CSF2, encoding granulocyte-macrophage colony stimulating factor (GM-CSF), and PTGS2, encoding prostaglandin synthase 2/cyclooxygenase 2 (PGS2/COX2). Prolonged CSF2 expression, high GM-CSF production, and GM-CSF activation of PTGS2 gene expression all are seen in type 1 diabetes (T1D) monocytes. Persistent phosphorylation activation of monocyte STAT5 (STAT5Ptyr) is also found in individuals with or at-risk for T1D. To examine whether elevated T1D monocyte STAT5Ptyr may be associated with aberrant inflammatory gene expression in T1D, blood monocytes from non-autoimmune controls and T1D patients were analyzed by flow cytometry for STAT5Ptyr activation, and by chromatin immuno-precipitation (ChIP) analyses for STAT5Ptyr’s ability to bind at CSF2 and PTGS2 regulatory sites in association with histone acetylation. In unstimulated monocytes, STAT5Ptyr was elevated in 59.65% of T1D, but only 2.44% of control subjects (p<0.0001). Increased STAT5Ptyr correlated with T1D disease duration . Unstimulated (p = 0.140) and GM-CSF-stimulated (p = 0.0485) T1D monocytes, had greater STAT5Ptyr binding to epigenetic regulatory sites upstream of CSF2 than control monocytes. Increased STAT5Ptyr binding in T1D monocytes was concurrent with binding at these sites of STAT6Ptyr (p = 0.0283), CBP/P300 histone acetylase, acetylated histones H3, SMRT/NCoR histone deacetylase (p = 0.0040), and RNA Polymerase II (p = 0.0040). Our study indicates that in T1D monocytes, STAT5Ptyr activation is significantly higher and that STAT5Ptyr is found bound to CSF2 promoter and PTGS2 enhancer regions coincident with histone acetylation and RNA polymerase II. These findings suggest that the persistent activation of STAT5 by GM-CSF may be involved in altering the epigenetic regulation of these inflammatory response genes in T1D monocytes.STAT5 proteins are adaptor proteins for histone acetylation enzymes. Histone acetylation at promoter and enhancer chromosomal regions opens the chromatin and allows access of transcription enzymes to specific genes in rapid response cell signals, such as in inflammation. Histone acetylation-mediated gene regulation is involved in expression of 2 key inflammatory response genes:  Myeloid antigen presenting cells (APC) are the primary immune regulatory cells inducing and enforcing peripheral self-tolerance. Granulocyte Macrophage Colony Stimulating Factor (GM-CSF) is a major differentiation and inflammatory response cytokine that is both produced by and acts on myeloid APC, including peripheral blood monocytes, interstitial macrophages, and myeloid dendritic cells CSF2 gene and to enhancer sequences found between and possibly within the IL10 to PTGS2 gene region on Chromosome 1 (mouse and human) CSF2 and PTGS2.Chronic high prostaglandin synthase 2 (PGS2/COX2) and GM-CSF expression by T1D patient monocytes and nonobese diabetic (NOD) mouse myeloid cells allow for the production of high levels of the pro-inflammatory prostanoid prostaglandin E2, PGE2. PGE2 blocks myeloid APC ability to prevent auto-reactive T cell escape from activation induced cell death PTGS2 mRNA in monocytes by adherence to glass or endotoxin contamination within 90 min ex vivo). High GM-CSF production is readily detectable in primary culture of T1D monocytes, but is less specific for T1D and autoimmune dysfunction than PGS2/COX2 High PGS2/COX2 expression seen in at-risk human subject monocytes is directly correlated to T1D risk and is indicative of a 1.5-fold increase in disease susceptibility within the high risk population CSF2 and PTGS2 genes. Using these tools, we investigate the mechanism of how STAT5Ptyr could affect epigenetic regulation of both GM-CSF and PGS2/COX2 expression. Our findings indicate that STAT5Ptyr in T1D but not non-autoimmune monocytes respond to GM-CSF stimulation by binding to the promoter of CSF2 and in an enhancer region associated with the Chromosome 1 region between IL10 and PTGS2. ChIP evaluation of GM-CSF-stimulated STAT5Ptyr binding at these sites show coincidence with histone modification and recruitment of epigenetic and transcription proteins that can modulate epigenetic gene expression in chromatin regions.In this report, we use a multiplex format chromatin immunoprecipitation (ChIP) assay that allows us to simultaneously detect several components of the cells epigenetic and transcriptional regulatory protein machinery associated on the same specific regulatory sequences upstream of All work done for this study involving the participation of human volunteers was done in accordance with IRB approved protocols  and only with informed consent. After giving written informed consent, healthy volunteers and T1D patients donated small blood samples either by venipuncture (up to10 ml) or finger prick (<0.5 ml), during routine visits to the pediatric and adult endocrinology clinics at the Shands at UF hospital on the University of Florida, Gainesville, FL campus. Information of the subject populations is given in The flow cytometric analyses were completed using a small volume, whole blood modification of our previously described intracellular STAT5Ptyr analysis using modification specific antibodies for tyrosine 694/699 phosphorylated STAT5 CSF2 promoter and PTGS2 enhancer regions in the human genome that are homologous to the regions in the mouse genome were designed using Net Primer  select for a region approximately 397 bp in length and include two STAT5 binding sites CSF2) promoter  generate a PCR product of length 1993 bp. DNA samples were purified using Qiagen Blood and Cell Culture DNA Mini Kit and amplified by PCR using Eppendorf 2.5X Master Mix (Fisher Scientific) and either CSF2 promoter or PTGS2 enhancer primers. PCR amplification products were visualized on 1% agarose gel (SeaKem Fisher Scientific) and purified using DNA Clean and Concentrator TM-5  then amplified again in a Big Dye PCR amplification reaction  and sequenced using an AB capillary sequence analyzer  and found comparable to sequences reported in these analyses are available in publically accessible NIH gene libraries.Twelve non-autoimmune controls and twelve T1D patient samples were subjected to sequence analysis. Population characteristics for this test population are given in t Primer Figure 16 cells per well and cultured for 1 hr at 37°C/5% CO2 to allow for monocytes adherence. The cultures were then washed with sterile 1× PBS  to remove non-adherent cells and re-fed with RPMI 10%FCS/1%PSA medium alone, or with 5.6 nM human recombinant GM-CSF , 500 U/ml human recombinant IL-4 (Invitrogen), 1 µg/ml dexamethasone (Sigma-Aldrich) or 1 ng/ml endotoxin (LPS) (Sigma-Aldrich). Dexamethasone (DEX) and LPS stimulations were included in the analyses as potential ‘negative’ and ‘positive’ controls, respectively, for STAT activation and epigenetic regulation of transcription, based on previous reports + flow cytometric analysis. Cultures were held 2 hr at 37°C/5%CO2 to allow time for activation by LPS or suppression by DEX, then washed with 1×PBS and then lysed in situ using ChIP Lysis Buffer  supplemented with a proteinase inhibitor cocktail . The lysates were sonicated on ice to shear chromatin complexes to approximately 1000 bp lengths and frozen at −80°C until used for ChIP analysis.Peripheral blood mononuclear cells (PBMC) were isolated out of 10 ml peripheral blood or umbilical cord blood samples from 4 non-autoimmune controls and 10 T1D patients, using a ficoll histopaque gradient (Sigma-Aldrich). The PBMC were washed with 1×PBS  and re-suspended in sterile RPMI medium , supplemented with 10% fetal calf serum , and 1% antibiotic-antimycotic mix  to a density of 1 to 5×10in situ with salmon sperm-Protein G coated sepharose beads (UpState/Millipore) and bead-free supernants aliquoted equally over 8 PCR microtubes in a strip/capped format . The aliquots were diluted with ChIP Dilution Buffer  to a total volume of 200 µl each. Salmon Sperm-Protein G sepharose beads (Upstate/Millipore) were pre-incubated with 1 µg/30 µl beads suspension in a 96-tube ‘Multiplex’ format using parallel ChIP isolations with one tube of each of the following UpState/Millipore ChIP certified antibodies: anti-STAT5Ptyr, anti-STAT6Ptyr, anti-RNA Polymerase II, anti-acetylated Histone H3, anti-P300/anti-CBP acetylase, and anti-SMRTe deacetylase. Thirty microliters of the antibody-bead mixes were added to 7 of the 8 tubes of sample, with the final tube being left unprecipitated to act as a total DNA control. After overnight incubation at 4°C, ChIP bead complexes were collected via centrifugation and washed successively with 200 µl of each of the following: Low Salt buffer , High Salt buffer , LiCl buffer , and TE buffer . The beads were then resuspended in freshly prepared 0.1 M sodium bicarbonate/1% SDS buffer (Fisher Scientific) and incubated for 45 min at rt to uncouple the ChIP complexes from the beads. The beads were removed by centrifugation and sodium chloride added to resultant supernatant for a final concentration of 500 mM NaCl (Fisher Scientific). The salted samples were then incubated for 4 hr at 65°C to reverse the formaldehyde crosslinking. After incubation, 0.5 M EDTA pH 8.0 (Sigma-Aldrich), 1 M Tris HCl pH 6.5  were added and the samples incubated for 1 hr at 45°C to digest the remaining proteins. The resultant chromosomal DNA samples were either analyzed as is or further purified by either phenol/chloroform extraction or with QIAGEN genomic DNA kit. Real time PCR (RLT PCR) analyses were done as previously described for single ChIP isolations using primers specific for the promoter region of the CSF2 gene and the enhancer region of the PTGS2 gene (ΔCtnon-specific IgG−ΔCtspecific Ab)6 cells were used to make ChIP extracts, a second, re-amplification round of RLT PCR with the same primers was often needed to verify the results. Such low sample