentration of 30 to 100 mg/ml.Make 1 L of refolding buffer pH=8.0  and chill to 4°C while stirring. Melt the volume of 50 mg of inclusion bodies slurry in 7 M GnHCl with 10 mM β-mercaptoethanol.Keep the inclusion bodies/GnHCl mixture at 37°C for 30 - 40 min and vortex every 5 min to ensure complete melting (slurry will become clear when completely melted).Centrifuge at max speed for 30 min in microcentrifuge at 4°C and transfer the supernatant to a 15 ml centrifuge tube. Do not to transfer any of the small blackish pellet.  Prepare the inclusion bodies for injection into the folding mixture:  It is ideal to make 5   10 injections each with 0.25   0.5 μM concentration so that the final concentration of the protein will be 2   3 μM.  Adjust volume with injection buffer .Inject 1 ml of diluted inclusion bodies every hour.  When injecting, stir at high speed, add 1 ml of diluted inclusion bodies drop by drop with 2 seconds between each drop.  When injection is done, stir at low speed.Continue overnight stirring at low speed at 4°C. Following Millipore s protocol, concentrate the 1 L of pre-filtered (0.22 μm filtered) refolding reaction using an Amicon Stirred Cell and YM10 NMWL 10K ultrafiltration membrane (Millipore) to ~10 ml. Concentrate to ≤2 ml using a Centriplus YM-10 10K MWCO (Millipore). Setup the AKTAFPLC at 4°C  according to GE Healthcare manuals.Purify the monomer form of KLRG1 by size exclusion chromatography using a Sephacryl S-200 16/60 high-resolution column  in 20 mM HEPES and 150 mM NaCl. .Concentrate to 1 ml using Centriplus YM-10 10K MWCO (Millipore).Use BirA enzyme according to Avidity s protocol to biotinylate the KLRG1 monomer.The free biotin is eliminated by size exclusion chromatography as in 2.KLRG1 molecule is tetramerized by mixing KLRG1 monomer with 4 fold molar excess PE conjugated streptavidin (BD Biosciences).          Click here for a larger figure.          Figure 1. Representative positive results from the AKTA FPLC size exclusion chromatography of the refolded KLRG1.  The graph shows the separation of the monomer (83.52 ml), dimer (56.36 ml), and multimer (36.26 ml) form of the refolded KLRG1.  The peak at 94.25 ml represents the buffer exchange.		In this protocol, it is shown how to purify, biotinylate, and tetramerize KLRG1. KLRG1 tetramer can be used to label KLRG1 ligands by flow cytometry. Although refolding conditions will have to be tested empirically for each protein, other C-type lectins could be tetramerized using a similar protocol. Using tetramerized proteins as probes, ligands to orphan C-type lectin receptors could potentially be identified.


According to this study, the strongest T cell receptor ligands in vitro do not necessarily induce the strongest T cell responses in vivo, suggesting that vaccine designers may need to reconsider their strategies. + T cell activation, effector, and memory responses to immunization with peptides exhibiting a range of TCR/pMHC half-lives and in vitro T cell activation potencies. Contrary to predictions from in vitro studies, we found that optimal in vivo T cell responses occur to ligands with intermediate TCR/pMHC half-lives. The diminished in vivo responses we observed to the ligand exhibiting the longest TCR/pMHC half-life were associated with attenuation of intracellular signaling, expansion, and function over a broad range of time points. Our results reveal a level of control over T cell activation in vivo not recapitulated in in vitro assays and highlight the importance of considering in vivo efficacy of TCR ligands as part of vaccine design.αβ T cell receptor (TCR) recognition of foreign peptides bound to major histocompatibility complex (pMHC) molecules on the surface of antigen presenting cells is a key event in the initiation of adaptive cellular immunity. In vitro, high-affinity binding and/or long-lived interactions between TCRs and pMHC correlate with high-potency T cell activation. However, less is known about the influence of TCR/pMHC interaction parameters on T cell responses in vivo. We studied the influence of TCR/pMHC binding characteristics on in vivo T cell immunity by tracking CD4 As an important part of immune system, T cells fight infections by recognizing signs of foreign invaders. A molecule on the surface of these cells—called the T cell receptor—recognizes and binds to protein components (peptides) from bacteria, viruses, and other pathogens that are displayed on the surface of other cells. The T cells can then use this information to orchestrate the fight against infection. Vaccination involves injecting into the body foreign peptides that mimic a pathogen, therefore tricking it into raising a T cell response against that pathogen that will be protective in the event of a real infection. We studied T cell responses in mice injected with one of several peptides to which the T cell receptor binds more or less strongly. Contrary to expectations, we found that the peptide that interacted most strongly with the T cell receptor did not provoke the strongest T cell response. This may be nature's way of preventing harmful inflammatory damage due to excessively strong T cell activation. Our work shows that peptides that bind the T cell receptor with medium strength may be best to use for vaccines. Current vaccine strategies seeking to design peptides that bind with maximum strength to the T cell receptor may need to be reconsidered in light of our findings. D) was found to be the more closely associated with potency when the association rate (ka) was large (>105 M−1s−1), whereas half-life or dissociation rate (kd) correlated well with potency when kon was small (103 M−1s−1) a), the strongest interactions, whether defined by kinetic or equilibrium parameters, yielded the most potent T cell responses.Activation of T cells by peptides bound to major histocompatibility complex (pMHC) molecules on antigen presenting cells (APCs) initiates the development of adaptive immunity to pathogens and is characterized by relatively low affinity but highly sensitive interactions between TCRs and pMHC + helper T cell differentiation + T cell response, especially given that the goal of many vaccine strategies is to elicit strong, persistent T cell responses to pathogens and tumors. The importance of CD4+ T cell help in vaccination protocols is underscored by the suboptimal responses elicited by minimal CD8+ T cell epitopes in many cases In addition to its influence upon the magnitude of T cell responses, TCR ligand potency is also thought to influence the outcome of in vitro CD4+ T cell vaccine design We examined the effect of TCR ligand potency upon in vivo CD4+ T cell responses using adoptively transferred 5C.C7 T cells activated by immunization with lipopolysaccharide (LPS) and moth cytochrome c (MCC) peptide, or related ligands of varying potency of in vitro T cell activation + T cell responses to Listeria monocytogenes expressing various OT-1 TCR ligands + T cell responses, we were able to examine a ligand that is stronger in vitro than the natural 5C.C7 TCR ligand MCC (88–103) A recent study of CD8k+ T cell activation. Amino acid substitutions of MCC (88–103) result in a panel of peptide ligands that bind to I-Ek equally well k (14.3 µM TCR), K3/I-Ek (1.8–14.3 µM TCR), MCC/I-Ek (1.8–14.3 µM TCR), and K5/I-Ek (1.8–14.3 µM TCR). SPR signal plots of all four pMHC complexes with 7.1 µM and 14.3 µM injected TCR are shown in a and kd constants, which were used to derive half-life and KD of the TCR/pMHC interactions  for MCC/I-Ek and K5/I-Ek are similar , while that for K3/I-Ek complexes (165 µM) indicates a lower monomeric binding affinity at 25°C , consistent with the inability to measure the interaction by SPR  in MCC- or K5-stimulated samples  and interferon-γ (IFN-γ) (+ 5C.C7 T