E. coli. Based on our ChIP-chip experimental data, forty-three binding sites were identified throughout the genome and the known PhoB binding pattern was updated by identifying the conserved pattern from these sites. From the gene expression microarray data analysis, 287 differentially expressed genes were identified in the presence of PhoB activity. By comparing the results obtained from our ChIP-chip and microarray experiments, we were also able to identify genes that were directly or indirectly affected through PhoB regulation. Nineteen out of these 287 differentially expressed genes were identified as the genes directly regulated by PhoB. Seven of the 19 directly regulated genes (including phoB) are transcriptional regulators. These transcriptional regulators then further pass the signal of phosphate starvation down to the remaining differentially expressed genes. Our results unveiled the genome-wide binding profile of PhoB and the downstream responses under phosphate starvation. We also present the hierarchical structure of the phosphate sensing regulatory network. The data suggest that PhoB plays protective roles in membrane integrity and oxidative stress reduction during phosphate starvation.The phosphate starvation response in bacteria has been studied extensively for the past few decades and the phosphate-limiting signal is known to be mediated via the PhoBR two-component system. However, the global DNA binding profile of the response regulator PhoB and the PhoB downstream responses are currently unclear. In this study, chromatin immunoprecipitation for PhoB was combined with high-density tiling array (ChIP-chip) as well as gene expression microarray to reveal the first global down-stream responses of the responding regulator, PhoB in  Bacillus subtilisCorynebacterium glutamicumEscherichia coliProchlorococcus marinusSinorhizobium melilotiand Vibrio choleraPhosphate participates in many important cellular processes E. coli, phosphate sensing had been reported to be performed by a seven-component apparatus In E. coliE. coli indicate that the expression of around 400 proteins varied in a comparison between excess and limited phosphate conditions To date, thirty-one responding genes composed of nine transcription units are known to be regulated by PhoB, while several other genes lack direct evidence of PhoB binding in E. coli to phosphate starvation through the PhoR/PhoB two-component system. This integrative genome-wide approach allowed us to identify 54 PhoB binding targets and 287 differentially expressed genes in the presence of PhoB activity during phosphate starvation. These results indicate that PhoB directly regulates a group of genes which contain distinct transcriptional regulators and further indirectly influences other genes. A specific group of genes involved in the functions of transportation and metabolism for membrane protection have also been identified.In this study, we combined ChIP-chip and gene expression microarray experiments, for the first time, to present the global responses of E. coli genomic DNA was constructed . These ten genes are likely to be directly regulated by PhoB. Promoter regions containing these target sites were amplified and cloned into the promoterless luciferase expression vector pGL3 to create the promoter::luciferase fusions. These fusion plasmids were transformed into the pression .in vitro. The purity of our PhoB-His fusion protein is shown in yhjC and ydfH were seen as having low-affinity binding in our in vitro experimental conditions. The other six binding targets also showed different affinities for PhoB binding as the shifts occurred at different concentrations of PhoB , meaning that all currently identified targets of PhoB share a significant conserved pattern . The seqetalSurprisingly, nearly half (20/43) of the binding targets were located within the coding regions and this percentage is relatively higher than that of other transcriptional regulators mentioned in the study by Shimada cof, yahA, and yddV  contained the Pho box within their upstream regions. Thus PhoB may pass the phosphate-limiting signal first to the six regulators which they then regulate the other 15 regulators, which in turn affect the remaining 265 differentially expressed genes. Under the hierarchical structures of phosphate signalling passages, feed forward loop (FFL) network motifs play a role in signal sensing and responding