volumes and re-amplication led to less conclusive results in the PTGS2 analysis of 2 control and 5 T1D samples, and as a result were not included in the data analysis presented.Monocyte cell extracts were analyzed as individual (not pooled) samples. Thawed extracts were pre-cleared of non-specific DNA and Protein G binding proteins by incubation ex vivo stimulation using intracellular flow cytometry. Intra-assay and inter-assay variance in this modified assay were assessed on samples drawn 24 hr prior to analysis and samples shipped on ice and then stored for over 72 hr post draw. There was an assay variance of <5% between individual control samples and intra-assay variance from 0 to 6.3% for a given control sample’s analysis. The assay variance between individual T1D samples was <13%, with an intra-assay 1 to 7.9% for a given T1D sample’s analysis  whole peripheral blood samples stored up to 24 hr at 4°C . However, there was a significant correlation of STAT5Ptyr levels increasing with disease duration in the T1D subject group (p = 0.0030, rCSF2 or PTGS2 upstream regulatory regions that directly associated with the increased GM-CSF production or COX2/PGS2 expression seen in at-risk/T1D individuals CSF2 promoter and the PTGS2 enhancer, it might enhance STAT5Ptyr response to cytokine activation by GM-CSF or IL-2, and/or hinder immunosuppression STAT6Ptyr activation by IL4 CSF2 and PTGS2 regulatory regions.Unlike our previous findings in NOD mice ex vivo stimulation with GM-CSF, and IL-4 (&CSF2 promoter, and a trend for epigenetic modification, and potential for transcription at both CSF2 and PGTS2 loci than non-autoimmune healthy controls. Unstimulated STAT6Ptyr binding in T1D monocytes was not outcompeted by STAT5Ptyr at either site, but was able to bind equally as well as STAT5Ptyr to this sequence. In comparison, little to no STAT5Ptyr and STAT6Ptyr binding were seen to either CSF2 or PTGS2 associated regulatory sites without stimulation in control monocytes ( & We used this assay to profile epigenetic modification and transcription potential in isolated monocytes from 10 T1D and 4 healthy, non-autoimmune control subjects without stimulation or after nd IL-4 &5. The CSF2 promoter.Exogenous GM-CSF significantly enhanced SMRT deacetylase associated with its own gene promoter in T1D monocytes compared with the same treatment in control monocytes (p = 0.0040, Mann-Whitney U test, PTGS2 enhancer region was conducted with the same samples, but low cell/DNA yields from the ChIP isolations only permitted analysis of 5 T1D samples and 2 controls, compromising the statistical analysis of the results. In the samples analyzed, exogenous GM-CSF treatment of T1D monocytes decreased STAT5Ptyr binding but enhanced histone H3 acetylation in T1D monocytes, though neither change was statistically significant. In controls analyzed, GM-CSF decreased SMRT/NCoR and enhanced CBP/P300 binding at the IL10-PTGS2 region, as well as increased histone H3 acetylation, but these changes were not found to be statistically significant Analysis of STAT5Ptyr binding at the CSF2 promoter in both controls and T1D subjects, but did not extinguish it in T1D monocytes (CSF2 promoter in T1D cells (IL10 -PTGS2 site in T1D monocytes. Both IL-4 and dexamethasone appeared to promote SMRT binding as seen at the CSF2 promoter, though these changes were not found to be statistically significant (IL-4 uniformly lowered STAT protein binding at the Mechanistic studies of inflammation activation indicate that changes in epigenetic regulation of genes seen in autoimmune T1D patients may be involved in their immune cell dysfunction CSF2 and PTGS2CSF2 and PTGS2 regulatory sites should be suppressed by the actions of anti-inflammatory, immunosuppressive cytokines such as IL-4 and IL-10. However, IL-4 is known to be inherently low in people at-risk for or with T1D PTGS2 remains resistant to its influence in vivo by STAT5 persistent binding at regulatory regions, both for CSF2 and PTGS2. STAT5Ptyr binding at the promoter of CSF2, could allow for continued expression of GM-CSF. However, persistence of STAT5Ptyr in T1D monocytes was not dependent on continuous GM-CSF stimulation. Previous studies have shown that GM-CSF can enhance the expression of IL-10- and IL-4- resistant COX2 CSF2 and PTGS2 regulatory regions. This STAT-STAT interaction may have the potential to promote histone acetylation and transcriptional activation of these genes by interfering with their STAT6Ptyr mediated suppression when little or no activation stimuli are present.Functional analysis suggests that the resistant STAT5 activation seen in autoimmune human myeloid cells has the potential to alter the epigenetic regulation of at least 2 genes important to promoting inflammation and myeloid APC dysfunction: Since STAT5 proteins are adaptor proteins for histone acetylation enzymes, not for methylation enzymes, it is less likely that STAT5Ptyr binding could affect methylation patterns than acetylation modifications. Methylation modifications at these sites were previously reported as unchanged in diabetic monocytes, enhancing the importance of other epigenetic modifications including acetylation for gene dysregulation seen in T1D monocytes CSF2 or the PTGS2 sites in auto-reactive cells. Furthermore, though both dexamethasone and IL-4 alter the amount of CBP/P300 histone acetylase binding and histone H3 acetylation at the CSF2 promoter in both control and T1D monocytes, neither abolished RNA Polymerase II recruitment at these sites in autoimmune cells, suggesting that gene expression could go forward even in these conditions.In terms of pharmacological intervention, dexamethasone should have been a promising potential treatment for this defective regulation based on previous reports CSF2 and PTGS2 gene expression, these data support previous findings that STAT5Ptyr activation and binding to epigenetic active sequences are elevated in untreated autoimmune monocytes compared with non-autoimmune monocytes While the data from our study do not prove that GM-CSF induced STAT protein binding causes the epigenetic regulation of CSF2 and the inflammation it promotes through PTGS2, may be abnormal self-perpetuating in T1D monocytes.GM-CSF stimulation of autoimmune monocyte STAT5Ptyr binding is found in direct association with increased binding of enzymes catalyzing histone acetylation/deacetylation, and the presence of RNA Polymerase II at these sites. Altered histone acetylation at these sites in GM-CSF stimulation, suggests that activation of


Mycobacterium tuberculosis latent infections that can persist for decades. Nitric oxide produced by infected lung macrophages promotes expression of genes associated with dormancy, and impaired nitric oxide production can lead to reactivation of latent disease. Recently, WhiB1 was identified as a nitric oxide-responsive transcription factor. Here it is shown that apo-WhiB1 binds to groEL2 (Rv0440) promoter DNA. Apo-WhiB1 inhibited transcription from the groEL2 promoter in vitro and the transcript start was located ∼181 bases upstream of the groEL2 start codon. Electrophoretic mobility shift assays with sub-fragments of the groEL2 promoter indicated that the complete Rv0439c-Rv0440 intergenic region was required for WhiB1 binding, suggesting that this region possessed more than one WhiB1-binding site. DNase I footprinting identified a WhiB1-binding region that overlapped the −35 element of the groEL2 promoter. The CRP-family transcription factor Cmr (Rv1675c) was shown to bind the groEL2 promoter and activate transcription in vitro in the presence or absence of cAMP. Therefore, it is suggested that WhiB1 acts to oppose Cmr-mediated cAMP-independent activation of groEL2 expression in the presence of nitric oxide by promoter occlusion.A central feature of TB pathogenesis is the formation of  Adaptation to these changing environments requires the reprogramming of M. tuberculosis gene expression coordinated by ∼190 transcription regulators responding to diverse signals.M. tuberculosis WhiB1 (a member of the WhiB-like (Wbl) protein family) was shown to be a nitric oxide-responsive transcription factor.M. tuberculosis to nitric oxide initiates the dormancy gene expression program that may contribute to the establishment of latent TB infections.M. tuberculosis gene expression is potentially significant in TB pathogenesis. Several states of the WhiB1 protein have been identified. The [4Fe–4S] form of WhiB1 (holo-form) was incapable of binding at the whiB1 promoter, whereas the reduced and oxidized forms of apo-WhiB1, as well as nitric oxide-treated holo-WhiB1 (nitrosylated-form) were able to specifically bind DNA.M. tuberculosis whiB1 gene is essential, the influence of this regulator in the reprogramming of gene expression is of considerable interest.whiB1 promoter itself.groEL2.The etiological agent of tuberculosis, M. tuberculosis has two chaperonin genes: groEL1 (Rv3417c) is located downstream of whiB3 (Rv3416), which, like whiB1, encodes a member of the Wbl protein family; and groEL2 (Rv0440). The groEL1 gene is dispensable, whereas groEL2 is essential.M. tuberculosis does not form such a structure but is dimeric and lacks ATPase activity.M. tuberculosis GroEL2 chaperonin deviates significantly from the established paradigm.groEL2 was increased upon heat shock in a process involving the repressor protein HrcA, and lowered in response to Mg(II)-starvation and in a cmr (Rv1675c) mutant 2 h into macrophage infection; cmr encodes a member of the cyclic-AMP receptor protein (CRP) family of transcription factors that is proposed to regulate cAMP-induced genes in macrophages.groEL2 expression.Expression of 22.1Mycobacterium smegmatis RNA polymerase were isolated as described previously.Escherichia coli from plasmid  and isolated by affinity chromatography on