cells induced in response to immunization with the various ligands is shown in the lower right panel of Upon ex vivo restimulation of CD4 (IFN-γ) . For 102 (IFN-γ) , showinglo phenotype (EM) + T cells. In contrast to what is predicted from biochemical data, the strong peptide ligand K5 induces a blunted in vivo 5C.C7 response that is of lower magnitude and function than the wild-type ligand MCC.No significant differences were found in levels of canonical activation markers such as CD25, CD62L, and CD44 during the initial expansion of 5C.C7 T cells in response to the various peptides . Howeverhenotype , suggesthenotype  and lymphenotype , these pk tetramer staining of lymph node cells from B10.A mice (containing no adoptively transferred 5C.C7 T cells). Seven days after immunization, we found low percentages of endogenous responder T cells to all four peptides  and analyzed the in vivo activated 5C.C7 T cells for levels of phosphatases and phosphorylated signaling intermediates  and S11.Strikingly, when the in vivo–activated 5C.C7 T cells were analyzed for levels of S473-phosphorylated Akt (pAkt), which reflects optimally active Akt kinase In contrast to the assumption that more potent T cell responses result from higher-avidity TCR recognition, here we report diminished T cell responses to a strong TCR ligand in vivo. We studied the in vivo activation of 5C.C7 T cells by immunization with LPS and a panel of peptides with a range of in vitro T cell activation potency, which results in fully functional effector and memory responses. Interestingly, we observed optimal T cell responses to the peptide ligands with intermediate TCR/pMHC half-lives. Contrary to what is predicted from in vitro data, the K5 ligand, which has the longest TCR/pMHC half-life, induces less in vivo division, accumulation, cytokine production of 5C.C7 T cells during the effector phase of the response, as well as persistence of fewer 5C.C7 T cells during the memory phase.EM development and greater homing to tissues CM development, or even impaired TEM development if the CD62L low population present in the MCC response is considered to be lymph node TEM cells -containing complex mTORC2 in these cells + T cell activation, in vitro ligand potency correlated with the magnitude of the in vivo T cell response, i.e. the highest-affinity ligand  induced the most robust response + T cell responses.In a recent study of in vivo CD8+ T cell response to pigeon cytochrome c (PCC) protein was recently examined by Malherbe et al. + T cell population that we tracked in response to strong TCR ligand. It is hard to compare the avidities of the responding TCRs since both the TCRs and the peptide ligands in question are different. Therefore, it is possible that the responses to the strongest ligand in our study exceeds an avidity threshold beyond which T cell attenuation occurs, and that this threshold is not reached by any of the TCRs in the Malherbe et al. study. It is also possible that the naïve repertoire in the Malherbe et al. experiments contains T cells with even higher avidity for PCC antigen than the clones favored by immunization with the TLR agonists. These clones may then be attenuated in response to high-avidity TCR engagement, such as we observed in our study. This could result in the underrepresentation of such clones at the peak of the primary response, and thus they may not be present during the CDR3 sequence analysis performed by Malherbe et al. The effect of adjuvant upon the avidity of TCRs in the polyclonal CD4+ T cell study mentioned above At the low potency end of the ligand spectrum, our results were in agreement with the CD8+CD25+Foxp3+ cells were depleted by injection of anti-CD25 antibody . Purified biotinylated pMHC complexes were immobilized on a streptavidin chip (BIAcore Inc.) to a level of 250–500 RU. Free biotin-binding sites were blocked with 10 mM biotin. Binding of 5C.C7 TCR was measured in PBS, pH 7.8 with 0.005% Surfactant P20 (BIAcore Inc.) by injecting a range of concentrations (0.9–14.3 µM) at a flow-rate of 30 µl/min. All data were background subtracted using a biotin-blocked streptavidin surface as a reference surface. Data were fitted to a Langmuir binding model using global fitting  to calculate ka and kd constants. To prevent aggregation artifacts, all proteins were purified no more than 12 h before analysis without subsequent concentration steps.Coding regions for the 5C.C7 TCR α and β chains were cloned by PCR into a modified version of the transfer vector pACGP67A (Pharmingen), which placed the 5C.C7 α and β in frame with c-jun (α) and c-Fos (β) heterodimerization motifs. Both chains were linked at the C-terminus to a 6XHis tag through a 3C-protease-cleavable site. TCR was expressed in insect cells as previously described 5C.C7 TCR transgenic RAG2−/−- mice were purchased from Taconic Farms and bred to B10.A CD45.1 (provided by W. Paul via the NIAID contract facility at Taconic Farms) to generate 5C.C7 RAG2−/− CD45.1 mice, which were used as donor cells in adoptive transfer experiments. Male B10.A recipient mice were purchased from Taconic and were used to initiate experiments at 6–9 wk of age.k tetramers with MCC and variant peptides were produced. Naïve 5C.C7 T cells were incubated with tetramers (50 µg/mL) for 2 h at RT in the presence of fresh 0.2% NaN3, equilibrated to 37°C or 25°C, and measurement of dissociation was initiated with the addition of anti-I-Ek antibody. Aliquots were removed in 10–20 s intervals and fixed immediately in 1% PFA. Levels of tetramer staining were assessed by flow cytometry. Apparent KD analysis was done as previously described Tetramer production, dissociation assays, and data analysis were done as described 2 in complete RPMI1640 . Single-cell suspensions were prepared from lymph nodes harvested from 5C.C7 RAG2-deficient mice (routinely >90% Vα11+Vβ3+ by flow cytometry) and stimulated in triplicate in 96-well round-bottom plates with irradiated  B10.A splenocytes and the indicated peptides for 60 h. Proliferation was monitored by the addition of 3H-methyl-thymidine (1 µCi/well). Cells were harvested onto glass-fiber filters using a Tomtec harvester, and filters were counted using a MicroBeta scintillation counter (Perkin-Elmer).Cells were cultured in a 37°C humidified chamber with 5% C0Peptides were synthesized and HPLC-purified (≥95%) by Biosynthesis, Inc. . Purified LPS was from Invivogen.Flow cytometry was done on a BD LSRII and data were analyzed with Flowjo software (Treestar). Antibodies to surface markers were from BD Pharmingen, eBioscience, or BioLegend. Antibodies to phospho-Akt, phospho-Stat, phospho-Erk, and PTEN were Alexa 647 conjugates from Cell Signaling Technology. SHP-1 antibody was from Santa Cruz Biotechnology.4 5C.C7 RAG2−/− CD45.1 T cells were transferred into B10.A recipients by tail vein injection, and the mice were immunized the next day with 20 µg peptide and 10 µg LPS at two sites on either side of the base of the tail. In some experiments the cells were CFSE-labeled prior to transfer. Proliferative capacity was calculated from CFSE profiles as described 1–5×102 of T cell area.5C.C7 RAG2−/− CD45.1 T cells were activated in vivo as described above. Six days after immunization with MCC or K5 peptide (5 mice per peptide), mice were sacrificed and the lymph nodes were embedded directly into OCT and frozen using dry ice and methanol. After temporary storage at −80°C, 10 µm sections were cut through the entire block (containing 50 lymph nodes) using a Leica Cryostat and stained with anti-CD45.1-biotin/streptavidin-PE to mark 5C.C7 T cells and anti-B220-FITC and anti-CD19-FITC to mark B cell areas. The stained sections (each containing 10 or more lymph nodes) were scanned with a Mirax slide scanner (epifluorescence). The number of 5C.C7 T cells was quantified from the T cell areas of 20 independent lymph nodes using Metamorph software. Total fluorescence was divided by the average individual T cell area to estimate the number of 5C.C7 T cells per mmTo enrich for 5C.C7 T cells originating from low precursor frequency before flow cytometry, CD4+ T cells were purified from lymph node suspensions by Dynal negative selection (Invitrogen).Lymph node suspensions were fixed immediately after harvest and then negatively selected for CD4+ T cells. After staining with antibodies to CD4 and CD45.1, cells were permeabilized with methanol and stained with phospho-antibodies.