Klebsiella pneumoniae CG43S3, we have previously shown that iron availability regulates capsular polysaccharide (CPS) biosynthesis and iron-acquisition systems. The present study investigated whether IscR is involved in this regulation. Compared with that in CG43S3, the amount of CPS was decreased in AP001 (ΔiscR) or AP002 (iscR3CA), a CG43S3-derived strain expressing mutated IscR mimicked apo-IscR, suggesting that only holo-IscR activates CPS biosynthesis. Furthermore, a promoter-reporter assay verified that the transcription of cps genes was reduced in AP001 and AP002. Purified IscR::His6, but not IscR3CA::His6, was also found to bind the predicted type 1 IscR box specifically in the cps promoter. Furthermore, reduced siderophore production was observed in AP004 (Δfur-ΔiscR) but not in AP005 (Δfur-iscR3CA), implying that apo-IscR activates iron acquisition. Compared with those in AP004, mRNA levels of three putative iron acquisition systems  were increased in AP005, and both purified IscR::His6 and IscR3CA::His6 bound the predicted type 2 IscR box in the fhuA, iucA, and sitA promoters, whereas IscR3CA::His6 displayed a lower affinity. Finally, we analyzed the effect of external iron levels on iscR expression. The transcription of iscR was increased under iron-depleted conditions as well as in AP001 and AP002, suggesting an auto-repression exerted by apo-IscR. Our results show that in K. pneumoniae, IscR plays a dual role in the regulation of CPS biosynthesis and iron-acquisition systems in response to environmental iron availability.IscR, an Fe–S cluster-containing transcriptional factor, regulates genes involved in various cellular processes. In response to environmental stimuli such as oxidative stress and iron levels, IscR switches between its holo and apo forms to regulate various targets. IscR binding sequences are classified into two types: the type 1 IscR box that is specific for holo-IscR binding, and the type 2 IscR box that binds holo- and apo-IscR. Studying  Klebsiella pneumoniae is a rod-shaped, gram-negative bacterium that causes community-acquired diseases including pneumonia, bacteremia, septicemia, and urinary and respiratory tract infections that occurr particularly in immune-compromised patients K. pneumoniae is the predominant pathogen responsible for pyogenic liver abscesses in diabetic patients K. pneumoniae, capsular polysaccharide (CPS) is considered the major determinant for K. pneumoniae infections. Pyogenic liver abscess isolates often carry heavy CPS loads that protect bacteria from phagocytosis and from being killed by serum factors Klebsiella CPS also promotes bacterial colonization and biofilm formation at infection sites K. pneumoniae is repressed in iron-replete conditions, and this regulation is controlled by an iron uptake regulator (Fur) K. pneumoniae CPS biosynthesis through transcriptional factors RmpA and RcsA and a small non-coding RNA, RyhB cps genes is directly regulated by RmpA and RcsA but appears to be indirectly regulated by RyhB. These findings indicate that environmental iron availability influences K. pneumoniae CPS biosynthesis through multiple regulators.Our previous studies have demonstrated that CPS biosynthesis in K. pneumoniaeK. pneumoniae CG43S3 2+) or ferric (Fe3+) iron and sulphide (S2−) iscRSUA operon. The switch between the [2Fe-2S] holo and apo forms of IscR is believed to be influenced by environmental conditions such as oxidative and nitric oxide stress and cellular iron levels To maintain iron homeostasis, Fur acts as a master regulator to control iron transport, storage, and metabolism in many gram-negative bacteria including Escherichia coliTranscriptomic analysis has identified 40 genes in 20 predicted operons, which are regulated by IscR under aerobic and anaerobic conditions in K. pneumoniae. We also analysed the expression of iscR in response to various iron levels.In this study, we investigated whether IscR participates in the regulation of CPS biosynthesis and the expression of iron acquisition systems in K. pneumoniae CPS biosynthesis, we determined the amounts of K2 CPS in CG43S3 (wild type [WT]) and AP001 (ΔiscR) strains. Compared with the WT, AP001 produced significantly lower amounts of CPS  which are thought to coordinate the [2Fe-2S] cluster To study whether IscR regulates s of CPS , suggestiscR mutant AP002 (iscR3CA) by replacing the three cysteines with alanines and tested whether this mutant, which is predicted to encode an IscR lacking an Fe-S cluster, affected CPS biosynthesis. As shown in iscR and iscR3CA were respectively cloned into pACYC184, to yield pIscR and pIscR3CA, for complementation analysis. Compared with AP001 [pACYC184], AP001 [pIscR] produced a significantly higher amount of CPS, whereas the introduction of pIscR3CA into the AP001 strain did not change the CPS amount  organised into 3 transcription units–namely, orf1–2, orf3–15, and orf16–17cps transcription, we used the reporter plasmids pOrf12 (Porf1-2::lacZ), pOrf315 (Porf3-15::lacZ), and pOrf1617 (Porf16-17::lacZ), each carrying a promoterless lacZ gene transcriptionally fused to the putative promoter region of the K2 cps gene cluster K. pneumoniae strains AP006 (ΔlacZ), AP007 (ΔlacZ-ΔiscR), and AP008 (ΔlacZ-iscR3CA). The measurements shown in orf1–2 and orf16–17 in AP007 and AP008 was lower than that in AP006 (P<0.01). Additionally, the promoter activity of orf3–15 was apparently decreased in AP008 but slightly decreased in AP007 compared with that in AP006. These results indicated that IscR activates the transcription of cps genes in an Fe-S cluster-dependent manner.The K2 cps transcription, the sequence of the putative IscR binding site was manually analysed in the three promoter regions of the K2 cps gene cluster. As shown in galF (orf1 in the K2 cps gene cluster). In addition, the putative IscR binding sequence in PgalF was highly homologous to the IscR-binding motif (5′-AxxxCCxxAxxxxxxxTAxGGxxxT-3′) reported by Nesbit et al. wzi (orf3 in the K2 cps gene cluster) or manC (orf16 in the K2 cps gene cluster), suggesting that IscR indirectly regulates the promoter activities of orf3-15 and orf16-17, which remains to be studied. On the other hand, we hypothesised that IscR binds directly to the promoter region of galF to activate gene transcription, and we confirmed this by performing an electrophoretic mobility shift assay (EMSA). As shown in the upper panel of 6 protein was able to bind PgalF-1 but not PgalF-2, in which the region containing the putative IscR binding site was deleted. In addition, compared with that of IscR::His6, the recombinant [2Fe-2S] clusterless IscR3CA::His6 had reduced PgalF-1 binding activity. Furthermore, no obvious interaction between the recombinant IscR proteins and PgalF-1*, the galF promoter lacking only the 25-bp predicted IscR box, was found .For further investigation of the mechanism of IscR regulation on K. pneumoniae against serum factors, we hypothesize that through modulation of CPS levels, IscR affects the ability of K. pneumoniae to resist the bactericidal effects of serum. To test this hypothesis, we treated K. pneumoniae strains with 75% normal human serum and determined their survival rates. Compared with the WT, the AP001 and AP002 strains had a slightly reduced survival rate  assay to assess siderophore secretion in K. pneumoniae strains. As shown in fur clearly increased halo formation on the CAS plate. However, the halo was reduced in the AP003 (Δfur) strain background by the further deletion of iscR, indicating the positive role of IscR in iron acquisition. Furthermore, no obvious difference in siderophore secretion was found between the AP003 and AP005 (Δfur- iscR3CA) strains, suggesting that the IscR regulation of iron acquisition does not require the [2Fe-2S] cluster.In 3CA into the AP004 (Δfur-ΔiscR) strain and performed a CAS assay. As shown in 3CA increased the halo phenotype on the CAS plate compared with that of the vector-only control. These results confirmed that IscR activates siderophore secretion in a [2Fe-2S] cluster-independent manner.To verify whether apo-IscR activates iron acquisition, we introduced pACYC184, pIscR, or pIscRK. pneumoniae strains. As shown in fhuA, iucA, and sitA) corresponding to three iron acquisition systems were increased more than 2-fold in the AP005 strain as compared with that in the AP004 strain. To further confirm this result, pACYC184, pIscR3CA, or pIscR were respectively introduced into the AP004 strain, to avoid the effects of Fur, and the transcription of fhuA, iucA, and sitA were measured. The introduction of pIscR3CA into AP004 apparently increased the transcription of fhuA, iucA, and sitA compared with that in the AP004 strain carrying pACYC184 only  to measure the expression of genes corresponding to the eight putative iron acquisition systems in the indicated 184 only . These r and sit .E. colifhuA, iucA, and sitA revealed consensus sequences of the E. coli type 2 IscR box. As shown in fhuA and −67 to −43 relative to the translation start site of iucA. The predicted type 2 IscR boxes in PfhuA and PiucA have 50% (13/26 bp) and 46% (12/26 bp) homology, respectively, with the consensus sequence. In addition, two putative type 2 IscR boxes (R1 and R2) located at −112 to −87 and at −53 to −28 relative to the translation start site of sitA were found in PsitA. The R1 and R2 sites contain 50% (13/26 bp) and 61.5% (16/26 bp) homology, respectively, with the consensus sequence.Apo-IscR has been demonstrated to bind the type 2 IscR box in IscR-regulated promoter sequences directly in 6 and IscR3CA::His6 were able to bind with the promoter regions of fhuA, iucA, and sitA, and IscR::His6 appeared to contain higher binding activities. Furthermore, IscR3CA::His6 did not bind PfhuA-2 and PiucA-2, which lacked a region containing a putative IscR box , and no obvious interaction was found. These results suggested that apo-IscR interacts directly with the promoters of fhuA, iucA, and sitA