a 1 ml Hi-Trap Chelating column  using the manufacturer's standard protocol.WhiB1 and 2.2Rv0439c-Rv0440 intergenic DNA (groEL2) or fragments thereof, or ahpC promoter DNA (∼1.6 nM) were incubated with 0–40 μM His6-WhiB1 in the presence of 40 mM Tris pH 8.0, 1 mM EDTA, 100 mM NaCl, 1 mM DTT, 10 mM MgCl2, 0.25 mg ml−1 bovine serum albumin and 1 μg calf thymus DNA, for 5 min on ice. For the analysis of Cmr–DNA interactions, the protein (0–8 μM) was pre-incubated with the groEL2 promoter DNA for 10 min at 25 °C in the same buffer as above in the presence or absence of 1 mM cAMP. The resulting complexes were then separated on 6% polyacrylamide gels. In vitro transcription reactions were assembled and quantified as described previously except that the indicated fragments of the Rv0439c-Rv0440 (groEL2) intergenic region were used.Electrophoretic mobility shift assays (EMSA) were as described previously.2.3Rv0439c-Rv0440 (groEL2) intergenic region (∼60 ng) was incubated with 20 μM His6-WhiB1 in the presence of 40 mM Tris pH 7.5, 50 mM NaCl, 10 mM MgCl2, 0.5 mM EDTA, 1 mM DTT and 0.25 mg ml−1 bovine serum albumin for 10 min on ice. The complexes were then digested with 1 unit of DNase I for 15–60 s at 25 °C. Reactions were stopped by the addition of 200 μl 0.3 M sodium acetate (pH 5.2) containing 20 mM EDTA, followed by phenol/chloroform extraction. The DNA was ethanol-precipitated and resuspended in loading buffer  for electrophoretic fractionation on 6% polyacrylamide–urea gels and autoradiographic analysis. Maxam and Gilbert G tracks of the DNA fragments were used to provide a calibration.Radiolabeled 33.1M. tuberculosis promoter regions were tested as targets for regulation by apo-WhiB1. Binding was detected in electrophoretic mobility shift assays (EMSA) when the Rv0439c-Rv0440 (groEL2) intergenic region was the target (whiB1 promoter itself (Ref. Rv0439c-Rv0440 (groEL2) intergenic region saturated over a narrow range of apo-WhiB1 concentration, consistent with cooperative interactions with the DNA, and implying the presence of more than one apo-WhiB1 binding site. Nevertheless, the apo-WhiB1 interactions were judged to be specific because binding was not detected when the ahpC promoter was used (rpfA promoter (Ref. Rv0439c-Rv0440 (groEL2) intergenic DNA was inhibited by excess (100- and 50-fold) unlabeled Rv0439c-Rv0440 (groEL2) competitor DNA (rpfA DNA was the competitor (Rv0439c-Rv0440 (groEL2) intergenic DNA (Rv0439c-Rv0440 (groEL2) intergenic region and that binding was modulated by the presence/absence of the iron–sulfur cluster.Several e target A. As preelf Ref. , bindingter Ref. , and wheenic DNA B, lane 33.2In vitro transcription assays were used to determine the consequences of apo-WhiB1 binding at the Rv0439c-Rv0440 (groEL2) intergenic region. Three templates were designed to distinguish which of the divergent Rv0439c and groEL2 promoters was active and potentially regulated by WhiB1  and −35 elements  separated by the optimal 17 bp  intergenic region were used as the targets in EMSAs. Whilst the mobility of the complete intergenic region (−116 to +181 bp relative to the groEL2 transcript start) was retarded by both apo- (groEL2 transcript start were not retarded by either apo- (not shown) or nitric oxide-treated WhiB1  is a member of the CRP family of transcription factors that is required for cAMP-induced protein expression, including GroEL2, in macrophages.groEL2 expression. Binding of Cmr to the groEL2 promoter was shown by Gazdik et al. (Ref. groEL2 promoter was unaffected by the addition of 1 mM cAMP (not shown). Transcription of groEL2 in vitro was enhanced ∼3-fold in the presence of 1 μM Cmr and this activation was independent of cAMP  and is also induced by cAMP.in vitro transcription reactions reported here show that Cmr activates groEL2 transcription in the absence of cAMP, suggesting that Cmr does not directly mediate the cAMP effect on groEL2 expression. Like the groEL2 promoter, expression of the other known target for WhiB1, whiB1, responds to cAMP.whiB1 expression in response to cAMP, but activation by cAMP-CRP is inhibited by apo-WhiB1, i.e. WhiB1 negatively auto-regulates expression.groEL2 expression observed in vivo arise, at least in part, from CRP-mediated regulation of whiB1. Nevertheless, it is clear that both of the WhiB1 targets identified so far are linked to cAMP-signaling. It has recently been shown that upon infection of macrophages, mycobacterium-derived cAMP promotes bacterial survival by subverting host signaling pathways.M. tuberculosis gene regulation.whiB1 and groEL2) have thus far been identified as WhiB1 targets both exhibited cooperative binding to sites overlapping the −35 elements of the promoters, resulting in repression of transcription. These observations suggest that promoter occlusion might be a common feature of gene regulation by WhiB1. However, it is apparent that apo-WhiB1 interactions with the groEL2 promoter region are complex and the complete Rv0439c-Rv0440 intergenic region is required to form a stable nucleoprotein complex. This suggests the presence of at least two WhiB1-binding sites within this region and that both the site overlapping the −35 element, that is identified here, and unidentified site(s) are required for detectable WhiB1 binding in EMSAs. Protein:DNA interactions of this type are not unprecedented but usually at least two regulator:DNA complexes are observed in EMSAs. These complexes result from binding of the regulator first to a high affinity site and then subsequent cooperative occupation of a low affinity site. In such cases impairment of the high affinity site abolishes binding to the low affinity site.Rv0439c-Rv0440 intergenic region is dependent on sites of similar affinity and that binding is highly cooperative.Until this report the only other known target for the nitric oxide-responsive transcription factor WhiB1 was its own promoter.M. tuberculosis groEL2. Previously, expression of groEL2 was shown to be controlled by HrcA (derepression in response to heat shock), by Cmr , and by Mg(II)-starvation (repression by a PhoPR-independent mechanism).groEL2 in response to signals – nitric oxide, oxidative stress, iron-starvation – that promote the formation of the DNA-binding apo- and nitrosylated-forms of WhiB1. An understanding of the precise interplay between these regulators and the consequences for groEL2 expression awaits further detailed biochemical analysis. However, it appears that several signals associated with the process of infection are sensed and transduced by integrated gene regulatory circuits to optimize expression of the essential chaperonin GroEL2.In conclusion, the work described here reveals new aspects of the regulation of


Igf1 locus, and proposed that they could regulate IGF-I gene activity. Here we investigate the biochemical and functional characteristics of these putative long-range transcriptional enhancers. Each element contained 2 or 3 individual Stat5b recognition sequences that could bind Stat5b in vitro, but with affinities that varied over a >100-fold range. Full transcriptional responsiveness to GH required that all Stat5b sites be intact within an individual enhancer. Replacement of a single lower-affinity Stat5b sequence with a higher-affinity one increased in vitro binding of Stat5b, and boosted transcriptional potency of the entire element to GH. As enhanced transcriptional activity involved changes in only one or two nucleotides within an enhancer DNA segment, there appears to be remarkable specificity and sensitivity in the ability of Stat5b to transform DNA binding activity into transcriptional function. Stat5b was able to stimulate the transcriptional activity of two enhancers in the absence of GH, indicating that individual Stat5b-regulated elements possess distinct functional features. We conclude that combinatorial interplay among multiple Stat5b-binding response elements with distinguishable biochemical properties is responsible for highly regulated control of IGF-I gene activity by GH.Many of the biological effects of growth hormone (GH) are mediated by insulin-like growth factor I (IGF-I), a 70-amino acid secreted peptide whose gene expression is rapidly induced by GH via the Stat5b transcription factor. We previously identified multiple evolutionarily conserved GH-activated chromosomal binding domains for Stat5b within the rat  Growth hormone (GH) plays a pivotal role in multiple physiological processes in mammals. It is essential for somatic growth, is a key contributor to normal tissue differentiation and repair, and is an important regulator of intermediary metabolism Like other members of the cytokine receptor family, upon ligand binding the GH receptor engages and stimulates the Jak - Stat signaling pathway Stats comprise a group of seven related proteins in mammals GH receptorStat5b in mice Socs2, Cish, and Igfals in rodents, in which functionally critical Stat5b binding sites are located within the proximal promoters, there are no Stat5b transcriptional response elements within either of the two promoters of the Igf1 gene Igf1 loci Despite clear evidence that multiple signaling pathways act downstream of the GH receptor, recently identified inactivating molecular lesions in the STAT5B gene in humans with impaired growth Igf1 locus, as a means to understand how they contribute to control of IGF-I gene transcription by GH. We find that each Stat5b element has distinct transcriptional regulatory properties, and that individual sites within each element have unique binding profiles for Stat5b. Taken together, our data define a framework for discerning how Stat5b acts in vivo as the key mediator of GH-regulated IGF-I gene transcription.Here we have evaluated the biochemical and functional characteristics of the multiple dispersed chromosomal Stat5b binding