+ cells were purified by positive selection (Miltenyi) from spleens of mice adoptively transferred and immunized as described, irradiated, and used to stimulate naïve 5C.C7 RAG2−/− T cells in vitro. Proliferation was assessed by 3H-thymidine incorporation as described.CD11ct test analysis with Prism software.Data were analyzed for significance using unpaired Student's Figure S1k half-life derived by tetramer dissociation and SPR.Correlation of 5C.C7 TCR/peptide-I-E Plot of the half-life of interaction of purified 5C.C7 TCR with I-Ek complexed with K3, MCC, and K5 peptides derived by SPR (d by SPR  versus td by SPR .(0.45 MB EPS)Click here for additional data file.Figure S2k tetramers interact more strongly with 5C.C7 T cells by equilibrium tetramer staining and apparent KD analysis.K5/I-E (A) Naive 5C.C7 T cells were stained for 2 h at 25°C with the indicated I-Ek tetramers (50 mg/mL). *p = 0.0190. Data are representative of three independent experiments and error bars show mean ± s.d. (B) Scatchard plot of tetramer binding at varying concentrations of tetramer, expressed in nM. Apparent KD analysis was done as described in D values from which are shown in (C). *p = 0.0364. Two-tailed p value is from unpaired t test.(0.82 MB EPS)Click here for additional data file.Figure S3Immunofluorescence of 5C.C7 T cells in lymph nodes 6 d after immunization with MCC or K5 peptide. B10.A mice adoptively transferred with CD45.1+ 5C.C7 RAG2−/− T cells were immunized with LPS and MCC or K5 peptide as described in p values are from unpaired t test.(7.04 MB TIF)Click here for additional data file.Figure S4Phenotype of in vivo stimulated 5C.C7 T cells. (A) B10.A mice with adoptively transferred CD45.1+ 5C.C7 RAG2−/− T cells were immunized with indicated peptides and LPS, and CD4+ T cells from lymph node were analyzed 2.5 d after immunization. Each plot represents samples pooled from three to five mice. Percent of CD25+ or CD62Llo (of CD4+CD45.1+ cells) is shown on plots. (B) CD5 levels on 5C.C7 T cells 2.5 d after immunization. Each histogram and bar represent samples pooled from three to five mice. (C) Lymph node samples analyzed at day 6. Error bars show mean ± s.d., n = 3 mice.(2.88 MB EPS)Click here for additional data file.Figure S5Reactivation of resting lymph node 5C.C7 T cells. B10.A mice with adoptively transferred CD45.1+ 5C.C7 RAG2−/− T cells were immunized with indicated peptides and LPS. (A) PBLs 45 d later immunization. *p = 0.0332, n = 4. (B) The mice in (A) were all reimmunized with MCC peptide and LPS, and lymph node CD4+s were analyzed 4 d later. **p = 0.0061, n = 4. (C) Lymph node CD4+s from the mice in (B) were restimulated ex vivo with DCs and MCC peptide and stained for intracellular IFN-γ. Two-tailed p values are from unpaired t test.(0.59 MB EPS)Click here for additional data file.Figure S6Endogenous responses to MCC and related variant peptides. B10.A mice were immunized with the indicated peptides and LPS, and lymph node cells were stained with PE-MCC/I-Ek tetramer 7 d later. (A) Plots are gated on CD4+CD44hiB220-propidium iodide. Percent of tetramer+ cells (of CD4+ T cells) is shown on plots and graphed in (B). Horizontal lines on the graph represent the mean. (C) Mean fluorescence intensity of tetramer in gated populations. Error bars show mean — s.d., n = 3 mice. The data are representative of two independent experiments.(1.81 MB EPS)Click here for additional data file.Figure S7MCC and K5 peptides persist equally in vivo. B10.A mice were immunized with the indicated peptides at 4, 2, 1, and 0 wk before adoptive transfer of CFSE-labeled 5C.C7 T cells. 5C.C7 T cell division was analyzed at 48 h after transfer as a measure of peptide antigen persistence over time. All histograms are gated on CD4+CD45.1+ 5C.C7 T cells. Data shown are representative of two mice per condition and two experiments.(0.99 MB EPS)Click here for additional data file.Figure S8No evidence of a role for regulatory T cells in blunted responses to K5 peptide in vivo. B10.A mice were injected intraperitoneally with 0.5 mg αCD25 antibody (clone PC61) to deplete regulatory T cells, or 0.5 mg rat IgG as a control.  Three days later, mice were bled and PBLs were analyzed for CD4+CD25+Foxp3+ cells to confirm depletion, and adoptively transferred with 5C.C7 T cells.  Analysis of CD4+Foxp3+ cells in lymph node samples 10 d after depletion shows decreased amounts of regulatory T cells persist throughout the course of the experiment. (E) Seven days after immunization with either MCC or K5 peptide (11 d after injection of PC61), CD45.1+ 5C.C7 T cells were quantified in lymph node samples. Data are representative of three independent experiments.(1.84 MB TIF)Click here for additional data file.Figure S9Subcutaneous immunization with peptide and LPS does not induce Foxp3 expression in 5C.C7 T cells. B10.A mice containing adoptively transferred CD45.1+ 5C.C7 T cells were immunized subcutaneously with LPS+the indicated peptide. Six days after immunization, Foxp3 expression was assessed in lymph node samples by flow cytometry. Each histogram represents an individual mouse, and the experiment shown is representative of two independent experiments. As a control, the bottom histogram shows the induction of Foxp3 that occurs in 5C.C7 T cells in response to low doses of i.v. MCC peptide.(1.59 MB EPS)Click here for additional data file.Figure S10No significant differences in ppErk, SHP-1, and PTEN levels between MCC- and K5-stimulated 5C.C7 T cells. B10.A mice containing adoptively transferred 5C.C7 T cells were immunized with LPS+MCC or K5 peptide. 2.5 d after immunization, lymph node samples were fixed immediately after harvest, methanol permeabilized, and stained with fluorophore-conjugated antibodies to ppErk, SHP-1, or PTEN (C). Histograms are gated on CD4+CD45.1+ cells (A). The graph in (C) shows that fewer K5 stimulated 5C.C7 T cells are present at the 2.5 d time point. The black histograms in (C) show the levels of the ppErk, SHP-1, and PTEN in endogenous CD4+ T cells.(0.86 MB EPS)Click here for additional data file.Figure S11Specificity controls for phosphoantibodies. B10.A mice containing adoptively transferred 5C.C7 T cells were immunized with LPS alone or LPS+MCC peptide. 2.5 d after immunization, lymph node samples were fixed immediately after harvest, methanol permeabilized, and stained with the indicated phospho-antibodies. In the right panels, the antibodies were incubated with a peptide corresponding to the reactive phospho-epitope (according to the manufacturer's protocol) prior to staining of lymph node samples. (A) pAkt (S473). (B) pStat3 (Y705).(0.92 MB EPS)Click here for additional data file.Figure S12pAkt and pStat staining of in vitro–stimulated 5C.C7 T cells. Lymph node cells from 5C.C7 RAG2−/− mice were stimulated with irradiated CD11c+ splenic DCs and the indicated peptide for 60 h. Cells were fixed, methanol permeabilized, and stained with antibodies to pAkt (S473) (A) or pStat3 (Y705) (B). Histograms are gated on CD4+ cells and percentages of phospho-Akt or phospho-Stat3 are shown.(0.82 MB EPS)Click here for additional data file.