via the predicted type 2 IscR boxes.To verify whether apo-IscR binds to these predicted type 2 IscR boxes, we performed an EMSA. As shown in IscR box . We also R2 site . To furtK. pneumoniae iscR expression, we grew AP006 in Luria-Bertani (LB) broth supplemented with increasing amounts of the iron chelator 2, 2-dipyridyl (Dip) and monitored the promoter activity of iscR using a LacZ reporter system iscR promoter (PiscR) activity by approximately 2-fold and 3.9-fold, respectively, indicating that the transcription of the iscRSUA operon was activated by iron limitation.To analyse whether environmental iron availability affects K. pneumoniae, Fur and RyhB reportedly play crucial roles in gene regulation in response to cellular iron levels iscR. As shown in fur and the further deletion of ryhB in AP006 strain had no obvious effects on PiscR activity, whereas the deletion of iscR in AP006 strain activated PiscR activity by approximately 4.5-fold. In E. coli, IscR has been demonstrated to exert negative auto-regulation which requires the [2Fe-2S] cluster iscR activity in an AP006-derived strain, AP008 (ΔlacZ-iscR3CA), expressing a mutated IscR predicted to be defective in cluster binding iscR activity in AP008 was increased approximately 4.5-fold. This increase was comparable to that in AP007 (ΔlacZ-ΔiscR). Our results suggested that IscR inhibits the transcription of the iscRSUA operon in a [2Fe-2S] cluster-dependent manner in K. pneumoniae.In iscRSUA transcription in response to iron availability, we monitored PiscR activity in the AP007 and AP008 strains in LB broth containing various levels of iron. As shown in iscR activity in AP006 was activated in LB broth supplemented with Dip, and the further addition of 100 or 250 µM FeSO4 reversed the activation. In the AP007 and AP008 strains, PiscR activity was increased compared with that of AP006. Nevertheless, the addition of Dip still activated PiscR activity, suggesting the presence of unknown factors, and the further addition of FeSO4 restored the effect.To investigate whether IscR is the sole regulator of iscR activity in AP011 (ΔlacZ-Δfur-ΔiscR) compared with that of AP007 at various iron levels and noted no obvious effect. These results indicated that in addition to IscR and Fur, other factors modulate the transcription of the iscRSUA operon in response to environmental iron availability.To analyse whether Fur is responsible for this regulation, we measured PK. pneumoniae strains usually carry large amounts of CPS to resist engulfment by phagocytes and serum bactericidal factors K. pneumoniaeK. pneumoniaecps gene cluster  the AP004 strain produced elevated amounts of CPS compared with that of the WT and in the AP004 background, expression of IscR or IscR3CA did not cause an obvious effect on CPS amount under iron-replete or iron-limited conditions (data not shown), and (iii) all three transcription units of the cps genes were obviously increased after fur deletion K. pneumoniae survival inside the host, so IscR may contribute in maintaining a higher basal expression level of the genes involved in CPS biosynthesis. On the other hand, in iron-limited conditions, the transcription of iron-acquisition genes is increased not only due to the de-repression of apo-Fur but also the activation of apo-IscR. Besides, in addition to iron level, oxidative stress has been demonstrated to signal Fur and IscR, and the reversible interconversion of Fe-S clusters makes them exquisite sensors of such stress However, we hypothesize herein that Fur plays a major regulatory role because (i) K. pneumoniae CG43S3. Moreover, Fur directly represses the transcription of genes corresponding to six iron acquisition systems  under iron-replete conditions fhuA, sitA, and iucA , suggesting that in K. pneumoniae, IscR and Fur do not share cross-regulation. Furthermore, E. coli RyhB reportedly binds directly to the upstream region of iscS mRNA to decrease the stability of iscRSUA but not iscR mRNA, thereby leading to a stable secondary iscR structure and resulting in active translation iscR activity.Because IscR and Fur are both iron-responsive regulators which share strong overlap among downstream targets, we next investigated whether IscR and Fur are cross-regulated. In d by Fur , which iiscS in K. pneumoniae, also identified a conserved sequence paired with RyhB (data not shown). However, the regulatory effect on PiscR activity mediated by RyhB was not obvious under our assay conditions  homology with the consensus sequence and is located at position −144 to −126 relative to the translation start site of iscR. Therefore, we hypothesized that SoxS may be involved in the regulation of iscR in response to oxidative stress and will investigate this possibility in future studies.An analysis of the upstream region of nditions . On the K. pneumoniae K2 cps gene cluster, we found a type 1 box in the promoter region of galF, and purified IscR::His6 could bind this motif in vitro . The resulting K. pneumoniae mutants are listed in Specific gene deletion of iscR was amplified by PCR using the primer pair GT138/GT139 , a DNA fragment containing the promoter and coding sequence of 38/GT139  and clon06/GT207 . The reciscR and approximately 1000 bp adjacent regions on either side was amplified by PCR using primer pairs GT241/GT242  and expressed as micrograms per 109 c.f.u. CPS was extracted and quantified as previously described K. pneumoniae strains by conjugation from E. coli S17-1 λpir. The bacteria were grown to logarithmic phase in LB broth or indicated medium, and the β-galactosidase activity was measured as previously described The promoter-reporter plasmids, pOrf12, pOrf315, pOrf1617, and piscRZ15 were individually mobilized into 6 c.f.u./ml. Next, 1 ml of the cultures was washed twice using phosphate-buffered saline (PBS) and resuspended in 1 ml PBS. A mixture containing 250 µl of the cell suspension and 750 µl of pooled human serum was incubated at 37°C for 15 min. The number of viable bacteria was then determined by plate counting. The survival rate was expressed as the number of viable bacteria treated with human serum compared with the number of viable bacteria pretreatment. The 0% survival of K. pneumoniae AP012  served as a negative control.Normal human serum, pooled from healthy volunteers, was divided into equal volumes and stored at −70°C before use. Bacterial survival in serum was determined as previously described iscR and iscR3CA were amplified using the primer pair GT215/GT216 . The resulting plasmids  were then transformed into E. coli BL21(DE3)[pLysS] , and overproduction of the recombinant proteins IscR::His6 and IscR3CA::His6, respectively, were induced by the addition of 1 mM IPTG for 3 h at 37°C. The cell pellets were washed and resuspended in cold binding buffer . The cells were then broken by sonication and the cell pellets were removed by centrifugation at 14000 rpm for 10 min at 4°C. The recombinant proteins were then purified from the soluble fraction of the total cell lysate by affinity chromatography using His-Bind resin  according to the manufacturer's instructions. The nonbinding proteins were washed away using binding buffer and the recombinant proteins were eluted by elution buffer . Finally, the purified proteins were dialyzed against TEGX buffer  containing 0.1 mM NaCl at 4°C overnight. Subsequently, the dialyzed protein was checked for purity by SDS-PAGE and stored for up to two weeks at 4°C. The purified protein was transparent and no obvious precipitation was observed after storage.The coding regions of 15/GT216  and clongalF, fhuA, iucA, and sitA were amplified with Pfu polymerase using specific primer sets . To obtain probes that lacked a putative IscR box, the DNA fragments were respectively amplified with Taq polymerase using the above-described primer sets , 10 mM KCl, 100 mM dithiothreitol, and 10 µg/ml BSA at 37°C for 30 min. The samples were then loaded onto a native gel of 5% nondenaturing polyacrylamide in 0.5× TB buffer . Gels were electrophoresed with a 20-mA current at 4°C and then stained with SYBR Green I dye (Invitrogen). The assay was repeated in at least 3 independent experiments.For the EMSA, the purified IscR::His−ΔΔCT method with 23S rRNA as the endogenous reference.Total RNAs were isolated from early-exponential-phase grown bacteria cells by use of the RNeasy midi-column (QIAGEN) according to the manufacturer's instructions. RNA was DNase-treated with RNase-free DNase I (MoBioPlus) to eliminate DNA contamination. RNA of 100-ng was reverse-transcribed with the Transcriptor First Strand cDNA Synthesis Kit (Roche) using random primers. qRT-PCR was performed in a Roche LightCycler 1.5 Instrument using LightCycler TaqMan Master (Roche). Primers and probes were designed for selected target sequences using Universal ProbeLibrary Assay Design Center (Roche-applied science) and listed in The CAS assay was performed according to the method described by Schwyn and Neilands P<0.01 were considered significant. The results of CPS quantification, β-galactosidase activity, serum survival rate, and qRT-PCR analysis were performed in triplicate and independently repeated at least three times, and the mean activity and standard deviation are presented.An unpaired t-test was used to determine the statistical significance and values of For isolation of normal human serum from healthy volunteers, the procedure and the respective consent documents were approved by the Ethics Committee of the China Medical University Hospital, Taichung, Taiwan. All healthy volunteers provided written informed consent.Figure S1Single-copy complementation of iscR but not iscR3CA in the AP001 strain restores native production levels of CPS. CPS levels of the K. pneumoniae strains, as indicated, grown in LB broth were determined as described in Materials and Methods (*P<0.01).(TIF)Click here for additional data file.Methods S1Supporting Materials and Methods.(DOCX)Click here for additional data file.