domains in the rat Fetal calf serum, Dulbecco's modified Eagle's medium, and phosphate-buffered saline were purchased from Mediatech-Cellgro . Transit-LT1 was from Mirus , and the QuikChange site-directed mutagenesis kit from Stratagene . Restriction enzymes, buffers, ligases, polymerases, and protease inhibitor tablets were from Roche Applied Sciences . Recombinant rat GH was purchased from the National Hormone and Pituitary Program, NIDDK, National Institutes of Health. Trypsin/EDTA solution was from Invitrogen . The BCA and 660 nm protein assay kits were from Pierce Biotechnologies  and AquaBlock EIA/WIB solution from East Coast Biologicals . QIA-Quick PCR purification kit was from Qiagen  and okadaic acid from Alexis Biochemicals . Primary antibodies were obtained from the following vendors: anti-phospho-Stat5 (clone 8-5-2) and anti-Creb, Millipore , anti-α−tubulin and anti-Flag (M2), Sigma-Aldrich , anti-Stat5b, Invitrogen. Goat-anti-rabbit IgG-IR800 and goat anti-mouse IgG-IR680 were from Rockland Immunochemical , and goat anti-mouse IgG1-Alexa 488 was from Invitrogen - Molecular Probes . Hoechst 33258 nuclear dye was from Polysciences . Oligonucleotides were synthesized at the OHSU DNA Services Core, at Oligos Etc , and at Eurofms MWG Operon . All other chemicals were reagent grade and were purchased from commercial suppliers.Igf1 promoter 2  were incubated in antibiotic-free Dulbecco's modified Eagle's medium with 10% fetal bovine serum at 37°C in humidified air with 5% COIsolation of nuclear and cytoplasmic proteins has been described previously CA, or Stat5bDN (500 ng). Two days later, cells were fixed in 4% paraformaldehyde for 15 min at 20°C and permeabilized with a 50∶50 mixture of methanol and acetone for 2 min followed by blocking in 0.25% normal goat serum for 2 h at 20°C. After addition of Flag M2 monoclonal antibody (1∶2000 dilution) or pStat5 antibody (1∶2000 dilution) in blocking buffer overnight, followed by a washing step, and incubation in goat anti-mouse IgG1-Alexa 488 (1∶1000 dilution) in blocking buffer for 2 h, images were captured with a Nikon Eclipse E800 compound microscope with CCD camera, using Nikon analysis software. Nuclei were stained with Hoescht dye.Cos-7 cells in 6 well plates were transiently transfected with expression plasmids for wild type Stat5b, Stat5bATGCAAATGGACGT ACGT-3′5′-TTTTAGAGGATCC. After incubation of proteins and DNA for 60 min at 4°C, products were separated by electrophoresis through non-denaturing 5% polyacrylamide gels in 1× Tris borate/EDTA  at 200V for 30 min at 20°C. After electrophoresis, gels were dried and the bands representing protein-bound DNA and free probe measured using the LiCoR Odyssey and version 3.0 analysis software. Quantitative DNA-protein binding studies were performed as described Electrophoretic gel-mobility shift assays and DNA competition experiments were performed as described t test, with the Bonferroni correction for multiple comparisons in the latter, and is indicated in each figure legend.Data are presented as mean±S.E. Statistical significance was determined using either paired or unpaired Student’s Igf1 locus that exhibited acute GH-stimulated binding of Stat5b in hepatic chromatin Igf1 promoters in a GH- and Stat5b-dependent way Igf1 locus is depicted in bona fide Stat5 binding sites, each consisting of the DNA sequence, 5′-TTC NNN GAA-3′ , with individual sites being separated by 6–251 base pairs of genomic DNA . Previous studies had found that no response to GH was seen in the absence of added Stat5b expression plasmid Functional studies were performed by reconstituting GH-activated signaling in cultured cells by expressing the mouse GH receptor and rat Stat5b, and using as transcriptional reporter genes recombinant plasmids containing a minimal 80 base pair fragment of proximal rat promoter . As seen3–4 GAA-3′ to 5′-GTC N3–4 GTA-3′ (modifications are in bold script). Results from promoter-reporter studies demonstrated that in all cases elimination of every Stat5b site in a genomic fragment markedly attenuated GH- and Stat5b-activated transcription . Analyses using semi-quantitative dose-response competition studies against the labeled R34 probe allowed us to calculate relative affinities of GH-activated wild-type Stat5b for each of the 14 Stat5 sites in the 6 elements. Results show a wide range of binding strengths, as illustrated by measured IC50’s of 1.4 to 158 nM . Similarly, when R35 was replaced by R43, which does not bind Stat5b Our third observation also relates to the apparently complex relationship between binding of Stat5b to individual DNA sites and the transcriptional competence of domains encoding two or three sites. Results from quantitative and semi-quantitative gel-mobility shift experiments revealed a wide range of binding affinities (spanning 2 orders of magnitude,  compare . Taken tGH orchestrates rapid and dramatic alterations in gene expression to yield potent biological effects on growth, metabolism, and tissue repair


Bacillus licheniformis and Rhodobacter sphaeroides that the corresponding genes are preceded by a highly conserved promoter motif and are the only detectable targets of ECF41-dependent gene regulation. In contrast to other ECF σ factors, proteins of group ECF41 contain a large C-terminal extension, which is crucial for σ factor activity. Our data demonstrate that ECF41 σ factors are regulated by a novel mechanism based on the presence of a fused regulatory domain.Bacteria need signal transducing systems to respond to environmental changes. Next to one- and two-component systems, alternative σ factors of the extra-cytoplasmic function (ECF) protein family represent the third fundamental mechanism of bacterial signal transduction. A comprehensive classification of these proteins identified more than 40 phylogenetically distinct groups, most of which are not experimentally investigated. Here, we present the characterization of such a group with unique features, termed ECF41. Among analyzed bacterial genomes, ECF41 σ factors are widely distributed with about 400 proteins from 10 different phyla. They lack obvious anti-σ factors that typically control activity of other ECF σ factors, but their structural genes are often predicted to be cotranscribed with carboxymuconolactone decarboxylases, oxidoreductases, or epimerases based on genomic context conservation. We demonstrate for  Bacteria populate complex habitats in which extracellular conditions can change very rapidly. In order to survive in such an environment, bacterial cells have to be able to sense and respond to these variations before cell damage actually occurs. Therefore, bacteria need signal transducing systems that enable them to sense these extracellular changes and respond by differential gene expression.A common mechanism to control gene expression at the level of transcription initiation is the use of σ factors, which constitute an essential subunit of the RNA polymerase (RNAP) holoenzyme and determine the promoter specificity. In addition to the primary σ factor, which is responsible for general expression of most genes in exponentially growing cells, most bacteria contain one or more alternative σ factors. These proteins are activated only under certain conditions and control expression of a specific set of target genes by recognizing alternative promoter sequences .70 family based on their relation to the primary σ factor of Escherichia coli, σ70   . Group 2 stimuli .70 proteins, the ECF σ factors only contain two of the four conserved domains, σ2 and σ4, which are sufficient for promoter recognition and interaction with RNAP. The bipartite promoter recognized by ECF σ factors typically contains a highly conserved “AAC” signature in the –35 region and a “CGT” motif in the –10 region  and Rhodobacter sphaeroides (α-proteobacteria). In both organisms, the ECF41 σ factor appears to control expression of a single transcript that is preceded by a highly conserved ECF41-specific promoter motif. A unique feature of ECF41 proteins is the presence of a large C-terminal extension containing a number of conserved signature motifs. We provide evidence that this C-terminal extension is involved in regulation of σ factor activity and we propose that it functions as a fused anti-σ factor-like domain.Here, we describe the characterization of one such uncharacterized group, ECF41. This group is widely distributed with about 400 proteins from 10 different phyla. Based on their genomic organization, the genes encoding these ECF41 σ factors are not transcriptionally linked to genes encoding proteins related to known anti-σ factors. Instead, they are located next to genes encoding carboxymuconolactone decarboxylases, oxidoreductases, or epimerases. To extract general features of ECF41-dependent gene regulation, we experimentally investigated ECF41 σ factors from two different organisms, http://mistdb.com. False positives (unclassified ECF σ factors) and redundant proteins (proteins from more than one sequenced strain per species) were removed leaving 373 sequences for further analysis. Multiple sequence alignments were performed using ClustalW  were analyzed for putative promoter motifs either manually or with the help of MEME , availabogo tool  at http:lgorithm , implemelgorithm  at http:Bacillus subtilis, B. licheniformis, and E. coli were grown in LB medium at 37°C with aeration. Rhodobacter sphaeroides was grown aerobically in Sistrom's minimal medium (B. subtilis and B. licheniformis mutants. Plasmid containing E. coli strains were grown with ampicillin (100 μg/mL) or kanamycin (50 μg/mL). Rhodobacter sphaeroides mutants were selected using tetracycline (1 μg/mL), spectinomycin (25 μg/mL), or kanamycin (25 μg/mL).l medium  at 30°C.Escherichia coli strain S17–1  and ligated to the Ω fragment derived from pHP45Ω  