Background Since its initial detection in April 2009, the A/H1N1pdm influenza virus has spread rapidly in humans, with over 5,700 human deaths. However, little is known about the evolutionary dynamics of H1N1pdm and its geographic and temporal diversification.Methods Phylogenetic analysis was conducted upon the concatenated coding regions of whole-genome sequences from 290 H1N1pdm isolates sampled globally between April 1 – July 9, 2009, including relatively large samples from the US states of Wisconsin and New York. Results At least 7 phylogenetically distinct viral clades have disseminated globally and co-circulated in localities that experienced multiple introductions of H1N1pdm. The epidemics in New York and Wisconsin were dominated by two different clades, both phylogenetically distinct from the viruses first identified in California and Mexico, suggesting an important role for founder effects in determining local viral population structures. Conclusions Determining the global diversity of H1N1pdm is central to understanding the evolution and spatial spread of the current pandemic, and to predict its future impact on human populations. Our results indicate that H1N1pdm has already diversified into distinct viral lineages with defined spatial patterns. The TMRCA of clade 2 suggests it emerged around the same time as clade 1  and circulated for ~3 – 6 weeks prior to detection .  In contrast to clade 1, clade 2 disseminated widely to Canada, France, Germany, China, and to multiple US states: Indiana, Kansas, Maryland, New York, Washington, and Wisconsin .  Clade 2 is characterized by two amino acid substitutions: M581L (PA) and T373I (NP) (Table 1).  Following week 8, no isolates collected globally were members of clade 2 , but further surveillance is needed to determine whether this clade may still be circulating at low levels.    Clade 3 was detected by surveillance several weeks after clades 1 and 2 .  However, the TMRCA  suggests that clade 3 emerged at a similar time as clades 1 and 2, and only was detected later.  Clade 3 is the most geographically diverse, containing isolates from Norway, France, England, Germany, Puerto Rico, China, Japan, Canada, Mexico, and the US states of New York, Wisconsin, and Arizona .  Although no amino acid changes have been fixed among all clade 3 isolates, at least 13 different amino acid changes were observed among clusters of isolates contained within this clade (data not shown).     Clade 4 is the only clade in this study that contains isolates from only one region: all 11 isolates are from East Asia.  Clade 4 is characterized by fixed amino acid changes in the PB2 (V649I), PB1 (I667T), NP (V100I), and NA (V106I), and NS2 (E63K) (Table 1).  Clade 4 was the last to be detected in this study, as the first isolate (A/Korea/01/2009) was not identified until May 2, 2009.  However, the TMRCA of clade 4  suggests that clade 4 circulated for ~2 – 7 weeks prior to detection, and likely in non-Asian countries as well .     Clade 5 is the second largest clade identified here, due primarily to the fact that nearly 88% (71/81) of isolates collected from Wisconsin belong to this clade .  More than 90% (71/77) of isolates in clade 5 were collected from Wisconsin, where the clade was first detected  and appears to have proliferated rapidly (Table S1).  Clade 5 also was identified several weeks later in Canada, China, and Japan .  Although clade 5 was detected <1 week after clade 3, the TMRCA of clade 5 ranges from March 28 – April 18, 2009 (95% HPD), more than one month later than clades 1, 2, and 3 .  Clade 5 is characterized by amino acid changes in the NP (V100I) and NA (V106I and N248D), relative to basal isolates, but these changes are also observed among clades 6 and 7 (Table 1).      Clade 6 is geographically diverse, given its size in this study (13 isolates) and relatively late TMRCA, ranging from April 4 – April 20, 2009 (95% HPD), which is ~1 – 3 weeks prior to detection .  Following the collection of the first clade 6 isolates on April 28, 2009 in the US, isolates were collected from Canada, China, Italy, and Japan .  In addition to the amino acid changes in the NP and NA that also are observed in clade 5, two unique amino acid substitutions in the HA are present among clade 6 isolates: K(-6)E and Q295H, but neither are epitopes (Table 1).     Clade 7 is the largest identified in this study, representing more than one-third of all isolates  (Table S1).  Almost 83% (67/81) of isolates from New York are members of clade 7.  Clade 7 also contains isolates from Canada, China, Japan, Germany, Italy, Luxembourg, Russia, and the US states of California, Maryland, Massachusetts, Ohio, and Wisconsin .  Approximately 40% of the isolates collected from Europe and Asia are members of clade 7 .  Clade 7 is characterized by fixed amino acid changes in the NP (V100I) and NA (V106I and N248D) that are also found in clades 5 and 6, as well as by unique amino acid changes in the HA (S206T) and NS1 (I123V) (Table 1).  Clade 7 was first detected April 24, 2009, and the TMRCA of clade 7 ranges from March 28 – April 16, 2009 (95% HPD), suggesting that clade 7 emerged approximately ~1 – 4 weeks prior to the date of first detection in this study and at a similar time as clade 5 .       Although none of the 12 HA amino acid substitutions that define clades were located in any of the four antigenic subsites of the H1 (13) (Table 1), amino acid changes of potential antigenic importance were observed among 4/290 isolates in this study.  In the Cb subsite, S79Y and S79F substitutions were identified in the isolates A/Wisconsin/629-D01521/2009 and A/Wisconsin/629-D01705/2009, respectively.  The G159E replacement, located within the Sa subsite, was observed in A/Bayern/62/2009.   And A/Wisconsin/629-D0223/2009 experienced the N160T replacement, located within the Sb subsite. Table 1.  Amino acid changes defining clades 1-7, identified using the MacClade program (30).  The asterisk (*) denotes changes that are present in a majority, but not all of the isolates from a clade.Our whole-genome phylogenetic analysis of 290 H1N1pdm isolates collected globally, including the intensively sampled populations in Wisconsin and New York, reveals at least 7 major clades of the H1N1pdm influenza virus that have co-circulated over time and space since April 2009.  Clearly, the evolution and spatial-temporal dynamics of the H1N1pdm virus are more complex than the global dissemination of a single viral lineage.  The TMRCA estimates suggest that the 7 major clades emerged between mid-February and late April of 2009, with clades 1, 2, and 3 appearing between mid-February and mid-March, and clades 5, 6, and 7 emerging between mid-March and mid-April. The global origins of clade 4 from Asia remain unclear.  Our estimate for the TMRCA of the entire H1N1pdm viral population ranges from late January – early March, and is consistent with previous estimates  Sample collection and viral isolation in Wisconsin. Patient specimens were collected at Children’s Hospital of Wisconsin (CHW), Dynacare Laboratories (DL), and their associated clinics in Milwaukee, Wisconsin.  Nasopharyngeal were transported in M4 or M6 viral transport medium  to CHW or DL microbiology laboratories to test for the presence of influenza A, influenza B, or respiratory syncytial virus (RSV) using two multiplex real-time RT-PCR assays.  The CHW test  RNA extraction and sequencing of Wisconsin and New York isolates. The extraction of the entire viral RNA genome from isolates from Wisconsin and from clinical samples from New York State was performed at the Wadsworth Center, New York State Department of Health, in Albany, NY, using the NucliSENS® easyMAG™ robot , RNeasy Kit  or QIAamp Viral RNA Kit with the QIAcube automated extractor (Qiagen).  The influenza A genomic RNA segments were simultaneously amplified from 5 uls of purified RNA using a multisegment RT-PCR strategy http://www.ncbi.nlm.nih.gov/genomes/FLU/FLU.html) available on GenBank http://beast.bio.ed.ac.uk), where individual clades supported by high Bayesian posterior probabilities (BPP > 90%) and long branch lengths were identified.      To further assess the reliability of our phylogenetic inferences, two additional trees were estimated .  First, a consensus phylogenetic tree was inferred using the Bayesian approach available in MrBayes program Phylogenetic analysis.  The sequence data used in this study came from 81 H1N1pdm isolates collected from Wisconsin between April 28, 2009 – May 30, 2009 and 209 H1N1pdm isolates that were collected globally between April 1, 2009 – July 9, 2009, which were downloaded from the National Center for Biotechnology (NCBI) Influenza Virus Resource  cells used for viral isolation were kindly provided by Dr. Xiyang Xu at the Centers for Disease Control and Prevention .  This project has been funded in part with federal funds from the National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health, Department of Health and Human Services under contract number HHSN272200900007C and by NIAID grants UO1-AI070428, U01-387 AI077988, and U01-AI066584.The authors have declared that no competing interests exist.