In Corynebacterium glutamicum, two putative genes, namely the bca (cg2954) and gca (cg0155) genes, coding for β-type and γ-type carbonic anhydrase, respectively, have been identified. We here analyze the transcriptional organization of these genes. The transcriptional start site (TSS) of the bca gene was shown to be the first nucleotide “A” of its putative translational start codon (ATG) and thus, bca codes for a leaderless transcript. The TSS of the gca gene was identified as an “A” residue located at position -20 relative to the first nucleotide of the annotated translational start codon of the cg0154 gene, which is located immediately upstream of gca. Comparative expression analysis revealed carbon source-dependent regulation of the bca gene, with 1.5- to 2-fold lower promoter activity in cells grown on acetate as compared to glucose as sole carbon source. Based on higher expression of bca in a mutant deficient of the regulator of acetate metabolism RamA as compared to the wild-type of C. glutamicum and based on the binding of His-tagged RamA protein to the bca promoter region, we here present evidence that RamA negatively regulates expression of bca in C. glutamicum. Functional characterization of a gca deletion mutant of C. glutamicum revealed the same growth characteristics of C. glutamicum ∆gca as that of wild-type C. glutamicum and no effect on expression of the bca gene.Carbonic anhydrase catalyzes the reversible hydration of carbon dioxide to bicarbonate and maintains the balance of CO Afterwards, the mixture was separated on a 2% agarose gel in 1x TAE buffer  at 70 volts and stained with ethidium bromide.The binding of purified His-tagged RamA protein with the ribed in ) was usefor RamA  as positgca deletion mutant of C. glutamicum, the upper and lower regions (each 423 bp) of gca were generated by PCR using primer pairs Del-gca-upper-fw / -rev and Del-gca-lower-fw /–rev, respectively. The two products were purified using NucleoSpin Gel and PCR Clean-up Kit and subsequently combined in a cross-over PCR  sequence for C. glutamicum [C. glutamicum [The TSS for  the gca . In acconscribed . Centereutamicum . Six bp utamicum .C. glutamicum strains carrying plasmids pET2, pET2-Pbca and pET2-Pgca, we performed a comparative expression analysis of the bca and gca genes in cells grown in minimal medium with 1% (w/v) glucose or 1% (w/v) acetate with initial pH values at 6.8 and 6.3. Cultures were harvested at the mid-exponential phase of growth (OD600 of about 5) and after cell disruption, the promoter activities were determined in the cell extracts by measuring the specific CAT activities. While the extracts from cells carrying the empty promoter-probe vector pET2 did not show any detectable CAT activity (< 0.01 U/mg protein), the extracts of the strains carrying the bca and gca promoters within pET2 showed activity and expression of bca also showed carbon source-dependent regulation  or acetate as carbon source at both pH values (6.3 and 6.8)  . These rbca promoter activity in cell extracts of the L-lysine-producing strain C. glutamicum DM1729 ΔilvB transformed with plasmid pET2-Pbca and grown in minimal medium containing 2% glucose. As can be seen in C. glutamicum wild-type. In accordance, L-lysine production by C. glutamicum DM1729 ΔilvB (pET2-Pbca) was in the same range as previously reported [C. glutamicum DM1729 ΔilvB, i.e., a final L-lysine concentration of about 32.3 mM after 24 h of incubation.We also tested the reported  for the bca gene in C. glutamicum with glucose and acetate (see above), we speculated the regulator of acetate metabolism RamA [bca expression was tested by comparative bca promoter activity analysis with the wild-type and a RamA-deficient derivative of C. glutamicum. For this purpose, plasmid pET2-Pbca was transformed into C. glutamicum ΔramA and the specific CAT activities of the resulting transformant and of C. glutamicum (pET2-Pbca) were determined in cell extracts after growth of the cells in minimal medium with either glucose 1% (w/v) or glucose plus acetate  and harvested at the mid-exponential growth phase (OD600 of about 5). As shown in bca promoter activity in the ΔramA mutant was about 1.6-fold higher in minimal medium with glucose and about four-fold higher with glucose plus acetate when compared to that in wild-type cells of C. glutamicum. The higher activity of the bca promoter in the absence of a functional RamA protein indicates that RamA acts as a negative transcriptional regulator for the expression of the bca gene in C. glutamicum.Based on the results of carbon source-dependent expression control of the ism RamA  to be inism RamA . The invbca promoter region and therefore, it was likely that the RamA protein binds to the bca promoter region. To analyze the binding of RamA to the bca promoter region, a His-tagged RamA protein was over-produced in E. coli BL21 (DE3) containing the pET28-ramA construct, identified by MALDI-TOF mass spectrometry, and used for EMSAs. For this purpose, the bca promoter fragment (Pbca) and its sub-fragments with only two, one or no RamA binding motifs , resulting in a truncated gca gene with an intragenic deletion of 422 bp, which was exchanged with the native chromosomal copy of gca. In minimal medium containing glucose 4% (w/v), the resulting strain C. glutamicum Δgca was observed to grow with the same growth rates and to the same final OD600 as the wild-type C. glutamicum (data not shown), indicating that the gca gene is dispensable under the conditions tested. This result is consistent with the previous observation that gca is not essential for growth of C. glutamicum under normal conditions [To test for a possible effect of Gca on growth and on expression of the nditions .bca was transformed into C. glutamicum Δgca and the activities of the bca promoter were determined by analysis of the specific CAT activities in crude cell extracts of cultures grown in minimal medium with glucose 1% (w/v). As shown in C. glutamicum Δgca were observed to be nearly the same as in the respective wild type derivative. This result indicates that there is no significant effect of the absence of a functional Gca protein on expression of the bca gene in C. glutamicum.Plasmid pET2-P2 is in chemical equilibrium with HCO3-, carbonic acid and carbonate. Of these, CO2 and its hydrated counterpart HCO3- are most important metabolites in living organisms as they serve as substrate or product in carboxylating and decarboxylation reactions, are involved in ion transport and internal pH regulation, regulate virulence and toxin formation in pathogenic bacteria and recently have been shown to be regulatory triggers of global transcriptional regulation and of microbial and mammalian production processes [2 is the product of respiration and as such in sufficient amounts present within metabolically active cells. However, for anaplerotic  carboxylation reactions, the physiologically most important reactant is HCO3- [2 (but not HCO3-) can diffuse out of the cell , the ingical pH  and thusrocesses , 79. Theto 5–10% , 80–82. C. glutamicum. This organism is an industrial workhorse widely used for the production of amino acids and a variety of other metabolites [2/HCO3- balance on the overall physiology of C. glutamicum can be presumed. Two CA genes, namely bca and gca, encoding β- and γ-CA, respectively, have previously been identified and bca has been shown to be essential under atmospheric conditions in C. glutamicum [The purpose of this work was to broaden our knowledge about the transcriptional organization of the CA genes as well as expression analysis in relation to media composition (carbon sources) and transcriptional regulation in abolites , 85, 86.utamicum .bca gene was identified to be the first nucleotide of its putative translational start codon (ATG). Thus, bca codes for a leaderless transcript which lacks the 5ˊ-untranslated region. Leaderless mRNAs starting with an AUG start codon have been reported in bacteria, archaea, eukaryotes [C. glutamicum [C. glutamicum revealed that about 33% of all mRNAs including that of the bca gene, in the cells are leaderless and that the translational start codon of these leaderless mRNAs generally is an AUG (about 79%) or GUG (about 21%) [E. coli, it has been shown that for translation initiation of leaderless mRNAs, the molar ratio of the initiation factors IF2 and IF3 plays a final role, indicating that the translation efficiency of these mRNAs can be altered, based on the availability of components of the translational machinery [C. glutamicum  . Howeverout 21%) . In E. cachinery , 88. Homctively; ), howevebca gene, we also determined the TSS for the gca gene, located at position -20 relative to the first nucleotide of the putative translational start codon of cg0154, a gene located upstream of the gca. Co-transcription of cg0154 and gca has previously been shown based on RNAseq analysis of C. glutamicum by Pfeifer-Sancar et al. [Apart from the r et al. .C. glutamicum, we investigated the expression of bca and gca in terms of their respective promoter activities in relation to glucose and acetate as carbon sources. This analysis revealed for both genes higher promoter activities when the cells were grown on glucose as compared to acetate. Furthermore, the activity of the bca promoter was observed to be about 15-fold higher than that of the gca promoter. The lower level of gca expression as compared to bca expression is consistent with the results of Mitsuhashi et al. [C. glutamicum cells the gca transcript level below the detection limit, suggesting that gca expression is either constantly very low or tightly regulated. Furthermore, it was also observed that expression of gca under control of the lac promoter restored the growth of bca mutant under normal environmental conditions [bca gene is subject to carbon source-dependent regulation as is the case for a variety of genes encoding key enzymes in central metabolism in C. glutamicum [To investigate a carbon source-dependent regulation of the CA genes in i et al.  who founnditions . Howeverutamicum , 90, 91.bca promoter activity and thus, the lower bca expression in acetate-grown cells than in glucose-grown cells of C. glutamicum might be attributed to a lower HCO3- demand and reduced need of anaplerosis by pyruvate or PEP carboxylation and using the glyoxylate cycle for anaplerosis when growing on acetate instead of glucose. This hypothesis is in agreement with carbon flux analysis of the central metabolism of C. glutamicum growing in minimal medium containing glucose and/or acetate [C. glutamicum grows on acetate but not on glucose [bca during growth on glucose may not only be confined to replenishment of oxaloacetate or other TCA cycle intermediates as Mitsuhashi et al. [C. glutamicum Δbca. It is, however, important to mention that C. glutamicum was found to be unable to take up and to grow on the TCA cycle intermediates fumarate, succinate and L-malate [C. glutamicum, aside of replenishment of TCA cycle intermediates, has to be experimentally proven.The lower  acetate  and with glucose . Howeveri et al.  observedL-malate . TherefoC. glutamicum [C. glutamicum  and/or effector(s) are involved directly or indirectly in the carbon source-dependent bca regulation. This argument is also reinforced by the fact that RamA affects and/or is affected by other transcriptional regulators such as GlxR, SugR and RamB [The transcriptional regulator RamA originally has been identified as the regulator of acetate metabolism in utamicum . Later iiewed in , 90, 95)erial in ). In addand RamB , 68, 91.S1 Table(DOCX)Click here for additional data file.