and TMBli003 (Δecf41Bli) were grown aerobically in LB medium at 37°C. Every 2 h, 30 mL samples were taken and mixed with cold killing buffer , harvested by centrifugation, and frozen in liquid nitrogen, before the pellets were stored at –80°C. The cells were resuspended in 200 μL killing buffer, immediately transferred to a precooled Teflon vessel and disrupted with a Micro-Dismembrator U (Sartorius) for 3 min at 2000 rpm. The resulting cell powder was resuspended in 3 mL prewarmed lysis solution  and total RNA was extracted twice with acid phenol  and once with chloroform  followed by isopropanol precipitation. Contaminating DNA was removed using the Baseline-ZERO DNAse  according to the manufacturer's protocol. RNA was quantified with a NanoDrop 1000 Spectrophoto-meter  and used for 5′RACE and Northern Blot analysis.Rhodobacter sphaeroides YSD354 (pIND4) and YSD333 (pYSD161) were grown aerobically in Sistrom's minimal medium  and harvested by centrifugation. The pellets were frozen in an ethanol/dry ice bath and stored at –80°C. Cells were resuspended in 2 mL lysis solution  and incubated at 65°C for 5 min. RNA was extracted three times with acid phenol prewarmed to 65°C followed by chloroform extraction and isopropanol precipitation. To remove contaminating DNA, the RNA was incubated with two units RQ1 DNase  in the presence of 80 units RNasin Plus RNase Inhibitor  for 30 min at 37°C. The RNA was finally purified with the RNeasy Mini Kit  and used for DNA Microarray analysis and 5′RACE.l medium  containiydfG was amplified by PCR with oligonucleotides listed in An ∼500 bp internal fragment of N-lauroyl sarcosinate, and 0.02% SDS). Hybridization was carried out overnight at 68°C in the same solution with 1 μg DIG-labeled RNA probe. The membrane was washed twice for 5 min at room temperature  and three times for 15 min at 68°C . The signal was detected with an antidigoxigenin antibody conjugated to alkaline phosphatase  and CDP-Star  according to the manufacturer's instructions. The signals were visualized using a LumiImager .For Northern Blot analysis, 10 μg of total RNA was separated under denaturing conditions on a 1% formaldehyde agarose gel and transferred to a positively charged nylon membrane  in a downward transfer using 20× SSC  as transfer buffer. The RNA was crosslinked by exposing the membrane to UV light. The blot was prehybridized at 68°C for 1 h with hybridization solution , 0.1% http://www.r-project.org/). Data were normalized using the affyPLM package with default settings  by 10°C/cycle to 10°C to optimize annealing of the hexamers. cDNA was synthesized using SuperScript II Reverse Transcriptase  according to the manufacturer's instruction. Temperature was increased from 20°C to 42°C in 22 cycles of 3 min with 1°C increment followed by incubation at 42°C for 1 h and inactivation at 70°C for 10 min. Remaining RNA was removed by alkaline hydrolysis and cDNA was purified using the PCR Purification Kit . A total of 3.2 μg cDNA was fragmented with 0.25 units RQ1 DNase  for 10 min at 37°C followed by inactivation for 10 min at 98°C. cDNA was labeled using the BioArray Terminal Labeling Kit with Biotin-ddUTP for DNA Probe Array Assays  according to the manufacturer's protocol. Labeled cDNA samples (3 μg/array) were hybridized to Affymetrix  custom arrays  accordinsettings . Differe at 0.05 .ydfG and RSP_0606 mRNAs were identified by rapid amplification of cDNA ends (RACE). A total of 15 μg of total RNA was incubated with 25 units tobacco acid pyrophosphatase  in the delivered buffer at 37°C for 60 min in the presence of 40 units Super RNaseIn RNAse inhibitor . As a control, 15 μg RNA was incubated under the same conditions, but without TAP. The reactions were phenol/chloroform extracted and ethanol precipitated. After dissolving the pellets in water, the RNA was mixed with 500 pmol RACE adapter (5′-GAUAUGCGCGAAUUCCUGUAGAACGAACACUAGAAGAAA-3′) and denatured at 95°C for 5 min. Ligation of the adapter was carried out at 17°C overnight with 100 units T4 RNA ligase  in the presence of 80 units Super RNaseIn RNAse inhibitor . Again, the reactions were phenol/chloroform extracted, ethanol precipitated, and the pellets were resuspended in water. One microgram RNA was used for reverse transcription with gene-specific primers , cells were lysed by sonication, and cell debris was removed by centrifugation. A total of 20 μg of the cleared lysate was separated by SDS-PAGE and transferred to a Polyvinylidene difluoride (PVDF) membrane using a Mini Trans-Blot Electrophoretic Transfer Cell  according to the manufacturer's instructions. The membrane was then incubated overnight at 4°C with blotto  to prevent nonspecific binding. Then, the membrane was incubated with the primary antibody  at room temperature for 1 h followed by four 10 min washing steps with blotto. Then the blot was incubated for 1 h with the secondary antibody (anti-rabbit IgG HRP conjugate [Promega] diluted 1:2000 in blotto). After four washing steps with blotto, the membrane was washed with TBS before the signals were detected with a LumiImager  using AceGlow  as chemiluminescence substrate.Bli-FLAG under control of a xylose-inducible promoter were introduced into B. subtilis 1A774, which contains a His6-tag fused to the β′ subunit of the RNAP, to form strains TMB1099 (wt Ecf41Bli), TMB1100 (Ecf41Bli 204), and TMB1101 (Ecf41Bli 167). As controls, the same constructs were transformed into B. subtilis W168 resulting in TMB695, TMB746, and TMB666. The RNA pull-down assays were performed as described previously  and cells were lysed by sonication. The cleared lysate was loaded on a column containing 0.5 mL Ni-NTA metal affinity beads. The beads were washed with each 10 column volumes of the above-mentioned phosphate buffer containing 5, 10, and 20 mM imidazole. Elution was carried out using 0.5 mL phosphate buffer with increasing imidazole concentration . Samples of the cleared lysate, washing steps, and elution fractions were run in duplicates on 10% and 12% SDS-PAGE gels and checked for presence of RNAP (coomassie staining) and Ecf41Bli-FLAG (Western Blot using anti-FLAG anti-bodies). For quantitative analysis, 5 μg of lysate as well as 5 and 10 μg of the 100 mM imidazole elution fractions were used and analyzed as mentioned above.Different versions of Ecf41eviously . In brieThe initial analysis of group ECF41  was baseGroup ECF41 shows a wide taxonomic distribution and proteins of this group can be found in 10 different phyla, but predominantly in the Actinobacteria and Proteobacteria . In addiStreptomyces contains large numbers of ECF41 σ factors with at least four copies per genome, which may reflect the complex lifestyle of these bacteria  can be divided into two main groups: the γ-carboxymuconolactone decarboxylases and the AhpD-like alkylhydroperoxidases H or FAD as cofactors. Since oxidoreductases can use a variety of different molecules as electron donor or acceptor, it is difficult to assign a specific function to these enzymes. In case of genes next to ECF41 σ factors, they were classified as oxidoreductase if their product carried at least one of the following Pfam domains: Oxidored_FMN, Flavodoxin_2, Pyr_redox/_2, FAD_binding_2/3/4, Amino_oxidase, Pyridox_oxidase, or FMN_red.Epimerases. The third group contained either an NmrA (PF05368) or an Epimerase (PF01370) domain. NmrA is a negative transcriptional regulator of AreA and involved in nitrogen metabolite repression in different fungi. The crystal structure of NmrA revealed a Rossmann-fold and similarity to members of the short-chain dehydrogenase/reductase family  the COE genes could be targets of ECF41-dependent regulation, (2) the COEs could be part of the signal transducing mechanism and function as an anti-σ factor, or (3) the C-terminal extension could be involved in controlling σ factor activity.B. licheniformis and RSP_0607 of R. sphaeroides. We named the genes encoding the ECF41 σ factors to ecf41Bli and ecf41Rsp and used these terms for the following analysis. The genomic neighborhood including the genes encoding the carboxymuconolactone decarboxylases YdfG and RSP_0606 is shown in To address these hypotheses directly and generalize our findings, we experimentally investigated ECF41 σ factors from two different organisms: BLi04371 of ydfG at different growth phases in a B. licheniformis wild-type and an isogenic Δecf41Bli deletion strain. Both strains show no difference in growth behavior  or the empty vector (YSD354) were grown aerobically to OD600 ∼0.3 and expression of the ECF41 σ factor was induced by adding IPTG. After 3 h of incubation, the cells were harvested, total RNA was prepared, and microarray analysis performed . The mRNA level for ecf41Rsp was ∼80-fold increased in cells overexpressing this protein. The only other more than twofold-induced gene was RSP_0606 (∼threefold), which encodes the associated carboxymuconolactone decarboxylase (data not shown). With this approach, we cannot discriminate between the expression level of the native and the ectopic copy of ecf41Rsp. Nevertheless, we propose that ecf41Rsp and RSP_0606 form a bicistronic operon due to an overlap of the structural genes. Taken together, these results indicate that Ecf41Rsp seems to control expression of only a single transcript that contains the ecf41Rsp and RSP_0606 genes.We also examined the transcriptome upon overexpression of Ecf41ydfG and RSP_0606 are preceded by such a unique sequence motif. We therefore mapped the transcriptional start site by 5′RACE in RNA samples from cells overexpressing the ECF41 σ factor (R. sphaeroides) or samples taken throughout the growth cycle (B. licheniformis)  . In bothiformis) . Upstreaiformis) : a –35 rB. licheniformis, we heterologously expressed Ecf41Bli-FLAG from a xylose-inducible promoter in B. subtilis, an organism lacking an ECF41 