Glucocorticoids are potent anti-inflammatory agents commonly used to treat inflammatory diseases. They convey signals through the intracellular glucocorticoid receptor (GR), which upon binding to ligands, associates with genomic glucocorticoid response elements (GREs) to regulate transcription of associated genes. One mechanism by which glucocorticoids inhibit inflammation is through induction of the dual specificity phosphatase-1  gene.We found that glucocorticoids rapidly increased transcription of DUSP1 within 10 minutes in A549 human lung adenocarcinoma cells. Using chromatin immunoprecipitation (ChIP) scanning, we located a GR binding region between −1421 and −1118 upstream of the DUSP1 transcription start site. This region is active in a reporter system, and mutagenesis analyses identified a functional GRE located between −1337 and −1323. We found that glucocorticoids increased DNase I hypersensitivity, reduced nucleosome density, and increased histone H3 and H4 acetylation within genomic regions surrounding the GRE. ChIP experiments showed that p300 was recruited to the DUSP1 GRE, and RNA interference experiments demonstrated that reduction of p300 decreased glucocorticoid-stimulated DUSP1 gene expression and histone H3 hyperacetylation. Furthermore, overexpression of p300 potentiated glucocorticoid-stimulated activity of a reporter gene containing the DUSP1 GRE, and this coactivation effect was compromised when the histone acetyltransferase domain was mutated. ChIP-reChIP experiments using GR followed by p300 antibodies showed significant enrichment of the DUSP1 GRE upon glucocorticoid treatment, suggesting that GR and p300 are in the same protein complex recruited to the DUSP1 GRE.Our studies identified a functional GRE for the DUSP1 gene. Moreover, the transcriptional activation of DUSP1 by glucocorticoids requires p300 and a rapid modification of the chromatin structure surrounding the GRE. Overall, understanding the mechanism of glucocorticoid-induced DUSP1 gene transcription could provide insights into therapeutic approaches against inflammatory diseases. Glucocorticoids are steroid hormones that exhibit potent anti-inflammatory effects through two main mechanisms. First, they inhibit the transcription of proinflammatory genes, such as cytokines, chemokines, and adhesion molecules via suppression of the transcriptional activation induced by AP-1 and NFκB DUSP1 opposes the inflammatory response by blocking crucial signaling pathways. DUSP1 is a member of a large family of multifunctional phosphatases that resides in the nucleus and specifically dephosphorylates and inactivates members of the MAPK family, such as JNK, p38 MAPK, and ERK Dusp1−/−) have prolonged activation of JNK and p38 MAPK, resulting in overexpression of inflammatory genes Dusp1−/− mice show exaggerated inflammatory responses Dusp1−/− macrophages, the ability of glucocorticoids to inhibit the expression of specific pro-inflammatory genes is significantly compromised Dusp1−/− animals support the critical role of DUSP1 in regulating anti-inflammatory responses mediated by glucocorticoids Mouse knockout studies support this role. Macrophages isolated from mice lacking DUSP1 gene  A549 cells were grown to confluence and treated with 0.5 µM DEX or DMSO, then cross-linked by using formaldehyde at a final concentration of 1% at room temperature for 5 minutes. The ChIP protocols were otherwise as previously described A549 cells were grown to confluence (4×150 mm plates per treatment) and treated with 0.5 µM DEX or DMSO, then cross-linked by using formaldehyde at a final concentration of 1% at room temperature for 5 minutes. Reactions were quenched with 0.125 M glycine for 5 min. Cells were washed with PBS, scraped in cell lysis buffer  supplemented with protease inhibitor cocktails , and incubated for 10 min at 4°C. Crude nuclei were collected by centrifugation at 600 x g for 5 min at 4°C then resuspended in 1.5 ml of ice-cold RIPA buffer . Chromatin was fragmented with 18 pulses of 20 sec at 30% power with a Branson Sonifier 250 sonicator at 4°C. To remove insoluble components, samples were centrifuged at 13,000 rpm for 15 min at 4°C, and the supernatant was recovered.For the first round of IP, two aliquots from each treatment were taken. 8 µg of rabbit polyclonal GR (N499) antibody was used to immunoprecipitate GR-bound chromatin or 8 µg of rabbit polyclonal IgG was used as a control for each aliquot, per treatment. The samples rotated overnight at 4°C. 120 µl of 50% protein A/G plus-agarose bead slurry (Santa Cruz Biotechnology) was then added to each immunoprecipitation and rotated for 2 h at 4°C. After incubation, beads were washed with 1 ml of solution per wash as follows: twice with RIPA, twice with RIPA containing 510 mM NaCl, twice with LiCl buffer , and twice with TE. Between each wash, samples were rotated at room temperature for 5 min, followed by 1 min centrifugation at 5,000 rpm. All washes were supplemented with protease inhibitors. Immmunocomplexes were eluted by adding 75 µl TE with 10 mM DTT and incubated at 37°C for 30 min. The samples were centrifuged at 800 x g for 2 minutes at room temperature, and the supernatant was transferred to a clean 1.5 ml tube. An additional 14 µl of RIPA was added to dilute the sample.For the second round of IP, 8 µg of p300  was used in all samples and rotated overnight at 4°C. Again, we added 120 µl of 50% protein A/G plus-agarose bead slurry and washed as described, modified only by replacing the final two TE washes with one wash in RIPA buffer. After removing the last wash buffer, 75 µl of proteinase K solution  was added to each IP. Reactions were incubated for 3 h at 55°C, then overnight at 65°C to reverse formaldehyde cross-links. DNA was purified using a QIAquick PCR purification kit (Qiagen) and eluted in 60 µl of Elution Buffer (Qiagen).To transfect A549 cells, we used Lipofectamine 2000 (Invitrogen) according to the technical manual in the 24-well format. Twenty-four hours post-transfection, cells were treated with either DMSO or 0.5 µM DEX for 18–24 h. Cells were then harvested, and luciferase activity was measured by a Dual-Luciferase Reporter Assay kit (Promega) according to the technical manual. pCI-p300 wild type and pCI-p300ΔHAT were provided by Mike Stallcup (USC) The human DUSP1 genomic region containing the predicted GR binding site was amplified by PCR using specific primers. The PCR fragment was restriction-digested and subcloned into the pGL4-TATA reporter. A mutagenesis kit (QuikChange) was used to make site-directed mutations per the manufacturer's instructions (Stratagene).2, 5% glycerol, 3 mM MgCl2, 0.2 mM spermine, 0.2 mM spermidine, 0.2% NP40). Cells were vortexed for 10 seconds to homogenize, then centrifuged at 500 x g in a microfuge for 5 minutes at 4°C. Pellets were resuspended in 100 µl DNase I Buffer without NP40, then equilibrated to room temperature for 10 min. Samples were digested with 5 units of DNase I (QIAGEN) for 5 minutes at 37°C, and reactions were stopped by addition of equal volume (100 µl) 2× Stop Reaction Buffer  with 200 µg/ml Proteinase K, and incubated at 65°C for 4 hours or overnight. DNA was purified using a PCR purification kit (QIAGEN), and samples were measured and diluted to use 50 ng/well for qPCR analysis.A549 cells were grown to confluence in 6-well plates then treated with DMSO or 0.5 µM DEX for 10 min. Cells were washed then scraped in 1 ml ice cold DNase I Buffer + NP40 , CBP (Santa Cruz Biotechnology), and a scramble sequence  were reverse transfected with HiPerFect transfection reagent (QIAGEN) according to the technical manual. For gene expression analyses, reverse transfection was done in a 24-well plate. Forty-eight hours post-transfection, cells were treated with either DMSO or 0.5 µM DEX for 5 hours, and total RNA was then isolated. For western blot analyses, cells were collected from a 24-well plate 48 hours post-transfection. For ChIP experiments, reverse transfection was done in 10 cm plates. Again, 48 hours post-transfection, cells were treated with either DMSO or 0.5 µM DEX, this time for 10 minutes.1 , β-Actin (C4) mouse monoclonal IgG1 , anti-rabbit IgG1-HRP , and anti-mouse IgG1-HRP .A549 cells transfected with p300, CBP, or scramble (control) siRNA were collected 48 hours post-transfection in RIPA buffer . Proteins were resolved and imaged as described [27. The following antibodies were used: p300 rabbit polyclonal IgG , CBP mouse monoclonal IgG2, 0.5% NP-40, 0.15 mM spermine, 0.5 mM spermidine). Cells were vortexed for 10 seconds to ensure thorough lysis before nuclei were collected by centrifugation at maximal speed in a microfuge for 5 minutes at 4°C. Nuclei were washed in ice-cold MNase Digestion Buffer without CaCl2  and re-centrifuged. Pelleted nuclei were suspended in ice-cold MNase Digestion Buffer with CaCl2 . Samples were treated with 1 unit of MNase  for 10 minutes at 25°C. Reactions were stopped by addition of 80 µl MNase Digestion Buffer with CaCl2, 20 µl MNase Stop Buffer , 75 µg Proteinase K, and 20 µl 10% SDS, and incubated at 65°C for 4 hours or overnight. Samples were then column purified using a PCR purification kit (QIAGEN). RNaseA (Millipore) was diluted to 0.1 µg/µl in TE, and 20 units were added to each sample for 2 hours at 37°C. Samples were then purified using a PCR purification kit (QIAGEN) and run on a 1.5% agarose gel. DNA fragments of 150 bp were purified using a gel extraction kit (QIAGEN).A549 cells were grown to confluence and treated with DMSO or 0.5 µM DEX for 10 minutes. Cells were cross-linked with 1% formaldehyde for 3 minutes at 22°C, and reactions were quenched by adding glycine to a final concentration of 0.125 M. Cells were then rinsed in PBS and scraped in ice-cold MNase NP-40 Lysis Buffer  at 4°C for 5 minutes. Nuclei were then isolated by centrifugation at 170 x g at 4°C for 5 min. The total nuclei from each of the DMSO or DEX treated samples were counted, and equal numbers of nuclei were used for in vitro transcription. The samples were split into two aliquots. One was incubated in 100 µl 2× in vitro transcription buffer , 4 mM each of ATP, GTP and CTP, 200 mM sucrose and 20% glycerol) plus 8 µl biotin-UTP (Roche), and the other in 100 µl 2× in vitro transcription buffer plus 8 µl UTP (negative control) for 30 minutes at 29°C. 6 µl of 250 mM CaCl2 and 6 µl of RNAse free DNase (Roche) (10 U/µl) were then added to stop the reactions. Total RNA was then isolated using Nucleospin RNA II (Macherey-Nagel).A549 cells were grown to confluence and treated with DMSO or 0.5 µM DEX for different times. Cells were then harvested and incubated in lysis buffer  were washed twice in solution A  for 5 min, once in solution B (0.1 M NaCl) for 5 min, and then resuspended in binding/wash buffer  plus 1 µl (40 U) RNasin per 100 µl of beads. 50 µl of beads (in binding/wash buffer) were then added to RNA, incubated at 42°C for 20 min, and then shaken for 2 h at room temperature. Afterward, the beads were isolated and the supernatant discarded. The beads were then washed once (5 min) with 500 µl 15% formamide plus 2× saline-sodium citrate (SSC) buffer, twice with 1 ml 2× SSC buffer, then resuspended in 30 µl RNase and DNase free water. 10 µl of beads were used for each reverse transcription (RT) reaction prior to qPCR.Total RNA was isolated from cells by using QIAshredder and RNeasy kits (Qiagen) or a NucleoSpin RNAII kit (Macherey-Nagel). To synthesize random-primed cDNA, 0.5 µg of total RNA (10 µl), 4 µl of 2.5 mM dNTP, and 2 µl of random primers (New England Biolabs) were mixed and incubated at 70°C for 10 min. A 4-µl cocktail containing 25 U of Moloney Murine Leukemia Virus (M-MuLV) Reverse Transcriptase (New England Biolabs), 10 units of RNasin (Promega), and 2 µl of 10× reaction buffer (New England Biolabs) was then added. The reaction was incubated at 42°C for 1 h, then 95°C for 5 min.Rpl19 expression was used for internal normalization. All primer sequences are presented in The resultant cDNA was diluted to 200 µl with water, and 2.5 µl was used to perform qPCR using EVA QPCR SuperMix Kit (Biochain) per the manufacturer's protocol. qPCR was performed in either a 7900 HT, 7500 HT, or StepOne PCR System (Applied Biosystems) and analyzed by using the ▵▵-Ct method as supplied by the manufacturer (Applied Biosystems). Previous studies have shown that DUSP1 gene expression is induced by glucocorticoids in A549 cells As our data revealed that glucocorticoids stimulate DUSP1 through transcriptional initiation, we wished to further probe the mechanism by which GR induces DUSP1 transcription. To this end, we used a chromatin immunoprecipitation (ChIP) scanning approach We next tested whether this GR binding region was a mediator of glucocorticoid response. We subcloned the genomic region extending approximately 200–300 bp upstream and downstream of our ChIP fragments (−1672 to −959) into an E4 TATA box containing pGL4 luciferase reporter to test whether the identified GR-binding regions are glucocorticoid-responsive . We tranTo further resolve binding in this region, we looked for direct GR binding sites by searching for a consensus GR binding sequence. Based on a compilation of known GR binding sequences , we searched for motifs matching the loose consensus sequence XGXACXxxxXGTXCX. We identified a 15 bp sequence that closely matched this consensus GRE in the −1672 to −959 region of the human DUSP1 promoter (GLS2). The search yielded four more degenerate potential binding sites  of DUSP1 was already sensitive to DNase I before DEX treatment . To test this, we used MNase to map the position of nucleosomes surrounding the DUSP1 gene. We treated A549 cells with DMSO or DEX for 10 min. Nuclei were isolated, cross-linked with formaldehyde, and digested with MNase. The MNase sensitivity of the chromatin was then analyzed using qPCR with primers designed to anneal to the DNA approximately every 50 bp from −1550 to −1050 of the DUSP1 TSS. Because the nucleosomal regions are less accessible to MNase, we recovered more DNA fragments from these regions. In contrast, the recovery of DNA fragments from linker regions was lower, as most of these fragments were digested by MNase. In untreated cells, three solid nucleosome positions are evident: from −1500 to −1350 (nucleosome 1), −1350 to −1200 (nucleosome 2), and −1200 to −1050 (nucleosome 3) . ConsistAn increase in the acetylation of histones H3 and H4 has been correlated with both gene transcription and active DNA control elements such as response elements and enhancers Next, we wished to determine the histone acetyltransferase (HAT) that acetylates H3 and H4 at the DUSP1 gene. Several coactivators for GR, including GCN5, PCAF, CBP/p300, have been shown to contain histone acetyltransferase (HAT) activity. We reason that if a coactivator is directly involved in glucocorticoid-activated DUSP1 gene transcription, it should be present on the DUSP1 