Intriguingly, while low concentrations of mTORC1 inhibitors completely inhibit growth of ccr4Δ, a ccr4Δ rpa12Δ rescues this growth defect suggesting that the sensitivity of Ccr4-Not mutants to mTORC1 inhibition is at least partially due to Pol I deregulation. Collectively, these data demonstrate a novel role for Ccr4-Not in Pol I transcriptional regulation that is required for bridging mTORC1 signaling to ribosomal RNA synthesis.Ribosomal RNA synthesis is controlled by nutrient signaling through the mechanistic target of rapamycin complex 1 (mTORC1) pathway. mTORC1 regulates ribosomal RNA expression by affecting RNA Polymerase I (Pol I)-dependent transcription of the ribosomal DNA (rDNA) but the mechanisms involved remain obscure. This study provides evidence that the Ccr4-Not complex, which regulates RNA Polymerase II (Pol II) transcription, also functions downstream of mTORC1 to control Pol I activity. Ccr4-Not localizes to the rDNA and physically associates with the Pol I holoenzyme while Ccr4-Not disruption perturbs rDNA binding of multiple Pol I transcriptional regulators including core factor, the high mobility group protein Hmo1, and the SSU processome. Under nutrient rich conditions, Ccr4-Not suppresses Pol I initiation by regulating interactions with the essential transcription factor Rrn3. Additionally, Ccr4-Not disruption prevents reduced Pol I transcription when mTORC1 is inhibited suggesting Ccr4-Not bridges mTORC1 signaling with Pol I regulation. Analysis of the non-essential Pol I subunits demonstrated that the A34.5 subunit promotes, while the A12.2 and A14 subunits repress, Ccr4-Not interactions with Pol I. Furthermore,  All cells communicate their environmental nutrient status to the gene expression machinery so that transcription occurs in proportion to the nutrients available to support cell growth and proliferation. mTORC1 signaling, which is essential for this process, regulates Pol I-dependent rRNA expression. We provide evidence that the RNA polymerase II regulatory complex, Ccr4-Not, also is a novel Pol I regulator required for mTORC1-dependent control of Pol I activity. Ccr4-Not disruption increases Pol I transcription due to an inability to decrease Pol I interactions with the transcription factor Rrn3 when mTORC1 signaling is reduced. Additionally, genetic and biochemical evidence supports a role for Ccr4-Not as a positive regulator of Pol I transcription elongation as well. Surprisingly, while Ccr4-Not mutations profoundly inhibit growth when mTORC1 activity is reduced, this phenotype is reversed by simultaneously impairing Pol I transcription. Overall, our data demonstrate that the evolutionarily conserved Ccr4-Not complex mediates environmental signaling through mTORC1 to control Pol I transcription initiation and, additionally, to regulate Pol I elongation. These studies further suggest that uncoupling Pol I from upstream mTORC1 activity by targeting Ccr4-Not sensitizes cells to mTORC1 inhibitors which is a concept that could have implications for anti-cancer drug development. Eukaryotic cells alter gene expression programs in response to changes in their environment, including nutrient availability and the presence of stress, by transmitting this information through nutrient-responsive signaling cascades to the transcriptional machinery . This prThe yeast rDNA exists as a multicopy array on chromosome XII with the individual 35S and 5S rRNA genes organized such that they are divergently transcribed and separated by non-transcribed sequences with only approximately half of the ~100–200 rDNA repeats expressed in a given cell . This poRRN3 mRNA translation [RRN3 is an essential gene required for Pol I transcription, this requirement can be bypassed if a chimera construct expressing Rrn3 fused to the A43 Pol I subunit is substituted [Another essential Pol I regulator is the transcription factor Rrn3  which is one of the few Pol I transcriptional regulators conserved at the amino acid level from yeast to mammals ,11. Rrn3nslation . Decreasnslation . Althougstituted . Cells estituted ,21. ThesWhile Pol I transcription initiation mechanisms are well defined, those affecting Pol I elongation remain are unclear. Studies have demonstrated that integral components of the Pol I holoenzyme, including the A49, A34.5, A12.2, and A14 subunits , are linked to Pol I transcription elongation –25. Mutaccr4Δ [The Ccr4-Not complex is composed of nine distinct subunits, including the Ccr4 mRNA deadenylase, Caf1, Caf40, Caf130, and Not1-5, and has both cytoplasmic and nuclear functions important for cell growth and proliferation. In the cytoplasm, Ccr4-Not acts to degrade mRNAs via the Ccr4 subunit which is the main deadenylase activity in yeast . In addiccr4Δ ,39. Nuclccr4Δ ,40. How ccr4Δ . WhetherCCR4, NOT1, or NOT2 genomic loci, we cultured cells in nutrient rich media (YPD) and performed chromatin immunoprecipitation (ChIP) coupled with quantitative PCR utilizing primers to the rDNA locus as outlined  or 143 copies (143C) [SPT15, to generate an 18S/SPT15 ratio. As expected, analysis of the 42C strain gave a low ratio whereas the analysis of the 143C strain resulted in a higher ratio  , we perfer ratio . InteresC strain . Becauseccr4 Δ increases overall rRNA transcription. Nascent rRNA synthesis is typically measured through incorporation of radiolabelled methionine or uridine via pulse-chase experiments of cells grown in nutrient defined media. However, both Pol I transcriptional activity, as well as the biological functions of Ccr4-Not, are downstream targets of nutrient regulated signaling pathways that may be differentially affected by growth under nutrient rich versus nutrient defined conditions [rpa49 Δ mutant cells. These samples were analyzed with primers specific to either ETS1 or ITS1 and normalized to the expression of the SPT15 housekeeping gene. RPA49 encodes the non-essential Pol I A49 subunit whose loss significantly impairs Pol I transcription [rpa49 Δ compared to wild-type , and rrp6 Δ (nuclear exosome mutant) backgrounds. Additionally, we also examined ETS1 and ITS1 expression in a catalytic mutant (pan2 Δ) of the Pan2-Pan3 complex which is an additional mRNA deadenylase complex that is partially redundant with Ccr4-Not [ccr4 Δ did not affect 5S expression, not4Δ severely impaired 5S rRNA levels  locus with wild-type and ccr4Δ cells and then monitored Rrn5 promoter association by ChIP. Rrn5 binding was not significantly increased in ccr4Δ suggesting that Ccr4-Not disruption did not augment Pol I transcription by elevating UAF promoter interactions (RRN6 (core factor subunit) genomic locus in wild-type and ccr4Δ cells and monitored Rrn6 binding. In wild-type cells, we detected a modest, but statistically significant, enrichment of Rrn6 over background which is likely due to the very low expression levels of core factor in cells  causes profound rapamycin sensitivity, consistent with previous studies linking Ccr4-Not to the mTORC1 pathway  did not alter Ccr4-Not co-association, deletion of the A34.5 (rpa34Δ) subunit reduced Ccr4-Not association to background levels (rpa12Δ) or the A14 (rpa14Δ) subunits increased Ccr4-Not association, suggesting these subunits normally limit Ccr4-Not interactions with Pol I  affecting ETS1 expression compared to rpa12Δ alone. Interestingly, while the ccr4Δ rpa12Δ reduced both ETS1 and ITS1 expression relative to ccr4Δ alone, ccr4Δ rpa12Δ restored the decreased ITS1 expression caused by the rpa12Δ mutation . Because 6AU depletes nucleotide pools, mutants deficient in either Pol I or Pol II elongation become hypersensitive to this compound while wild-type cells eventually adapt by upregulating IMP dehydrogenase expression [ccr4Δ is already 6AU sensitive in the presence of 15 μg/mL 6AU  or caffeine (8 mM) to reduce mTORC1 signaling and mimic nutrient stress. These same cultures were also spotted to plates containing the DNA replication inhibitor hydroxyurea (100 mM) as well as to control plates that were incubated at 37°C to assay their sensitivity to heat stress. After three days growth, the effects of the various stress conditions on the individual mutants were assessed. While the ccr4Δ exhibited severe sensitivity to even modest mTORC1 inhibition or DNA replication stress, the rpa12Δ was not sensitive to any of these agents  and pJCN101 (ccr4-1) as templates in a PCR reaction with CCR4 primers containing a C-terminal mono-FLAG sequence. The resulting PCR fragments were cloned into the Xba I and EcoRI sites of the p416ADH expression vector [All chemical reagents utilized in this study, including rapamycin, caffeine, and 6AU were purchased from Fisher Scientific or Sigma-Aldrich. The n vector .600 = 1.0–1.5) at 30°C in either YPD or SC media for the experiments outlined in this study. All yeast spotting assays were performed utilizing cultures grown overnight in the appropriate growth media. Cell numbers were then normalized based on their OD600 reading, five-fold serially diluted and spotted to the appropriate plate media. Cells were typically incubated for three to five days at 30°C unless otherwise specifically stated. For 6-azauracil assays, the indicated strains were transformed with a URA3 containing plasmid and cultured overnight in SC-ura media before spotting to either control SC-ura media or SC-ura media containing 15 μg/mL 6-azauracil. Plates were then incubated for six days at 30°C.All yeast strains were derived from the BY4741 genetic background. Yeast strains and plasmids are listed in 600 = 1.0–1.5 before cross-linking with 1% formaldehyde for 15 minutes followed by a five minute quench with 125 mM glycine at room temperature with gentle shaking. ChIP extracts and immunoprecipitations were performed exactly as described previously utilizing 400–500 μg of ChIP extract [(Input Ct-IP Ct) and expressed as % IP/Input. Primer sequences are listed in Yeast strains were cultured in either YPD or the appropriate SC media to an OD extract . 