σ factor, and measured the activity of the target promoter PydfG trans-criptionally fused to lacZ by β-galactosidase assays  and subsequent determination of β-galactosidase activity did not reveal any Ecf41Bli-dependent activation. Even expression of a highly active truncated version of Ecf41Bli (see below) did not result in any promoter activation (data not shown). In addition, we performed a genome-wide analysis on the presence and conservation of the ECF41-dependent promoter motif upstream of orthologous genes in a wide range of ECF41-harboring organisms. While this in silico analysis has been successfully used to identify candidate promoters and core regulons for other regulators including ECF σ factors  and RSP_0606-ecf41Rsp deletion mutant (YSD331) or Ecf41Rsp1–206 overexpression strain  as well as B. licheniformis wild-type (MW3) and Δecf41Bli (TMBli006) or ΔydfG (TMBli003) deletion strains, respectively. No significant differences were observed in the presence of the oxidative stress generating compounds H2O2, cumene hydroperoxide, t-butyl hydro-peroxide, or paraquat (data not shown).The analysis of the function of ECF41-dependent target genes could provide some hints for the physiological role of this novel group of ECF σ factors. The only detectable target gene of the ECF41 σ factor in both ompounds . But besompounds . Additio to H2O2 . Based ohttp://www.biolog.com/PM_Maps.html for details). We compared phenotypic differences between the R. sphaeroides wild-type (2.4.1) and RSP_0606-ecf41Rsp deletion strain (YSD239) as well as a strain expressing the highly active variant of Ecf41Rsp . Overall, only very few phenotypes can be linked to the expression or deletion of the ECF41 σ factor. Besides resistance to spectinomycin due to the resistance cassette, strain YSD239 showed only a positive phenotype against the sulfonamide anti-biotic sulfadiazine. As expected, strain TMR003 displayed relative resistance to aminoglycoside antibiotics  due to the resistance cassette of the overexpression plasmid. Surprisingly, gained phenotypes were observed for the carbon sources adonitol, d-mannitol, d-sorbitol, and glucose, suggesting a metabolic function of the ECF41 σ factors in utilization of additional carbon sources. But none of these additional phenotypes not due to the presence of a resistance cassette could be verified by serial dilution spot tests (data not shown). Taken together, the mutant and overexpression strains do not show any reproducible growth differences compared to the wild type under all the conditions tested. Therefore, we were not able to identify an ECF41-related phenotype.We subsequently performed a phenotype microarray (PM) analysis. This high-throughput approach allows testing hundreds of different conditions in parallel in order to identify phenotypes associated with genetic alterations . Our PM Bli. Therefore, we expressed Ecf41Bli separately (TMB455) and simultaneously with YdfG (TMB456) in B. subtilis and measured the activity of PydfG fused to lacZ by β-galactosidase assays, but did not observe any significant differences (data not shown). Therefore, we do not have any indication that the corresponding COEs are involved in ECF41-dependent signal transduction and focused our attention on the C-terminal extension. To study a possible function of this extension, we investigated the effect of C-terminal truncations of Ecf41Bli on promoter activation and RNAP interaction.The activity of ECF σ factors is normally regulated by a cognate anti-σ factor. The genes encoding these two proteins are usually located next to each other on the chromosome and cotranscribed . As mentecf41Bli-FLAG and tested their ability to activate the target promoter PydfG. The different mutant proteins were named according to their length  of Ecf41Bli resulted in clearly decreased target promoter activation. Furthermore, expression of variant 167, which lacks the whole extension, completely lost the ability to activate the target promoter  led to ∼120-fold higher PRSP_0606 activity than the full-length protein, whereas deletion of the whole extension (169) resulted in a total loss of promoter activation. Our collective data therefore strongly suggest that the C-terminal extension of group ECF41 proteins might represent a fused regulatory domain that is involved in controlling σ factor activity.To test if similar results can be observed for Ecf41function . ExpressBli interacts with RNAP, we performed in vivo RNAP pull-down assays. Ecf41Bli-FLAG was expressed from a xylose-inducible promoter in a B. subtilis strain carrying a His6-tagged β′-subunit of RNAP. The His6-tag was used for rapid purification of RNAP holoenzyme (Bli-FLAG copurifies with RNAP (6-tag (data not shown) indicating that enrichment of Ecf41Bli-FLAG from B. licheniformis is due to interaction with RNAP.Bacterial σ factors form a complex with the RNAP core enzyme and recruit the resulting holoenzyme to the corresponding target promoters . To demoloenzyme . The sucloenzyme . Westernith RNAP . The samBli truncations , which is consistent with its inability to activate the target promoter  to interact with RNAP, additional factors must account for the strongly increased promoter activation of the partly truncated version (204), despite its weaker interaction with RNAP compared to the wild-type protein.Taken together, C-terminal truncations of Ecf41Taken together, our data demonstrate that the ECF41 σ factors seem to have a completely new way of signal trans-duction presumably not involving a second protein functioning as an anti-σ factor. Instead, the C-terminal extension, which is not present in other ECF σ factors, affects both the target promoter activation and binding to RNAP  and 6. T16-CGTC) upstream of these COE genes that is recognized by the corresponding ECF41 σ factor (A recent classification identified a large number of novel groups of ECF σ factors with unique features compared to “classical” ECF σ factors , includiσ factor .4 is absolutely required for σ factor activity, in contrast to other ECF σ factors described so far. But most of the C-terminal part of the extension clearly plays a negative regulatory role: even partial deletions result in a strongly increased activity of the target promoters in both organisms studied (The most important finding of our study concerns the regulatory role of the C-terminal extension of group ECF41, which is not present in any other group of ECF σ factors . Based o studied , suggestE. coli, σ70. While this σ factor does not need to be activated, binding of free σ70 to DNA is inhibited by region 1.1, presumably by interaction with the σ4 DNA-binding domain (E. coli σ32 and B. subtilis σK (While our data clearly establish a regulatory role of the C-terminal extension for the activity of the ECF41 σ factor, the exact molecular mechanism will be the subject of further investigations. One possibility would be that the C-terminal extension functions as a sensory domain. In the absence of a suitable trigger, it could keep the σ factor domains inactive through intra- or intermolecular interactions. These interactions could prevent the binding of the ECF41 σ factor to RNAP or promoter DNA. The presence of an input signal could then result in a conformational change that releases the σ factor domains from the inhibitory grip of the extension, thereby initiating transcription of the COE genes. Such a mechanism involving intramolecular interactions has for example been described for the primary σ factor of g domain . A similtilis σK . AnotherK and σE, which are involved in the sporulation process in B. subtilis, are known to be expressed as inactive precursors. Activation is achieved by regulated proteolytic processing of the N-terminus of these proteins (Bli is already sufficient to activate the target promoter, showing that the full-length protein is not completely unable to initiate transcription. But the resulting activity can be drastically increased by truncation of the ECF σ factor. Since these expression and promoter activity experiments were carried out heterologously in B. subtilis, which does not possess an ECF41 σ factor, it is unlikely that another protein is involved in the signal transduction.Alternatively, though maybe less likely, a stimulus sensed by the C-terminal regulatory domain could also result in a conformational change that exposes a protease recognition site. After regulated cleavage, the truncated and thereby activated σ factor would then mediate transcription initiation. Alternative σ factors such as σproteins . But in proteins , the N-tFuture studies will be necessary to unravel both the physiological role and the mechanistic details underlying ECF41-dependent signaling. But the data presented in this initial study clearly demonstrate the value of our ECF classification  and can