mechanisms. Each FFL network motif contains three genes. Two of the three are transcription regulators, one of which regulates the other, and they jointly regulate the third target gene. At least four sets of gene pairs, cusR/cusC, prpR/prpB, feaR/feaB, and yedW/yedX, have the potential to form feed forward loops with PhoB regulation. For example, PhoB regulates cusR and both PhoB and CusR regulators regulate cusC. Thus, the phoB, cusR, and cusC form a FFL network motif. A previous study has demonstrated in silico that these FFLs can enhance the signal transduction processes or delay the response or adjust the sensing mechanisms through transcriptional regulation in vivo investigations. Overall, the data suggest that PhoB specifically regulates a relatively small group of genes, which influence a large group of downstream genes during phosphate starvation.It is worth noting that our gene expression data showed 287 genes affected by PhoB while only 19 out of these 287 genes were considered to be directly regulated PhoB targets . There ayhjC and yegH may be positively regulated by PhoB based on our reporter gene assay. However, they both showed down regulation in the presence of PhoB from our microarray datasets. The opposite results indicate that other regulators may have inhibitory roles in the coding regions to block the up-regulation of PhoB in our experimental condition. In addition, post-transcriptional modifications, such as mRNA degradation or small RNA regulation, may also be other reasons for reductions in PhoB up-regulation. Secondly, only about 35% of PhoB binding targets showed differential expression , Copper/silver efflux system (cusCFBA), multidrug-efflux systems , neutral amino-acid efflux system (eamB) and others , ycfS  and mltD (predicted membrane-bound lytic murein transglycosylase) were observed to be activated in transcriptional expression. The mipA gene also has a PhoB binding signal in its upstream region and is considered as a directly regulated target.Murein or peptidoglycan can help E. colipagP. Palmitoylated lipid A may also be synthesized during phosphate starvation since PagP transfers palmitate from phospholipid to lipid A precursor to generate palmitoylated lipid A, which protects bacteria from host defences and is likely related to bacterial virulence For the modification of lipid A, the hexa-acyl pyrophosphate Lipid A is known to be modulated through the Pho regulon in wzxC (colanic acid exporter), wcaJ (predicted UDP-glucose lipid carrier transferase), wcaK (predicted pyruvyl transferase), wcaL (predicted glycosyl transferase) and wcaM (predicted colanic acid biosynthesis protein) are involved in the colanic acid biosynthesis and transportation pathway and were observed to be up-regulated in the wild-type strain relative to the PhoB knock-out strain.Additionally, colanic acid is an extracellular polysaccharide and has been shown to increase tolerance to heat and acid conditions puuCBE , puuP (putrescine importer), puuA (gamma-Glu-putrescine synthase), and puuD (gamma-Glu-GABA hydrolase). In our study, all of these genes showed down-regulation and their transcription repressor, PuuR, in turn was up-regulated in the presence of PhoB activity. The repressed putrescine degradation pathway indicates that, during phosphate starvation, membrane stabilization is more important than growth since E. coli cells enter the stationary phase.As for the linear polyamine, putrescine, its role is related to membrane stabilization and optimal growth. However, a high concentration of polyamines will inhibit cell growth and protein synthesis. Therefore, the polyamine degradation pathway exists in bacteria for balancing the concentration ahpCF was identified to be up-regulated indirectly by PhoB. This suggests that PhoB plays a protective role for the oxidative stress which occurs during phosphate starvation.Previous studies described that although cells stop growing, bacteria will still undergo aerobic respiration during phosphate starvation yeaE gene encoding the methylglyoxal reductase was up-regulated in the wild-type E. coli strain compared to the PhoB knock-out strain. This is another indication that PhoB has a protective role for oxidative stress produced during phosphate starvation.It is known that methylglyoxal is synthesized to enhance the phosphate turnover during phosphate starvation We have presented that during phosphate starvation, PhoB