10% of 2O in the presence of DNase I and digested for 30 minutes at 37°C. Samples were then phenol/chloroform extracted, precipitated, and resuspended in 50 μL DEPC-dH2O. For cDNA synthesis with the Im-Prom II cDNA synthesis system (Promega), 1 μg of total RNA was utilized with random hexamer primers, per the manufacturer’s instructions, in a total volume of 20 μL. After synthesis, purified cDNA were diluted to a final volume of 100 μL and then 1:5 dilutions were utilized for qPCR analysis with gene-specific primers listed in SPT15 housekeeping gene using the formula 2(SPT15 Ct- Target gene Ct). Wild-type was set to a value of 1 while all yeast mutants were expressed relative to wild-type. All results were presented as the average and standard deviation of four or more independent experiments and the statistical significance determined by Student’s t-test.Total RNA was isolated by resuspending yeast pellets in Tri-Reagent (Sigma-Aldrich) in the presence of chloroform and glass beads. Samples were homogenized by vortexing and then centrifuged for two minutes at maximum speed. The soluble fraction was removed, precipitated with isopropyl alcohol, and centrifuged for 15 minutes at 4°C to pellet the nucleic acids. Pellets were resuspended in 50 μL of DEPC-treated dHnot4Δ cells expressing Rrn3-1XMyc/7X-histidine were cultured as described above in YPD, and then cell pellets were weighed and equivalent amounts of pellet were lysed in denaturing buffer  by bead beating at 4°C for six minutes in the presence of glass beads. Cell lysates were clarified by centrifugation at 15,000 rpm at 4°C for 15 minutes. From the clarified supernatant, 50 μL were removed and used as the total extract. The remainder of the supernatant was rotated with 50 μL of Ni2+-conjugated agarose beads at room temperature for two hours followed, by three washes in denaturing buffer before resolving samples by 8% SDS-PAGE and immunoblotting. All immunoblots were developed utilizing Immobilon ECL reagent (Millipore) and exposure to film. Quantification of immunoblots was performed using ImageJ. Antibodies used for immunoblot and ChIP analyses were as follows: α-HA , α-Myc , α-β-actin (Abcam), α-ubiquitin , α-G6PDH  and general IgG .Yeast whole-cell extracts were prepared as previously described and 500 μg of extract was utilized for immunoprecipitations in extract buffer  containing protease and phosphatase inhibitors . Immune S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S1 Supplemental References(DOCX)Click here for additional data file.


Moreover, we report that serum stimulation causes the prompt accumulation of ADP-ribose polymers on the same region and that this modification is required for chromatin decondensation and for the exchange of negative for positive transcriptional regulators. Finally we provide evidence that the inhibition of PARP activity along with serum stimulation impairs c-MYC induction by preventing the proper accumulation of histone H3 phosphoacetylation, a specific chromatin mark for the activation of Immediate Early Response Genes. These findings not only suggest a novel strategy by which PARP-1 regulates the transcriptional activity of promoters but also provide new information about the complex regulation of c-MYC expression, a critical determinant of the transition from quiescence to proliferation.Poly(ADP-ribosyl)ation is a post-translational modification of various proteins and participates in the regulation of chromatin structure and transcription through complex mechanisms not completely understood. We have previously shown that PARP-1, the major family member of poly(ADP-ribose)polymerases, plays an important role in the cell cycle reactivation of resting cells by regulating the expression of Immediate Early Response Genes, such as c-MYC, c-FOS, JUNB and EGR-1. In the present work we have investigated the molecular mechanisms by which the enzyme induces c-MYC transcription upon serum stimulation of quiescent cells. We show that PARP-1 is constitutively associated  Poly(ADP-ribosyl)ation has been implicated in several distinct processes regulating chromatin structure and transcriptional activity, both at global and gene-specific levels Poly(ADP-ribose)polymerases, also termed ADP-ribosyltransferases with diphtheria toxin homology (ARTDs) according to a new nomenclature The mechanisms by which PARP-1 regulates transcription in different biological contexts seem to be very complex and are still object of investigation PARP-1 affects chromatin function also by modulating the activity and localization of DNA-methyltransferase 1 (DNMT1) The enzyme activity is induced by several different signals, including binding to damaged DNA or to particular DNA structures, interaction with other proteins, heat shock and post-translational modifications brought about by signal transduction mediators Human primary foreskin fibroblasts (FB1329) FB1329 human fibroblasts were made quiescent by serum starvation for 36 hours and then transfected with Lipofectamine 2000 (Life Technologies) according to the manufacturer's instructions. siRNA (purchased from Eurofin MWG Operon) were used at the final concentration of 100 nM. The siRNA oligonucleotide sequences specific for human PARP-1 (Accession number NM_001618.3) 5′- AAG CCT CCG CTC CTG AAC AAT-3′ (nucleotides 2413-2433);5′-AAG ATA GAG CGT GAA GGC GAA-3′ (nucleotides 2671–2691) used for the experiment reported in The oligonucleotide sequence targeting GFP used as a control was:5′- GGC UAC GUC CAG GAG CGC ACC-3′. 72 hours post transfection, cells were collected or reactivated by serum addition.The stably knocked-down cell line for PARP-1 was obtained by using a MicroRNA-30-adapted shRNAmir retroviral vector (Open Biosystems). Briefly a 97mer containing the miR-30 back-bone plus sense and antisense sequence targeting nucleotides 1750–1771 of mouse PARP-1 (NM_007415.2) was cloned into the MSCV-LTRmiR30-PIG (LMP) according to the manufacturer's instruction. Production of empty (Control) and shPARP-1-expressing retroviruses were performed as described previously 5′-TGCTGTTGACAGTGAGCGCAGGGACGAACTCCTATTACAATAGTGAAGCCACAGATGTATTGTAATAGGAGTTCGTCCCTTTGCCTACTGCCTCGGA-3′Total proteins were extracted using lysis buffer , resolved by SDS-PAGE and transferred to a nitrocellulose membrane. The immunodetection was performed using anti-PARP1  or anti-TUBULIN  antibodies. After antibody binding, the filters were incubated with HPR-conjugated secondary antibodies (BioRad), and the blots were developed using SuperSignal West Pico kit . The relative protein amounts were quantified with ImageJ software.Total cellular RNA from quiescent and reactivated cells was isolated and purified by High Pure RNA isolation kit (Roche) according to the manufacturer's instructions. 1 µg of total RNA was reverse-transcribed using iScript cDNA synthesis kit (BioRad). Quantitative real-time PCR (qPCR) amplifications were carried out in triplicate for each sample with 1 µl of diluted (1∶6) total cDNA using sybr green GoTaq qPCR MasterMix (Promega). Data were collected and analyzed by comparative cycle threshold method. Standard curves were done to test the efficiency of each primer pair, which sequences are reported in ChIP experiments were carried out as previously described 6 cells per sample were rinsed with phosphate-buffered saline (PBS) and then with ice-cold CSK buffer . Cells were scraped from the plates, pelleted by centrifugation at 1500 rpm, and lysed in CSK-Triton buffer (CSK buffer containing 0.5% Triton X-100 supplemented with protease inhibitors) for 10 min on ice. Nuclei were collected after centrifugation at 3000 rpm for 5 min at 4°C and dissolved in 150 µL CSK-Triton buffer additioned with 50 mM MgCl2, divided into three aliquots and treated with different amount of DNaseI  for 4 min on ice. Then the reactions were stopped by diluting samples with Proteinase K Buffer . Total DNA was recovered by phenolic extraction. Purified DNA samples were analyzed by PCR using primer pairs specific for c-MYC, Rhodopsin (RHO) and GAPDH promoters. RHO promoter was chosen as a reference since it was virtually not digested by DNAseI in our conditions. The PCR products were detected on agarose gel and quantified by ImageJ software.For the accessibility assays approximately 4×10ChIP experiments for histone modifications were performed using Magna ChIP Protein A Magnetic Beads (Millipore) according to the manufacturer's instructions and using chromatin amounts corresponding to 50 µg of DNA. The antibodies used were: anti-Acetyl-Histone H3 , anti-trimethyl-Histone H3 (Lys4) , Anti-Histone H3   and Normal Rabbit IgG . Pulled-down DNA was analyzed in triplicate by qPCR and the values were normalized respect to GAPDH promoter (used as control region). Each experiment was repeated at least twice and a representative result is shown. The negative control values (not shown) were lower than the 10% of the immunoprecipitated samples.All the results plotted were reported as mean value ± the Standard Deviation (SD). Results obtained by qPCR were analyzed by CFX manager software (Bio-Rad) which retains the propagation of the errors between the values. Experiments with a statistical significance  were conducted a minimum of three times with independent biological replicates. The significance of differences between specific and control samples was determined using a two tails paired Student's t test. The T Test was performed using the Excel software. Differences were considered to be statistically