Thermotogales lineage can produce hydrogen by fermenting a wide range of carbohydrates. Previous experimental studies identified a large fraction of genes committed to carbohydrate degradation and utilization in the model bacterium Thermotoga maritima. Knowledge of these genes enabled comprehensive reconstruction of biochemical pathways comprising the carbohydrate utilization network. However, transcriptional factors (TFs) and regulatory mechanisms driving this network remained largely unknown. Here, we used an integrated approach based on comparative analysis of genomic and transcriptomic data for the reconstruction of the carbohydrate utilization regulatory networks in 11 Thermotogales genomes. We identified DNA-binding motifs and regulons for 19 orthologous TFs in the Thermotogales. The inferred regulatory network in T. maritima contains 181 genes encoding TFs, sugar catabolic enzymes and ABC-family transporters. In contrast to many previously described bacteria, a transcriptional regulation strategy of Thermotoga does not employ global regulatory factors. The reconstructed regulatory network in T. maritima was validated by gene expression profiling on a panel of mono- and disaccharides and by in vitro DNA-binding assays. The observed upregulation of genes involved in catabolism of pectin, trehalose, cellobiose, arabinose, rhamnose, xylose, glucose, galactose, and ribose showed a strong correlation with the UxaR, TreR, BglR, CelR, AraR, RhaR, XylR, GluR, GalR, and RbsR regulons. Ultimately, this study elucidated the transcriptional regulatory network and mechanisms controlling expression of carbohydrate utilization genes in T. maritima. In addition to improving the functional annotations of associated transporters and catabolic enzymes, this research provides novel insights into the evolution of regulatory networks in Thermotogales.Hyperthermophilic bacteria from the  Carbohydrates constitute the most abundant single class of organic substances found in nature. Plant cell walls constitute ~70% of the worldwide biomass production by land plants, but only ~2% of this biomass is currently utilized by humans  involved in carbohydrate metabolism , 18 DNA-binding transcriptional factors, (TFs), and 1 RNA-binding regulator (GlpP).Initially, we used the subsystems-based bioinformatics workflow (see Methods for details) to identify the repertoire of known and putative genes involved in carbohydrate utilization in T. maritima sugar catabolic, transport, and regulatory genes in 10 additional Thermotogales species with complete genomes ; GalR, GlpP, and UgtR (present in 9 genomes), as well as CelR and ChiR (present in 8 genomes). Three regulators  were found only in 6 Thermotoga genomes, whereas four regulators  are conserved in 5 closely-related Thermotoga spp. Finally, GloR, GluR, InoR, and RhaR have orthologs in a small subset of Thermotoga spp.Next, we identified orthologs for the T. maritima. In addition, we inferred a regulon for the predicted fructose regulator FruR, which is present in two Thermotoga species but not in T. maritima. The glycerol regulon operated by the RNA-binding antiterminator protein GlpP was not studied by comparative genomics.To infer novel TF regulons, we applied the comparative genomics–based approach implemented in the RegPredict Web server (see Methods). As a result, we revealed DNA motifs and reconstructed regulons for all 18 DNA-binding TFs associated with sugar catabolism in T. maritima are illustrated in Figure Thermotogales genomes is available in the RegPrecise database , a xylose-specific ABC transporter, a set of hydrolytic enzymes specific for xylan and xylan oligosaccharides, and two xylan-induced ABC transporters. XylR belongs to the ROK  protein family. All six predicted XylR-binding sites have been experimentally validated in vitro in our previous study of ROK-family regulators  is homologous to a repressor of the 2-keto-3-deoxy-gluconate (KDG) metabolism and pectin utilization in enterobacteria  but is predicted to control the uronate isomerase uxaC, encoding a bifunctional enzyme in the catabolism of glucuronate and galacturonate. Additional candidate KdgR-binding sites were identified in the promoter regions of two XylR-regulated gene clusters, xynB/xlo and xynA/xtp, that are induced during the growth of T. maritima on xylan. Glucuronoxylan is a linear polymer of β-linked xylose residues with many of the xylose units substituted with glucuronate residues. The xtp gene cluster encodes α-glucuronidase AguA that can release glucuronate residues from glucuronoxylan. Thus, we propose that the two Thermotoga loci under dual regulation by XylR and KdgR are involved in the xylan and glucuronoxylan utilization.The predicted regulator KdgR in Thermotoga genomes  in T. petrophila, T. naphtophila, and T. neapolitana. A conserved UxaR binding site was identified upstream of the kdgA-kdgK-uxuB-uxuA operon encoding enzymes of the glucuronate utilization pathway. These results suggest that the pectin/galacturonate and glucuronate catabolic genes are co-regulated by the transcription factor UxaR in Thermotoga spp. The UxaR regulon predicted by in silico genomic analysis was experimentally validated in T. maritima .The predicted regulator UxaR (GntR family) was attributed to a conserved 17-nt DNA motif found in multiple copies within the pectin/galacturonate utilization gene loci in five T. maritima  includes two operons encoding a set of mannan and mannosides hydrolytic enzymes and a mannan-induced ABC transporter. Both candidate binding sites of ManR and its mannose effector have been previously experimentally tested in T. maritima. The conserved part of the CelR regulon (present in five Thermotoga genomes) includes glucan and β-glucoside degradation enzymes, cellobiose phosphorylase, and a barley glucan–induced ABC transporter. The latter transporter, termed celEFGKL, is also present and regulated by orthologous CelR regulators in three other Thermotogales genomes  encoded within the predicted xyloglucan utilization gene cluster. The LacI-family transcription factor GloR was predicted to function as a local regulator of the glo locus. The common intergenic region of gloR and gloE genes contains distinct operator sites for CelR and GloR regulators. The BglR regulator (ROK family) controls another β-glucosides utilization operon encoding laminarinase, β-glucosidase, and cellobiose- and laminaribiose-specific ABC transporters in Thermotoga spp. A BglR-binding site was experimentally validated, and it was found that the BglR regulator from T. maritima responds to two effectors, cellobiose and glucose  has an extensive regulon including 22 genes from six genomic loci in s Figure . In T. mT. maritima control the glucose and trehalose utilization pathways  catabolic genes nagAB. Single DNA binding sites of both IolR and ChiR regulators were experimentally confirmed  is encoded within the ribose utilization operon. The conserved part of the RbsR regulon in eight Thermotogales genomes includes a ribose-specific ABC transporter and a ribokinase. In T. maritima, T. neapolitana, and T. lettingae, the ribose operon also includes genes encoding two predicted D-arabinose catabolic enzymes and transketolase.The predicted regulator AraR (GntR family) is homologous to the L-arabinose-responsive repressor from T. martima and three other Thermotoga spp. The reconstructed RhaR regulon includes a rhamnose-induced ABC transporter, several glycoside hydrolases, and a set of rhamnose catabolic enzymes. The predicted galactose regulator GalR (LacI family) controls the galactan/galactoside utilization pathways in nine Thermotogales genomes. In T. maritima, the GalR regulon includes two galactose catabolic enzymes, two β-galactosidases, one galactan hydrolase, and two lactose-inducible ABC transporters. The predicted fructose utilization regulon FruR (DeoR family) was found in only two Thermotoga spp. (T. naphthophila and RQ-2). Finally, three other TF regulons reconstructed in Thermotoga genomes control yet uncharacterized sugar catabolic pathways .The predicted rhamnose regulator RhaR (DeoR family) was identified within the rhamnose utilization gene cluster in T. maritima cells grown on various mono- and disaccharides. First, the ability of T. maritima to grow on 17 different carbon sources was tested in minimal media with a 10-mM concentration of the particular carbohydrate in 500-ml bottles under anoxic conditions at 80°C. Growth phenotype testing revealed that T. maritima is able to grow on the monosaccharides rhamnose, galactose, glucose, fructose, arabinose, xylose, ribose, and mannose and the disaccharides trehalose, cellobiose, and maltose as a sole carbon and energy source . The obtained sugar-specific transcriptomes were compared with ribose and other sugars to calculate the differential expression of all genes from regulons reconstructed in T. maritima  were observed for two xylan utilization gene clusters, xynB<>xloEFGKL-xyl3-axeA and xynA-cenC<>xtpFGKLE-aguE. The xylose catabolic gene cluster xylR-adhB-xylFUEKB demonstrated modest upregulation on xylose (~2- to 6-fold), whereas the xylose isomerase xylA had approximately 10-fold higher transcript levels. The xylan oligosaccharide utilization operon xtpN-bgaL was induced approximately 6-fold. In vitro, the XylR regulator responded to 0.02 mM of xylose and also to >0.2 mM of glucose  and 3- to 11-fold induced by glucose. The obtained gene expression profiles for the BglR regulon are corroborated by the previous in vitro DNA-binding assays with the BglR repressor that was affected by both cellobiose and glucose, although the glucose disaccharide was a more effective inducer , whereas the responses of the celEFGKLR, cel12AB, and TM0312-13 operons to cellobiose were more moderate (5- to 9-fold). In contrast, the putative xyloglucan oligosaccharide utilization gene cluster gloR<>gloEFGKL-cel74-fucAI, which was predicted as a part of the CelR regulon, was uninduced on cellobiose or other sugars tested. The latter fact can be explained by transcriptional repression of the xlo gene locus by another LacI-family predicted regulator, GloR, that has a yet-unknown sugar effector. Based on these results, we concluded that cellobiose is a physiological effector that induces the CelR regulon in T. maritima.Genes from the BglR-controlled treTR operon encoding a putative trehalose utilization enzyme and the trehalose-responsive repressor was highly upregulated in T. maritima cells grown on trehalose  and TM1847 (gluR). This DNA region is present in two other T. maritima MSB8 isolates that have recently been resequenced [genomovars DSM 3109  with probes designed for treE, gluE, gluR, and bglA (used as a negative control)  Figure . All thrT. martima cells grown on arabinose compared to cells grown on ribose. The TM0280-abfA and araA