promoter upon DEX treatment. To determine which of these factors are directly involved in glucocorticoid-activated DUSP1 gene transcription, we treated A549 cells with DMSO or DEX for 10 minutes, then ChIPed for GCN5, PCAF, CBP and p300 in the areas surrounding the DUSP1 GRE. Of these, p300 was present before treatment with glucocorticoids (relative to ChIP with IgG control), with levels increasing approximately 16 fold after only 10 minutes of DEX treatment . In contWe used RNAi to decrease the expression of p300 to further investigate its role in DEX-induced histone hyperacetylation. As shown in To further confirm the importance of p300-induced histone acetylation in DEX-induced DUSP1 gene transcription, we compared the ability of wild type (WT) p300 and a p300 mutant with reduced histone acetyltransferase (HAT) activity (p300ΔHAT) to coactivate DEX-induced pDUSP1 reporter gene expression . We founFinally, we performed ChIP-reChIP experiments to determine if p300 and GR are recruited to the same genomic region of DUSP1. GR antibodies were first used to pull down GR-associated DNA fragments. Antibodies against p300 were then used to isolate GR-associated DNA fragments that also associate with p300. Compared to ChIP-reChIP using IgG control antibodies, ChIP-reChIP using GR and then p300 showed significant enrichment of the DUSP1 GRE 23 fold, . These rMultiple mechanisms contribute to the anti-inflammatory effects of glucocorticoids. One of them is the ability of glucocorticoids to induce the transcription of DUSP1. Thus, understanding the regulatory mechanism of glucocorticoid-activated DUSP1 gene transcription is vital for future therapeutic interventions against inflammatory diseases. In this study, we systematically investigated the mechanisms of GR-regulated DUSP1 gene transcription. First, we determined that GR regulates the expression of DUSP1 through transcriptional initiation ; then, wA recent study using genomic conservation analyses identified multiple GREs upstream of the human DUSP1 gene In addition to GLS2, we identified three other GLS elements that may play accessory roles in the regulation of DUSP1 gene transcription. Interestingly, only one of these three is required for a complete glucocorticoid response, and any of these three can play the accessory role. It is unclear whether the mechanisms governing the functional interactions between GLS2 and GLS1, 3, or 4 are the same. This will require detailed studies of the role of these accessory elements in glucocorticoid response. At present, we cannot be sure if they directly associate with GR. It is possible that GLS2, which fits the consensus sequence of a canonical GR binding site, serves as an essential site for the binding of GR while the other GLS elements, which are more divergent, can only bind to the receptor when GR:GLS2 interacts. Alternatively, these GLS elements may bind to other transcriptional regulators. The requirement of functional interactions between GR and other DNA-binding transcriptional regulators to confer a complete glucocorticoid response has been previously reported. These functional interactions are required in GREs of rat PEPCK Chromatin structure plays a critical role in the regulation of gene transcription. Based on our findings from DNase I accessibility experiments, the DUSP1 genomic region up to ∼2 kb upstream of the TSS is already opened up prior to treatment with glucocorticoids . GlucocoThe analyses of histone acetylation status in the DUSP1 gene promoter showed that the genomic region up to 2 kb upstream of the TSS is already acetylated in its basal state. These results are in agreement with the DNase I accessibility findings discussed above and are not surprising, as DUSP1 gene expression is not silent in the absence of glucocorticoid treatment. The fact that glucocorticoid treatment further increases the acetylation status of histone H3 and H4 indicates that HAT(s) are correlated with the glucocorticoid response on DUSP1 gene expression. We found two HATs, p300 and CBP, whose recruitment to the DUSP1 GRE is increased upon DEX treatment. RNAi knockdown of CBP fails to affect basal or glucocorticoid-stimulated DUSP1 gene expression, both of which are affected by p300 RNAi . AlthougWe found that p300 is present in the DUSP1 gene promoter in the basal state, and glucocorticoid treatment further augments its occupancy. It is possible that p300 is recruited to the promoter region near the DUSP1 GRE through existing DNA-binding transcriptional regulators. Upon glucocorticoid treatment, GR binds to the GRE and further recruits p300 to the GRE. Interestingly, the results from our RNAi experiments indicate that p300 has dual roles on DUSP1 gene expression. RNAi knockdown of p300 increases basal gene expression of DUSP1, but also limits the ability of glucocorticoids to potentiate DUSP1 gene transcription. Thus, p300 likely acts as a corepressor for basal DUSP1 expression, but as a coactivator for GR-stimulated DUSP1 gene transcription. Other results in this report support this model. Supporting the corepressor role, p300 overexpression reduced the activity of a reporter gene containing the −1672 to −959 region of DUSP1 gene. This suggests that the element(s) responsible for repressive effects of p300 is located in this genomic region. Notably, it has been reported that p300 can reduce the transcriptional activity of certain DNA-binding transcription factors Glucocorticoids are an essential tool in combating inflammatory disorders, however their use is limited by multiple serious side effects. There is a conception that glucocorticoids' anti-inflammatory effects are attributed to GR-regulated transcriptional repression, while the side effects reflect transcriptional activation by GR. However, recent studies clearly demonstrate the importance of glucocorticoid-activated DUSP1 gene expression in mediating the anti-inflammatory effects of glucocorticoids. Thus, an important topic in glucocorticoid pharmacology will be how to dissociate transcriptional activation of GR-induced anti-inflammatory actions from the undesired side effects. To do this, it is critical to identify the specific components of the mechanisms governing GR-activated transcription of DUSP1 and other genes involved in the induction of unwanted side effects.Figure S1DNaseI accessibility of α-fetoprotein gene in response to glucocorticoids. An example of a gene whose chromatin structure is not opened up in response to glucocorticoids. α-fetoprotein (AFP) gene schematic shows the location of three primers used (black boxes labeled A1–A3) for qPCR analyses. The regions of the AFP gene (relative to the TSS) amplified by these primers are: A1 (−729 to −652), A2 (−405 to −324), and A3 (−183 to −99). DNase I accessibility assay and calculations were performed as described in (0.29 MB TIF)Click here for additional data file.Figure S2Basal and glucocorticoid-induced histone H3 and H4 acetylation of the DUSP1 gene promoter. A AcH3/H3 ChIP experiments presented in (0.45 MB TIF)Click here for additional data file.Figure S3Effects of p300 and CBP siRNA on basal DUSP1 gene expression. RNAi knockdown experiments presented in (0.16 MB TIF)Click here for additional data file.Figure S4Effects of p300WT and p300ΔHAT overexpression on basal activity of pDUSP1 reporter gene. pDUSP1 reporter assay presented in (0.15 MB TIF)Click here for additional data file.Table S1Primers.(0.04 MB DOC)Click here for additional data file.