significant at P<0.05 (*) and P<0.01 (**).The regulation of c-MYC gene expression is very complex and occurs at different levels ranging from transcriptional to post-translational control 2/ME1a1 and located between P1 and P2 transcriptional start sites, is required for the maintenance of an open chromatin configuration at the promoter region and plays a central role in the regulation of the activity of both promoters A key function in the transcriptional up-regulation of c-MYC is played by the promoter region, which has been demonstrated to be the target of a plethora of signaling pathways and transcription factors. c-MYC transcription starts from two major promoters, P1 and P2, with a predominance of P2-initiated transcripts in vivo to the c-MYC promoter region, gave support to our hypothesis that the enzyme is involved in c-MYC induction by acting at the promoter level.To determine the role of PARP-1 in c-MYC induction, we first explored the enzyme occupancy of the promoter region, by performing ChIP assays. To this end we designed three pairs of primers useful for amplifying three fragments which cover, respectively, the GQ element (fragment 1), the region comprised between P1 and P2 transcriptional start sites (fragment 2) and the region downstream of P2 promoter (fragment 3). Chromatin samples were collected from quiescent or serum-stimulated cells and immunoprecipitated with a PARP-1-specific antibody. As reported in In light of our previous finding that PARP enzymatic activity increases after serum stimulation and is necessary for the induction of IEG expression For this purpose, we performed ChIP experiments using a specific antibody recognizing poly(ADP-ribose). Quiescent and serum-stimulated cells, treated or not with the PARP inhibitor PJ-34, were collected for ChIP assays. The presence of the c-MYC promoter region in the poly(ADP-ribosyl)ated chromatin fraction was assessed by qPCR. The results, reported in The c-MYC regulatory region contains a number of DNaseI hypersensitive sites, indicative of an accessible chromatin conformation, some of which are strictly associated with the activation of the gene transcription In light of the ability of poly(ADP-ribosyl)ation to alter chromatin structure The DNAseI hypersensitive sites represent highly accessible DNA regions that could be prone to dynamic changes in the binding of transcription factors. Thus we asked whether the observed chromatin changes were associated to changes in transcription factors binding on the c-MYC promoter. To this aim, we analyzed the occupancy of the region of interest by a couple of factors known to modulate c-MYC expression in an opposite manner. In particular, we focused on CTCF and SP1, which act respectively as a negative and a positive regulator of c-MYC expression and whose consensus sequences lie in the same region that we have observed to become poly(ADP-ribosyl)ated Taken together, these findings suggest that poly(ADP-ribosyl)ation changes the chromatin conformation at c-MYC promoter and is involved in the transcription factor exchange required for the gene induction see The reorganization of chromatin structure involves a complex and not completely understood interplay between several types of post-translational modifications of histone proteins and the activity of chromatin remodelling complexes As potential targets of PARP activity in the induction of c-MYC promoter, we considered two other marks of chromatin relaxation: acetylation and phosphoacetylation of histone H3. Acetylation of core histones is generally enriched at the 5′ of actively transcribed genes These results indicated that poly(ADP-ribosyl)ation mediates the serum-dependent up-regulation of c-MYC through a mechanism involving the modulation of other histone modifications, in particular H3 phosphoacetylation.We have previously demonstrated, by means of PARP inhibition and PARP-1 knock-down, that the enzyme activation is required for resting cells to emerge from quiescence. This function involves the ability of PARP-1 to mediate the accumulation of IEG products in response to mitogen stimulation in vitroin vitro conversion of the c-MYC GQ structure into its B-DNA form 2 element that has been previously recognized as biologically relevant for c-MYC transcription. This element is critical for the maintenance of an open chromatin configuration at the dual c-MYC P1/P2 promoter in episomal assays and participates in the complex regulation of initiation and elongation of P1 and P2 transcripts We show that PARP-1 occupies c-MYC promoter in both quiescent and serum-stimulated cells. One of the bound regions covers the GQ-element, a sequence able to adopt a four-stranded secondary structure named guanine (G)-quadruplexes. This structure, which is in equilibrium with the double-stranded B-DNA form, prevents the binding of transcriptional activators Concerning how PARP-1 activity may induce chromatin decondensation, transcriptional accessibility of the promoter and co-factor exchange, the simplest hypothesis would be that the enzyme directly modifies chromatin structural proteins or transcription factors. Unfortunately this case cannot be directly verified at this moment since antibodies for specific poly(ADP-ribosyl)ated proteins, to be used in ChIP assays, are not yet available. It is worth mentioning that both SP1 and CTCF have been shown to physically interact with and to be targets of PARP-1. However, as regards SP1, the poly(ADP-ribosyl)ation of this factor impairs its binding to different targets promoters While it is conceivable that PARP-1 causes histone poly(ADP-ribosyl)ation at c-MYC promoter, our data also revealed the existence of a functional interplay between PARP activation and other histone modifications, in the induction of c-MYC upon mitogenic signalling. In this regard, previous results showed that PARP activity is induced downstream of receptor tyrosine kinase stimulation by direct interaction with ERK2 MAP kinase Our data are consistent with a model in which acetylation and phosphoacetylation at c-MYC promoter not only are directly stimulated by MAPK-activated acetylases and kinases but also are modulated by the MAPK-PARP-1-poly(ADP-ribosyl)ation pathway . The abiWe would like to point out that, despite PARP-1 activity has been clearly linked to the inhibition of the histone demethylase KDM5B and to the accumulation of the permissive chromatin mark H3K4me3 Interestingly, we have observed the presence of PARP-1 and ADP-ribose polymers on c-FOS promoter as well (unpublished results). Moreover, a very recent work reported that PARP-1 is required for c-FOS promoter activation in HeLa cells in response to ERK MAP kinase signaling The present work suggests an additional function of PARP-1 in the regulation of chromatin organization and transcriptional activity, involving its ability to modulate the phosphoacetylation of target promoters. Moreover, it provides new information about the regulation of c-MYC promoter, a very complex and still poorly understood issue. The molecular mechanisms underlying the transcriptional induction of early response genes are of basic importance for the proper regulation of cell quiescence, a fundamental process for controlling differentiation, preserving stem cell function and preventing tumorigenesis.Figure S1PARP-1 required for serum-induced accumulation of c-MYC mRNA. A, PARP-1 levels assessed by western blot in quiescent FB1329 fibroblasts transfected with the control (siGFP) or the specific (siPARP-1) siRNAs. TUBULIN was used as a loading control. Left panel shows the results of a representative experiment; right panel shows the averages and the standard deviations (SD) of densitometric values of PARP-1 signals normalized respect to TUBULIN, derived from three independent experiments. B, c-MYC expression assayed by RT-qPCR in quiescent (0) and serum-stimulated (for 30 or 60 minutes) siRNA-transfected fibroblasts. c-MYC expression levels were normalized relatively to TBP expression and reported as fold increase respect to the control quiescent sample. The error bars represent the SD of three technical replicates.(TIF)Click here for additional data file.Figure S2Poly(ADP-ribosyl)ation in cells stably knocked-down for PARP-1. Mouse C3H10T1/2 fibroblasts expressing the PARP-1 short hairpin RNA vector (shPARP-1) or the empty vector (Control) were made quiescent by serum deprivation for 72 hrs. After serum stimulation, cells were collected and nuclei were isolated by incubation with buffer A . After centrifugation, nuclei were re-suspended in RIPA buffer  and quantified by Lowery assay (Bio-Rad). 20 µg of nuclear extracts were resolved on SDS PAGE and transferred on a nitrocellulose membrane. The immune-detection was performed using anti-Poly(ADP-ribose) , anti-PARP1  or anti-H3  antibodies. The total H3 was used as a loading control. Min p.s.a. means minutes post serum addition.(TIF)Click here for additional data file.Figure S3PARP-activity is required for histone modifications at c-MYC promoter. Acetyl histone H3 (AcH3) and phospho-acetyl histone H3 (pAcH3) levels at c-MYC promoter assessed by ChIP-qPCR. Chromatin samples were obtained from quiescent (-FBS) or 30 minutes serum-stimulated (+FBS) fibroblasts treated or not with PJ-34, and immuno-precipitated with an antibody specific for AcH3, pAcH3 or total H3. Values of modified histones were normalized respect to those of total H3 in the same promoter region (fragment fr.2+3). The error bars represent the SD of three technical replicates.(TIF)Click here for additional data file.Table S1Primers for RT-qPCR.(DOCX)Click here for additional data file.Table S2Primers for ChIP and DNAseI accessibility.(DOCX)Click here for additional data file.