genes demonstrated the highest induction levels (61- to 161-fold), the arabinoside transporter genes araEFG were modestly induced (9- to 16-fold), whereas expression of the arabinose catabolic genes araMDBW was increased only 2- to 6-fold. Large differences in the induction levels between the TM0280-abfA and araMDBW genes can be attributed to the putative transcriptional terminator upstream of araM . Unexpectedly, the ribose operon genes were almost equally highly induced in cells grown on xylose. The ribose operon is predicted to be controlled by the LacI-family regulator RbsR, whose molecular effector is not yet experimentally determined. The unusual induction patterns for ribose utilization genes may be explained by the dual specificity of the RbsR repressor to ribose and xylose. Alternatively, since the xylose utilization feeds the pentose-phosphate pathway, it is possible that ribose 5-phosphate, an intermediate of this pathway, serves as a physiological effector of the RbsR regulon in T. maritima.Transcription of all genes from the ribose utilization operon was highly upregulated in T. maritima includes 14 genes organized in three transcriptional units that are predicted to be regulated by RhaR. Transcription of all three RhaR-regulated operons was highly elevated in T. maritima cells grown on rhamnose compared to cells grown on ribose. The rhamnose catabolic genes from the rhaMADBC operon were induced between 27- and 37-fold, the rhaR-agu4C operon showed ~12-fold induction, whereas the rtpEFGKL-gusB-TM1061 operon genes demonstrated the gradual decrease in their induction level from 60-fold for the first gene to ~5-fold for the latter gene. These expression results suggest that the RhaR regulator in T. maritima responds to the rhamnose monosaccharide.The rhamnose oligosaccharide utilization gene cluster in T. maritima includes 15 genes organized into two operons, both of which were upregulated during growth on galactose. Genes encoding putative transporters for galactose oligosaccharide and galactosides (ganEFG and ltpEFGKL), as well as two β-galactosidases (lacA and ganA), demonstrated the highest levels of induction on galactose compared to ribose (8- to 20-fold). The galactose catabolic genes  and the putative galactose repressor gene galR were modestly induced by galactose (~4-fold). These results are in agreement with the previous microarray expression profiling of T. maritima that have demonstrated induction of all above-mentioned GalR regulon genes in cells grown on lactose but not on maltose .The GntR-type transcriptional regulator UxaR was predicted to bind a conserved DNA motif in regulatory regions of four pectin and galacturonate utilization operons in  Figures . The Uxa Figures . We perfy Figure . The resT. maritima when used as a single carbon source (data not shown). Thus, we assessed gene expression in cells grown on pectin, a galacturonate-containing polysaccharide, which can support the growth of T. maritima  showed more than 17-fold upregulation , arabinose (TM0276-284), and galactoside (TM1190-1204) utilization pathways were significantly upregulated in T. maritima cells grown on pectin. These genes clusters do not belong to the UxaR regulon but were predicted to be regulated by BglR, AraR, and GalR, respectively. Induction of these three regulons by pectin may be explained by the presence of glucose, arabinose, and galactose residues in this polysaccharide.Galacturonate does not support the growth of n Figure . The extEscherichia coli and Bacillus subtilis is nearly comprehensive, the projection of this knowledge to the sequenced genomes of bacteria from distant taxonomic groups is still a challenge. The major difficulties for bioinformatics-based propagation of TF regulons include duplications and losses of TFs and their binding sites, rapid diversification of specificities of TFs toward DNA sites and sugar effectors, non-orthologous replacements of entire regulatory systems, and frequent horizontal transfers. Comparative genomic analysis of specific sugar utilization pathways has led to substantial progress in the identification and reconstruction of their cognate TF regulons in diverse bacterial lineages . We propose that the entire fructose utilization operon was laterally transferred between these two lineages that likely share the same ecological niche.All sugar utilization regulons reconstructed in Thermotogales are also remarkable  is missing only in a single Thermotoga spp., including the inositol and ribose utilization regulons in the RQ-2 strain and the glucose utilization regulon GluR in T. petrophila. In another case, the RhaR regulator was lost in a single strain of Thermotoga sp. RQ-2, however, the rhamnose utilization operons are still retained in the genome. Many other cases of the absence of orthologous regulators for sugar catabolic pathways in the genomes of more distant Thermotogales can be explained by their control by non-orthologous TFs that have yet to be characterized , each controlling between one and seven operons. In contrast to other model bacteria  that employ various transcriptional mechanisms for global catabolite repression of sugar metabolism, our genomic-based analysis did not reveal any global regulons for sugar utilization genes in Thermotoga. This observation correlates with the genome-wide transciptome data for T. maritima obtained in this and previous studies  systems that are essential components of global carbon catabolic repression mechanisms in both Gram-negative and Gram-positive bacteria . For the remaining ~50 T. maritima proteins, the previously reported gene expression profiles revealed their upregulation during the growth on specific poly- or monosaccharides used as a single carbon source     was grown in 500-ml serum bottles containing 200 ml of minimal medium . Hybridization, washing, and scanning were performed according to the manufacturer's instructions. Two biological replicates were utilized for each growth condition. Probe level data were normalized using the Robust Multiarray Analysis algorithm without background correction as implemented in NimbleScan™ 2.4 software (Roche-NimbleGen). Transcriptome data have been deposited in NCBI's Gene Expression Omnibus and are accessible through GEO Series accession number GSE47615 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE47615).T. maritima MSB8: cbpA (Tmari_1862), treE (Tmari_1861), gluE (Tmari_1858), and gluR (Tmari_1855) (Genbank Accession CP004077). Genomic RNA was isolated from cells grown on minimal medium supplied with either trehalose, D-glucose, or D-ribose and collected at the same optical density using the RNeasy minikit . Reverse transcription was performed on 10 μg of total RNA. The reverse transcription mixture (60 μl) contained 10 μg total RNA, 75 μg random primers, 1 × 1st Strand buffer, 10 mM dithiothreitol, 0.5 mM deoxynucleoside triphosphates, 30 U Superase, and 1,500 U Superscript II. The mixture was incubated in a thermocycler  at 25°C for 10 min, 37°C for 1 h, and then 42°C for 1 h. The reaction was followed by incubation at 70°C for 10 min to inactivate the superscript. The RNA was then degraded by adding 20 μl of 1 N NaOH and incubating at 65°C for 30 min. After the incubation, 20 μl of 1 N HCl were added to neutralize the solution. QIAquick PCR purification kits were used to clean up the cDNA synthesis product. Following the purification, the cDNA was quantified and then directly used in quantitative PCR (qPCR) reactions. The 50 μl of qPCR reaction volume contained 25 μl Sybr green tag master mix (Qiagen), 0.2 μM forward primer, 0.2 μM reverse primer, and cDNA as a template. Each qPCR reaction was run in triplicate in the Bio-Rad thermocycler  with the following settings: 95°C for 15 min, 94°C for 15 s, 52°C for 30 s, and 72°C for 30 s; the denaturation, annealing, and extension steps were repeated for 40 cycles. In order to determine the binding affinity of each primer set, a standard curve was calculated for each primer and reaction efficiency obtained from it. Using the standard curve, the relative cDNA quantity was obtained for each gene by normalizing it to the quantity of gap (TM0688) cDNA in the same sample. The gap gene was used as an expression control as it demonstrated no differential expression across all gene expression arrays in the present study. Normalized transcript levels are provided in Figure Individual transcript levels were measured for four genes from the ATCC-derived strain of E. coli BL21 strains containing pMH2T7-derived plasmid that harbor the T. maritima TM0439 (uxaR) gene with the arabinose promoter and the His6 tag were a kind gift from Dr. S. Lesley at the Joint Center for Structural Genomics . Recombinant UxaR protein containing an N-terminal His6 tag was overexpressed in E. coli in 50 ml volume culture and purified using Ni2+-chelating chromatography, as previously described , whereas the complementary oligo was unlabeled. The double-stranded labeled DNA fragments were obtained by annealing the labeled oligonucleotides with unlabeled complementary oligonucleotides at a 1:10 ratio. The labeled 30-bp DNA fragments (1 nM) were incubated with increasing concentrations of the purified UxaR protein (10–500 nM) in a total volume of 100 μl of the binding buffer containing 20 mM Tris-HCl (pH 7.5), 100 mM NaCl, and 0.3 mg/ml BSA at 37°C for 30 min. Poly(dI-dC) was added to the reaction mixture as a non-specific competitor DNA at 1 μg to suppress non-specific binding. The fluorescence-labeled DNA was detected with the FLA-5100 fluorescent image analyzer. A DNA fragment containing the Rex regulator binding site was used as a negative control (Ravcheev et al., The interaction of the purified recombinant UxaR protein with their cognate DNA-binding sites in 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.