is involved in triggering the membrane component rearrangement for membrane integrity. In addition, PhoB also indirectly affects genes participating in protecting cells from oxidative stress and genes that balance the level of methylglyoxal. These results together suggest that PhoB protects the bacterium by enhancing membrane integrity and reducing oxidative damage to the cell membranes. We have identified several predicted transcription factors that are regulated by PhoB. Further studies of these predicted transcription factors are needed in order to understand the complex interplay between genes and regulators in the bacterial signalling and regulatory networks during phosphate starvation.In summary, our genome-wide approach for characterizing the roles of PhoB by ChIP-chip and gene expression array provides a comprehensive global binding profile of PhoB. We have presented a hierarchical structure of transcriptional regulators of the phosphate-sensing network as well as the potential membrane protective roles of PhoB.phoB knock-out derivative from the BW25113 strain was requested from Keio collection phoB disruption was then transferred into MG1655 strain by P1 transduction phoB gene was constructed from the BW25113 strain using an epitope tagging approach Bacterial strains used in this study are shown in 2HPO4 and 0.4% glucose. 600nm of 1.0 was selected since phosphate was used up and PhoB had a higher activity for ChIP-chip assay. For the gene expression microarray experiments, MG1655 and MG1655_PhoB_KO were compared under the same conditions as the ChIP-chip assay. To compare the promoter activity of the upstream regions, promoter::luciferase gene fusion plasmids were constructed, and luminescence was measured for both MG1655 and MG1655_PhoB_KO strains at the same time point as the two above experiments  minimal medium with 200 μM Keriments . The ChI2HPO4 and 0.4% glucose, cultures were diluted in 1∶100 ratio in MOPS minimal medium containing 200/1000 μM K2HPO4 and 0.4% glucose and grew at 37°C. At each time point, cultures were collected, centrifuged at 12,000 g for 5 min, and then 1 ml supernatants were added to 160 μl reaction solution ). After 10 min incubation of supernatants with the reaction solution, the light absorbance at 880 nm was measured. By interpolation of the standard curve, the phosphate concentration was determined.To determine the concentration of orthophosphate, an ascorbic acid method described previously was applied in biological triplicates with slight modifications etal. 600 value of 1.0 and treated with 1% formaldehyde for 10 min. To quench the reaction, glycine was added at the final concentration of 0.125 M for 5 min. Cells were centrifuged at 12,000 g at 4°C for 20 min and washed two times with the washing buffer (10 mM Tris-HCl (pH 7.4), 0.1 M NaCl, 1 mM EDTA and 0.5% Tween-20). The washed cells were then lysed with the lysis buffer (10 mM Tris-HCl (pH 7.4), 0.1 M NaCl, 1 mM EDTA and 0.5% Tween-20, 8 kU/ml lysozyme, 1 mM PMSF, and protease inhibitor cocktail (Sigma)) for 30 min at 4°C. The lysates were sonicated (Bioruptor) to result in DNA fragments ranging from 100 bps to 1000 bps with the average size of 500 bps. After sonication, the lysates were centrifuged at 12,000 g for 20 min at 4°C and the resulting supernatants were used for immunoprecipitation.To identify the genome-wide DNA-binding profile of PhoB, ChIP assays were performed on MG1655_PhoB_FLAG and MG1655. The ChIP assay protocol was modified from Byung-Kwan Cho To eliminate the non-specific bindings between the magnetic beads coated with Protein G (Invitrogen) and the anti-FLAG antibody, the magnetic beads were pre-incubated with 0.05 mg/ml anti-FLAG antibody (Sigma). Similarly, for the purpose of eliminating the non-specific bindings between our lysates and the beads, lysates were also pre-cleared by incubating them with the beads without the anti-FLAG antibody. To immunoprecipitate the PhoB-FLAG-DNA complex, beads pre-incubated with the antibody were added in both lysates from MG1655_PhoB_FLAG and MG1655 strains at 4°C overnight. The beads were washed once with IP buffer (10 mM Tris-HCl (pH 7.4), 0.1 M NaCl, 1 mM EDTA, and 0.05% [v/v] Tween-20 and 1 mM fresh PMSF), twice with ChIP wash buffer I (10 mM Tris HCl (pH 7.4), 300 mM NaCl, 1 mM EDTA, 0.1% Tween-20 and 1 mM fresh PMSF), three times with ChIP wash buffer II (10 mM Tris-HCl (pH 7.4), 500 mM NaCl, 1 mM