Basic transcriptional machinery in eukaryotes is assisted by a number of cofactors, which either increase or decrease the rate of transcription. Mediator complex is one such cofactor, and recently has drawn a lot of interest because of its integrative power to converge different signaling pathways before channeling the transcription instructions to the RNA polymerase II machinery. Like yeast and metazoans, plants do possess the Mediator complex across the kingdom, and its isolation and subunit analyses have been reported from the model plant, Arabidopsis. Genetic, and molecular analyses have unraveled important regulatory roles of Mediator subunits at every stage of plant life cycle starting from flowering to embryo and organ development, to even size determination. It also contributes immensely to the survival of plants against different environmental vagaries by the timely activation of its resistance mechanisms. Here, we have provided an overview of plant Mediator complex starting from its discovery to regulation of stoichiometry of its subunits. We have also reviewed involvement of different Mediator subunits in different processes and pathways including defense response pathways evoked by diverse biotic cues. Wherever possible, attempts have been made to provide mechanistic insight of Mediator's involvement in these processes. The process of transcription in eukaryotic organism is an immensely complex and highly orchestrated phenomenon, and is mediated by a plethora of proteins wherein primary role is played by RNA polymerase II (RNAP II) . These interactions can bring changes in the structure of the resultant complex which may affect transcription. Thus, Mediator acts as a docking site for many other transcriptional regulators and plays critical role in relaying regulatory signals from them to RNAP II machinery  and med20a plants revealed 84% overlap in the down-regulated genes, implying that Mediator complex is as important as the RNAP II for gene expression . This dilemma could be explained by taking into account the modular nature of Mediator complex and by assigning the “division of labor” principles to each module. The head module subunits might be involved in the more basic functions of the Mediator complex, whereas the tail module subunits residing on the periphery might be controlling gene-specific functions by contacting the specific transcription factors.Even after two decades of its discovery, it is still debatable whether Mediator complex is a general transcription factor or just a cofactor of gene expression. Although, initially identified as an entity that supports activator-dependent transcription, now, according to several evidences, Mediator complex can also be categorized as general transcription factor. The human Mediator complex can support basal level transcription of many genes by playing important roles in assembly of PIC and transcription initiation  and abscissic acid (ABA) have more significant impact on the transcription of MED genes as compared to other hormones including auxin and jasmonic acid (JA)  .A large portion of total protein coding genes in eukaryotes requires the presence of Mediator complex even to sustain basal level of transcription. This proves unequivocally that Mediator constitutes important part of the basal transcriptional machinery. However, drastic morphological changes in mutants of individual Mediator subunits suggest that Mediator could also act as selective gene regulator both in metazoans and plants  and CENTER CITY (CCT), respectively, mediate the embryo pattern formation, albeit in a transient manner  is a positive regulator of flowering and affects multiple genes working upstream and downstream of FT , and together binds to the promoter of FLC gene  and superoxides  of the kinase module plays important role in specification of stamen and carpel in Arabidopsis  . The arrest of cell division in Atmed14 plants may result from the endoreduplication of the chromosomal DNA. Mechanistically, AtMED14 may interact with SMP1, SMP2 which encode step II splicing factors as both the mutants show similar phenotypes , and AtMED5b (RFR1), have been shown to negatively regulate plant height and lignin content , and AtMED25, have been reported to control iron homeostasis is plants , and provides resistance against the leaf-infecting necrotrophic fungi, Alternaria brassicicola, and Botrytis cinerea , which mediates the H2B ubiquitination, thus establishing a link between Mediator and the chromatin remodeling. The induced expression of both MED21 and HUB1 in response to chitin treatment, an important constituent of fungal cell wall, suggests their probable role in defense signaling . This leads to proteasome-dependent degradation of AtMed19a and shift the balance from SA-mediated disease resistance to ET/JA-mediated transcriptomic changes making the plants more vulnerable to bitrophs  of powdery mildew pathogen, MED16 are compromised for SA- and JA-dependent defense responses  proteins and defensins. Moreover, the expression levels of the important SAR (systemic acquired resistance) markers like PR1, PR2, PR5, GST11, EDR11, SAG21 are severely reduced in Atmed16 mutant  plays a pivotal role and takes the center stage in the SA-mediated defense pathways  differ considerably toward controlling the expression of defense genes. The Atmed16 mutation differentially affects the expression of different positive and negative regulators of SAR, whereas Atmed14 mutation inhibits expression of similar genes. Moreover, defense-related transcriptomic change in the case of Atmed14 is much smaller as compared to that in the case of Atmed16.Three tail module subunits, AtMED14, AtMED15, and AtMED16 have been reported to be involved in defense signaling as well  gene whose products are involved in the biosynthesis of defense active bio-compounds like hydroxycinnamic acid amides in plants.The kinase module component, AtCDK8, has recently been reported to be a positive regulator of disease response , AtPRP40a has been recently named as AtMED35 of the Mediator complex. It interacts both with the phosphorylated and the unphosphorylated forms of the largest subunit of RNAP II. In Arabidopsis, it has its characteristic high expression level in roots and cauline leaves as compared to the other parts. The mutant does not show any phenotype, probably because of its redundancy with AtPRP40b and AtPRP40c , alternative splicing and epigenetic regulation Table . Hence, cis-element bound transcription factor and the RNAP II-mediated transcriptional machinery relaying the information from the transcription factor to the transcription apparatus. Recently, a couple of reports in plants have made the picture more complicated as the interaction between the transcription factor and the Mediator complex is not a simple binary one-one interaction. The Arabidopsis Mediator subunit, MED25 can interact with several transcription factors , that function in the same pathway. The mutants of all these three genes show increased sensitivity to salinity stress  were discovered in Arabidopsis, but later AtMED32 and AtMED33 were found to be AtMED2 and AtMED5, respectively. As some of their functions are not directly related to Mediator functions or transcription , concern has been expressed regarding how truly these proteins represent Mediator subunits.Many of the Arabidopsis Mediator subunits were characterized earlier, but not in consideration of its Mediator membership. Over the time, several Mediator subunits have been characterized in Arabidopsis and many more may follow. In most of the cases, phenotypes of a particular Mediator subunit mutant has been described, but its association with transcription factors and the set of genes under its control are yet to be discovered in majority of the cases. What is lacking more is the understanding of how the Mediator subunits interact with components of the basic transcriptional machinery resulting in the regulated transcription.Recently, many of the hitherto unknown but interesting functional aspects of Mediator has been unveiled in other organisms further broadening the horizon of its roles. Mediator not only takes part in the recruitment of RNAP II on the promoters of the active genes but also in transcription elongation and termination, chromatin remodeling, alternative splicing, small, and long non-coding RNA biogenesis, heterochromatin formation. All these developments are taking place in the arena of yeast and metazoan biology. Except characterization of few Mediator subunits, studies involving the Mediator complex as a whole or the mechanistic dexterity of Mediator complex in general or gene-specific regulation has not been addressed with proper emphasis and interest in plants. So, besides characterization of the every Mediator subunits in model species, attention should also be focused to address how the Mediator controls different steps of transcription in terms of mechanical intricacies.Presence of more than one paralog has been reported for some Mediator subunits. Another level of complicacy may arise regarding which paralog remains with the complex, which most probably is controlled in a temporal and spatial manner. The presence of more than one paralog at a time in the Mediator complex has not been reported by any group. The more interesting question which has just been started to be answered is how stable is the Mediator structure in terms of its subunit composition. We postulate that the structure of Mediator complex changes depending on the composition of Mediator subunits, which again is controlled by different biotic and abiotic stimuli. Mediator complex isolation and its structural comparison from different stages of growth and development hold the key to the questions of how the structural shifts due to changes in Mediator composition are translated into transcriptomic changes of a species in response to intrinsic and extrinsic factors. Armed with the tools of modern molecular biology like TAP, MudPIT, LC-MS/MS, and HT-ChIP; the aforementioned questions are anticipated to be answered at an accelerated speed in near future.