Mycobacterium tuberculosis kills approximately 2 million people each year and presents an urgent need to identify new targets and new antitubercular drugs. Thymidylate synthase (TS) enzymes from other species offer good targets for drug development and the M. tuberculosis genome contains two putative TS enzymes, a conventional ThyA and a flavin-based ThyX. In M. tuberculosis, both TS enzymes have been implicated as essential for growth, either based on drug-resistance studies or genome-wide mutagenesis screens. To facilitate future small molecule inhibitors against these proteins, a detailed enzymatic characterization was necessary.M. tuberculosis enzymes had very low kcat values. One possible explanation for the low catalytic activity of M. tuberculosis ThyX is that its true biological substrates remain to be identified. Alternatively, this slow-growing pathogen, with low demands for TMP, may have evolved to down-regulate TS activities by altering the turnover rate of individual enzyme molecules, perhaps to preserve total protein quantities for other purposes. In many organisms, TS is often used as a part of larger complexes of macromolecules that control replication and DNA repair.After cloning, overexpression, and purification, the thymidylate-synthesizing ability of ThyA and ThyX gene products were directly confirmed by HPLC analysis of reaction products and substrate saturation kinetics were established. 5-Fluoro-2′-deoxyuridine 5′-monophosphate (FdUMP) was a potent inhibitor of both ThyA and ThyX, offering important clues to double-targeting strategies. In contrast, the folate-based 1843U89 was a potent inhibitor of ThyA but not ThyX suggesting that it should be possible to find ThyX-specific antifolates. A turnover-dependent kinetic assay, combined with the active-site titration approach of Ackermann and Potter, revealed that both M. tuberculosis provides a framework for future development of cell-active inhibitors and the biological roles of these TS enzymes in M. tuberculosis.Thus, the present enzymatic characterization of ThyA and ThyX from  Mycobacterium tuberculosis, the causative agent of tuberculosis An estimated 2 million deaths occur annually due to infections of N5,N10-methylene-5,6,7,8-tetrahydrofolate (mTHF) as the methyl donor and reductant in the reaction, yielding dihydrofolate (DHF) as a by-product  is an attractive target for drug development -product  [7]. EveM. tuberculosis also appears to carry a flavin-dependent thymidylate synthase (FDTS or ThyX) Helicobacter pylori and Thermotoga maritima, M. tuberculosis ThyX is expected to catalyze the reductive methylation of dUMP to afford TMP using mTHF as the methyl donor in the reaction (M. tuberculosis ThyX binds NADP+ and FAD and has no direct similarity to ThyA M. tuberculosis ThyX tritium-release activity thyX gene is absent in humans and transposon site hybridization (TraSH) experiments indicate that Mycobacterium thyX is an essential gene for optimal growth of the pathogen In addition to the conventional ThyA, reaction  [13],  was purchased from American Radiolabeled Chemicals . To prepare a 20 mM stock solution of methylene tetrahydrofolate (mTHF), solid tetrahydrofolic acid was dissolved in a buffer containing 125 mM TES, 60 mM MgCl2, 2.5 mM EDTA (pH 8.0), 190 mM 2-mercaptoethanol, and 16 mM formaldehyde. All, more dilute, mTHF solutions were made from this stock solution. mTHF solutions were stored under argon at −20°C. 1843U89 was synthesized as previously reported FAD, NADPH, tetrahydrofolic acid, Pefabloc SC, DNAseI, FdUMP, dUMP, TMP and protein standards for size-exclusion chromatography were purchased from Sigma-Aldrich .  UTP. Labeled RNA was quantified by the 32P-incorporation assay following the protocol from Promega. RNA  was incubated with varying concentrations of purified enzyme in 10 mM HEPES pH 7.5, 3 mM MgCl2, 40 mM KCl, 5% glycerol, 200 mM 2-mercaptoethanol, and 1 unit of RNasin at 37°C for 15 minutes. The unbound RNA was digested by addition of 3 units of RNase T1 and incubation at 25°C for 10 minutes. Non-specific interactions were reduced by addition of 5 mg/ml heparin and incubation at 25°C for 10 minutes. The RNA-protein complexes were resolved on a 5% TBE polyacrylamide gel and visualized by autoradiography.RNA binding experiments was measured as previously described M. tuberculosis ThyA, or 3 µg of M. tuberculosis ThyX, was added to a mixture of 60 µL mTHF , 15 µL of 140 µM dUMP, 3 µL of 350 µM FAD (for ThyX assay only), and 1 µL of 35 mM NADPH (for ThyX assay only) and water to give reaction volume of 105 µL. The reaction times varied from 30 to 120 minutes. To isolate the products, the reaction mixture was spun through a Microcon YM-10 centrifugal filter  at 13,000×g for 5 minutes. An additional 100 µL of water was added and the spin was repeated. The deproteinized reaction mixture was run on an HPLC using a Beckman reversed phase Ultrasphere ODS 5 µm C-18 column  with isocratic flow with 5 mM potassium phosphate (pH 7.0), 5 mM tetrabutylammonium dihydrogen phosphate, and 5% (v/v) acetonitrile, while monitoring elution at 260 nm. Standard solutions of dUMP, TMP, and mTHF were also run to assign the elution positions of reactants and products.Approximately 150 ng of M. tuberculosis ThyA or 1 µg of M. tuberculosis ThyX, in 1 µL, was added to 20 µL mTHF (mTHF concentrations in the reactions ranged from 5.7 µM–1143 µM for ThyA assay or 1.25 µM–20 µM for ThyX assay), 1 µL of 200 µM FAD (for ThyX assay only), and 1 µL of 2 mM NADPH (for ThyX assay only). The total reaction volume was brought to 30 µL with the addition of water. To initiate the reaction, 5 µL of 13.1 µM–140 µM [5-3H]-dUMP (0.4 Ci/mmol) was added. After a 25 minute reaction period, at room temperature, the reaction was terminated by the addition of 20 µL of a stop solution (a 3∶1 ratio of 2 N TCA∶4.3 mM dUMP). To remove unreacted substrates, 200 µL 10% (w/v) activated charcoal in water was added to the reaction mixture. The reaction tubes were incubated on ice for 15 minutes then spun at 13,000×g at 4°C for 10 minutes. A 100 µL aliquot of the supernatant was assayed by liquid scintillation counting to determine the amount of tritium-containing water produced by the reaction. Specific activities and Vmax values were determined in the presence of saturating concentrations of substrates.In a standard tritium-release assay, 50 ng of Km values of dUMP, mTHF, and NADPH and the kcat values of thymidylate synthase activity. See the legends for max[A][B])/([A][B]+KmA[B]+KmB[A]+KmBKsA). kcat values were determined from Ackermann-Potter plots using the equation, Slope = (kcat[S])/([S]+Km) The tritium release, at fixed time points, revealed initial reaction velocities needed to calculate the Ki values of the inhibitors for the ThyA and ThyX enzymes. Inhibitor and corresponding substrate concentrations were varied as listed in the legends of max[B])/((1+[I]/Ki)(KmB+[B])), competitive: v = (Vmax[B])/(KmB(1+[I]/Ki+[B]), or mixed inhibitor: v = (Vmax[B])/(KsA(1+[I]/Ki)+[B](1+[I]/Kis)).The tritium-release assay was also used to determine the