EDTA, 0.1% [v/v] Tween-20 and 1 mM fresh PMSF), once with ChIP wash buffer III (10 mM Tris-HCl (pH 7.4), 250 mM LiCl, 1 mM EDTA, 0.1% [v/v] Tween-20 and 1 mM fresh PMSF) and once with TE buffer (10 mM Tris-HCl (pH 7.4) and 1 mM EDTA). After removing the TE buffer, beads were incubated twice with elution buffer (50 mM Tris-HCl (pH 7.4), 10 mM EDTA and 1% SDS) at 65°C for 15 min and the two resulting eluted solutions were combined.After incubating the combined eluted samples with proteinase K (Sigma) to the final concentration of 10.5 U/ml at 42°C for 2 hours, the reverse cross-link procedure was performed by incubating at 65°C overnight to unlink the covalent bonds formed by formaldehyde between peptides and DNA. Samples were then treated with RNase A (Sigma) to the final concentration of 26 μg/ml, followed by purifying DNA from the RNase A-treated samples using the PCR purification kit (Qiagen).E. coli K12 MG1655 (Cat. No. 05542901001) was used for our ChIP-chip assay. The instructions of the NimbleGen's protocol (version 2.0) were followed for all procedures. Immunoprecipitated samples were amplified by whole genome amplification kit (Sigma) twice and pooled together. The amplified samples from MG1655_PhoB_FLAG were labelled with Cy5 dye while the control samples from MG1655 were labelled with Cy3 dye. After the hybridization step, the arrays were washed and then scanned with an Axon scanner (GenePix 4000B).The NimbleGen 385 K high density tiling array for 2-ratios (Cy5/Cy3). The enriched regions were then identified by the CMARRT package The scanned TIF image files were then processed by NimbleScan software to generate the intensity pair files. The R package Ringo E. coli K12 MG1655 sequences of all the enriched regions were extracted from NCBI RefSeq (accession no. NC_000913). We used the MEME program E. coli K12 MG1655 reference sequence (accomplished by using the –bfile <background model file> option in MEME). In addition, sites on both strands were allowed (accomplished by using the –revcomp option). The sequence logo To find the position weight matrix (PWM) of PhoB binding sites, E. coli Genome 2.0 array was used to investigate gene expression status in the presence and the absence of PhoB activity. The E. coli K12 MG1655 and MG1655_PhoB_KO strains grew in MOPS minimal medium containing 200 μM K2HPO4 and 0.4% glucose. At an OD600 of 1.0, cultures were treated with 10 mg/ml lysozyme and 10% SDS at 4°C for 5 min to lyse bacterial cells. Then, the protocol for total RNA purification using TRIZOL reagent (Sigma) was followed. The Affymetrix standard protocol was then applied for cDNA synthesis, fragmentation, biotin labelling and hybridization. The raw CEL files were normalized by a robust multi-array average approach The Affymetrix 2HPO4 and 0.4% glucose at the same condition as the experiments for ChIP-chip assay and gene expression microarray (The promoter regions of the PhoB targets identified in ChIP-chip experiments were amplified by PCR from the MG1655 strain using the primers listed in croarray . The luc(6x) fusion protein was transformed into BL21. Overnight cultures were diluted 1∶500 into 250 mL LB cultures containing 100 μg/μl ampicillin. The cultures were grown at 37°C until an OD600 of 0.4∼0.6, then treated with 1 mM IPTG to induce PhoB-His(6x) expression and then grown at 37°C for another 2 hours. After centrifugation, the pellets were resuspended in 10 ml lysis buffer . The cells were lysed for 30 min at 4°C and then the lysates were cleared by centrifugation at 14000 g for 30 min at 4°C. After applying the lysate to the Ni-sepharose column , the column was washed two times by 4 ml wash buffer . The elution was performed by applying 1 ml elution buffer  to the column four times. The eluted samples were dialyzed in the storage buffer ). The concentration of PhoB-His fusion protein was determined by the Bradford assay (Bio-Rad) using the bovine serum albumin (BSA) as the standard.In order to overexpress the PhoB-His fusion protein, the PhoB coding region was cloned into a pET21d (+) plasmid. This constructed vector expressing the PhoB-His2, 0.1 mM DTT, and 20 mM acetylphosphate) at 37°C for 75 min 2, 100 μg/ml BSA, and 0.5 μg/ml poly dI-dC with various amounts of PhoB-His(6x) fusion protein . The y-axis in this figure shows the relative light unit (RLU). The phoB-deprived strain, MG1655_PhoB_KO, shows the basal level activity of phoB promoter