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Genome occupancy of Myc-Max is thought to be driven by Enhancer box (E-box) DNA elements, CACGTG or variants, to which the heterodimer binds in vitro sequence preferences, Myc-Max genomic occupancy measured by ChIP-Seq is largely, although not completely, independent of sequence specificity.By analyzing ChIP-Seq datasets, we demonstrate that the positions occupied by Myc-Max across the human genome correlate with the RNA polymerase II, Pol II, transcription machinery significantly better than with E-boxes. Metagene analyses show that in promoter regions, Myc is uniformly positioned about 100 bp upstream of essentially all promoter proximal paused polymerases with Max about 15 bp upstream of Myc. We re-evaluate the DNA binding properties of full length Myc-Max proteins. Electrophoretic mobility shift assay results demonstrate Myc-Max heterodimers display significant sequence preference, but have high affinity for any DNA. Quantification of the relative affinities of Myc-Max for all possible 8-mers using universal protein-binding microarray assays shows that sequences surrounding core 6-mers significantly affect binding. Compared to the We quantified the affinity of Myc-Max to all possible 8-mers and compared this with the sites of Myc binding across the human genome. Our results indicate that the genomic occupancy of Myc cannot be explained by its intrinsic DNA specificity and suggest that the transcription machinery and associated promoter accessibility play a predominant role in Myc recruitment.The online version of this article (doi:10.1186/s13059-014-0482-3) contains supplementary material, which is available to authorized users. In vitro, the basic helix-loop-helix leucine zipper (bHLHZip) domain of Myc binds preferentially, albeit very weakly, to double stranded DNA containing the palindrome CACGTG and this is considered the canonical E-box [c-Myc was initially identified as a proto-oncoprotein and subsequently demonstrated to be a global regulator of transcription -6. In vial E-box ,8. Myc pal E-box -11. A cral E-box . These ial E-box . Howeveral E-box ,14.Many studies attempting to identify Myc target genes found that the sets of genes regulated by Myc displayed great variation depending on the cell types and conditions used . A signiMYC was the first gene shown to be regulated by elongation [Regulation of gene expression is controlled predominantly through the action of DNA-binding transcription factors that affect both initiation and elongation. A prominent feature of metazoan genomes is the promoter proximal paused Pol II that is found engaged in transcription about 30 to 80 bp downstream of the transcription start site (TSS) on most expressed genes -19. The ongation  and Myc ongation -24 and congation -27.in vitro.The mechanism of Myc regulation of transcription has been assumed to involve Myc-Max heterodimers binding to E-boxes near TSSs and then influencing the function of the transcription machinery. The discovery that Myc globally regulates essentially all expressed genes ,6 hints MYC, provides further evidence that Myc and Max occupancy is tied to Pol II, including polymerases within the transcribed regions and downstream of the Poly(A) addition site .Using the UCSC Genome Browser  and ChIPe Figure C. For the Figure . In compSeveral straightforward bioinformatic tools were used to obtain a global view of the correlation of Myc and Max compared to Pol II and CTCF. The average occupancy around the TSS of 20,886 genes in HeLa cells was calculated and plotted. Promoter proximal paused Pol II peaked on average 83 bp downstream of the TSS. Myc and Max on average peaked upstream of the TSS at -20 and -35, respectively  , but instead gave only a smeary band below the position of a tightly bound complex (arrow)   Figure D. MaxL d) Figure E. This i) Figure F. Compete Figure G and H. e Figure G and H. e Figure F. In thein vivo occupancy of Myc and Max to the location of E-boxes, we ran into difficulty because of the existence of a large number of reported non-canonical E-boxes. Without quantification of the relative affinity of Myc-Max for all these sites it was difficult to correlate them with in vivo occupancy. Because of this, protein-binding microarray (PBM) assays using ‘all 10-mer’ universal array designs [L heterodimer and the MaxL homodimer across all possible 8 bp sequences . After normalization, relative Myc-Max occupancy for each of the 32,896 8-mers exhibited a 56-fold range, from 0.018 to 1  of each of the top 12 6-mers was generated that graphically displays the range of intrinsic affinities across the genome . This enrichment is further shown by means of a receiver operating characteristic (ROC) curve  can distinguish between two classes of sequences . Although the area under the ROC curve is better than expected by chance (0.637 vs. 0.5), the ROC analysis shows that the in vitro 8-mer occupancies cannot be used to accurately predict whether an accessible genomic region will be bound by Myc in cells. Here, the ROC plot shows that at a false positive rate of 0.1, the true positive rate is only 0.25. To make only 10% false positive predictions of Myc in vivo binding using the in vitro 8-mer scores, we would only be able to capture 25% of the true Myc ChIP-Seq peaks. This means that the vast majority of sites occupied by Myc are associated with low scoring 8-mers, as graphically indicated in Figure Several analyses were performed to compute the correlation between the 8-mer sequence preferences determined by PBM and the actual genomic occupancy of Myc, as measured by ChIP-Seq. The ChIP-Seq Peak algorithm  was used regions ) . Had the cellular occupancy correlated well with the affinity for the underlying DNA sequences, there would have been a cloud of black dots clustered around the blue curve in Figure in vitro occupancy scores is clearly found even for the highest occupancy sites . The top 1,487 peaks of extra Max  were selected for further analysis  could then stabilize this interaction with specific sequence elements playing only a minor role  that bridges Myc with the transcription machinery is not clear, but Mediator is found in a similar position to Myc upstream of the promoter proximal paused Pol II. Like Mediator, Myc and Max peaked upstream of the TSS. Both the Myc and Mediator peaks extended into the downstream region occupied by Pol II. This could be due to crosslinking between the N-terminal transcription activation domain of Myc with Mediator, which has been shown to interact with both Pol II ,50 and MOur results have clarified the DNA binding preferences of the Myc-Max heterodimer, widening the range of core binding sites bound specifically by Myc-Max and suggesting that non-specific interactions could also be significant. In examining binding to all possible 8-mers it became apparent that sequences flanking the core 6-mers were important. Changing the flanking sequences of the canonical E-box 6-mer from GCACGTGC to TCACGTGA resulted in a four- or five-fold reduction in Myc-Max and Max binding. A similar reduction has been previously noted for Pho4p but not Cbf1p, two yeast bHLH proteins ,55. Despin vitro. Previously, these preferences were determined using EMSAs that routinely included unlabeled carrier DNA. Unfortunately, this leads to an inaccurate determination of the actual range of affinities for different sequences. Many reporter assays have been performed where the presence of a CACGTG E-box sequence enhances the expression of the reporter  does not mean that sites matching the motif are present in all of the DNA sequences analyzed. In fact, very significant enrichment for a particular motif can be observed even when only a very small fraction of the analyzed sequences contain the motif. A second problem arises because of the length of the sequences analyzed and the resolution of actual binding site. Reasonable quality ChIP-Seq datasets like those analyzed here can determine individual binding sites to within 25 to 50 bps. If the fragments analyzed by a motif discovery algorithm are more than about 100 bp , a particular motif can be present in the sequence analyzed but not be bound . In example, , which dE. coli lac repressor paradigm [The model we propose for Myc might be applicable to other transcription factors. Significant overlap of occupancy between Myc and other factors such as AP-1 and AP-2 can be observed and two recent ENCODE reports showed co-association of many transcription factors binding to ‘surprisingly plastic’ DNA sequences ,59. Indeparadigm , in whicparadigm . In addiparadigm . The humparadigm . TBP on paradigm . The metin vitro for different sequences covers only a little over two orders of magnitude (Kd = 10-8 to 10-10 M). Even taking into account the fact that much of the human genome is not accessible due to chromatin structure, we found that Myc occupancy was not well correlated with affinity for underlying DNA in accessible regions. We found instead a strong correlation of Myc (and Max) occupancy with that of Pol II and on average Myc was located about 100 bp upstream of the promoter proximal paused Pol II. We propose that Myc is a general factor brought to promoters predominately by protein-protein interactions and like TBP, its recruitment to promoters does not strictly require sequence specific binding.We combined an extensive determination of the DNA binding properties of Myc-Max with genome-wide occupancies of Myc, Max, and Pol II and conclude that the affinity of Myc-Max for specific DNA sequences cannot be the main determinant of Myc genomic occupancy in cells. Our results indicate that the range in affinities The alignment files of HeLa, GM12878, and K562 cell lines were downloaded from human ENCODE Project at UCSC . The rawThe complete set of human RefSeq genes was downloaded from the UCSC table browser  on 1 JanThe number of reads within 10,000 bases of the TSS of each RefSeq gene was tabulated without binning. The average value of the lowest 2,000 of 20,000 data points was subtracted from each position, and then data were normalized so that the area under each curve was equal. For Figure Heat maps were generated using the program R . Genes wFor Figure A peak finding algorithm (ChIP-Seq Peak)  was applL (gi:21704261) and MaxS (gi:21704263) were amplified from HeLa cDNA and individually cloned into pET21a . All proteins were expressed in BL21 star E. coli after overnight induction of MaxL and MaxS at 18°C or 4 h induction of Myc at 37°C. Soluble Max proteins were purified over Ni-NTA and Mono Q for MaxS or Mono S for MaxL as described for Gdown1 [Coding sequences of Myc (gi:29839758), Maxr Gdown1 . Yields 32P-ATP and then annealed to generate dsDNA. Each reaction contained 0.01 nM labeled dsDNA and 0.01 pM, 0.1 pM, 1 pM, 0.01 nM, 0.1 nM, 1 nM, 10 nM, or 100 nM protein. For competition assays 0.01 nM labeled CACGTG probe, 1 nM of the indicated protein and the indicated amounts of non-labeled competitor DNA were used. After 30 min at room temperature, Ficoll  was added to each reaction and the samples were run on a 6% polyacrylamide gel in 0.5X Tris/glycine buffer at 10 mA for 2 h. The gels were then analyzed by silver staining or phosphorimaging.Binding reactions were 25 mM HEPES (pH 7.6), 100 mM KCl, 1 mM DTT, 0.1 mM EDTA, and 0.01% Triton. For reactions analyzed on silver stained gels, 200 ng of protein and 0, 0.1, 0.3, 1, or 3-fold molar excess of unlabeled dsDNA were used in each reaction. For the reactions using radiolabeled probes, each strand of the indicated probe was end-labeled with γ-in vitro occupancies for Myc-Max and Max-Max across all 8-mers are provided in Additional file in vitro occupancy scores were obtained by normalizing the observed intensity for each 8-mer to the intensity of the highest ranked 8-mer . The raw PBM data as well as the 8-mer intensities have been deposited in GEO (GSE58570) and the genome track for the top 12 6-mers has been deposited in GEO (GSE43227).Protein-binding microarray (PBM) experiments were performed as described previously . Brieflyin vitro Myc 8-mer occupancies were compared using the Wilcoxon rank-sum test . A receiver operating characteristic (ROC) curve was used to assess the enrichment of sites with high in vitro 8-mer occupancies in the Myc ChIP-Seq peaks. A ROC curve is a plot of false positive rate (1-specificity) versus true positive rate (sensitivity) computed for different 8-mer occupancy cutoffs. Each point on the ROC curve corresponds to one cutoff. Sequences with 8-mer occupancy above or below the cutoff are predicted positives  or predicted negatives , respectively. In Figure in vitro occupancies was tested using Spearman’s rank correlation coefficient (cor.test with the method parameter set to Spearman).Statistical analyses were performed in R. Fisher’s exact test (dhyper) was used to compare the frequency of CACGTG sites in Myc-occupied regions and in all accessible DNA (DNase I hypersensitive regions ). A simi