without PhoB. The wild type MG1655 strain represents the activity of phoB promoter under wild type PhoB positive regulation. This figure displays that our PhoB-FLAG fusion protein in the MG1655_PhoB_FLAG strain has the same activity as the wild type PhoB protein.B_KO see . The tra(TIF)Click here for additional data file.Figure S2The genome-wide profile of PhoB binding regions across the E. coli genome. (A) An overview of the results from ChIP-chip experiments at an OD600 value of 1.0. The log2 fold change (y-axis) is the log2 ratio (grey line) of the normalized Cy5 signal (MG1655_PhoB_FLAG) divided by the normalized Cy3 signal (MG1655) after averaging our triplicate results. These ratios were plotted against their locations on the 4.64 Mb E. coli chromosome (x-axis). (B) Expansion of PhoB binding peaks on the previously known regulatory sites. The detected peaks were centered with 5000 bps flanking regions and these peaks were located in the promoter regions of (i) phoE, (ii) phoB, (iii) ugpB, (iv) pstS, (v) phoA, and (vi) phnC. The log2-ratios (grey line) and the smoothed ratios (black line) on y-axis were calculated from the normalized Cy5 signal (MG1655_PhoB_FLAG) divided by the normalized Cy3 signal (MG1655) after averaging the triplicate results. (C) The most significant pattern was found in the 43 PhoB ChIP-chip peaks. The DNA sequences from all 43 PhoB ChIP-chip peaks (see http://regulondb.ccg.unam.mx/MatrixAlignment/results/). The first five bases of the known pattern was trimmed to produce an 18 bp pattern that can be compared with our pattern. Panels C and D in this figure show high similarity between these two patterns.eaks see  were com(TIF)Click here for additional data file.Figure S3Purity data for purification of PhoB-His fusion protein. This figure displays the (A) SDS-PAGE and (B) the western blot results to demonstrate the purity of our purified PhoB-His fusion protein. The lanes from left to right are Marker (M), cell lysate (CL), flow-through (FT), the first washed fraction (W1), the second washed fraction (W2), the first eluted fraction (E1), the second eluted fraction (E2), the third eluted fraction (E3), the forth eluted fraction (E4), and the pooled and enriched fraction (P). The expected size of PhoB-His fusion protein is 27.9 kDa.(TIF)Click here for additional data file.Figure S4Novel PhoB bindings located in the coding regions may participate in gene regulation. The three genes, yahA (i), cof (ii), and yddV (iii), were shown to be differentially expressed in our gene expression microarray analysis. From our ChIP-chip analysis, significant PhoB binding peaks were detected and the putative PhoB binding motifs were also identified within the coding regions (panel A). For further investigation of PhoB bindings, the in vitro binding assays were carried out by gel mobility shift assay Click here for additional data file.Figure S5A non-preferred binding region of PhoB. In addition to PhoB binding regions, there is a long region in which the PhoB binding signals are lower than background noises. This region ranges from the genomic location of 1292000 to 1306000 bps. The y-axis represents the log2 ratio (grey line) and the smoothed ratio (black line) of the normalized Cy5 signal (MG1655_PhoB_FLAG) divided by the normalized Cy3 signal (MG1655) after averaging our triplicate results. In order to show the boundary of this non-preferred binding region, the genomic region from 1291000 to 1207000 bps is plotted. Genes located in this region are shown at the bottom of the plot. The pseudogene, insZ, located between tdk and adhE is not shown. All genes, tdk, insZ, adhE, ychE, oppABCDF and yciU were up-regulated with PhoB activity.(TIF)Click here for additional data file.Table S1List of differentially expressed genes identified from the microarray analysis.(DOCX)Click here for additional data file.Table S2List of plasmids used in this study.(DOC)Click here for additional data file.Table S3List of oligonucleotides used in this study.(DOC)Click here for additional data file.


CUX1, inverselycorrelates with patient survival in breast cancers. Cell-based assays andmolecular studies have revealed that transcriptional regulation byCUX1 involves mostly the proteolytically processed p110isoform. As there is no antibody specific to p110 CUX1 only, an alternatestrategy must be employed to identify its targets.Overexpression of the Cut homeobox 1 gene, in vivo. Thepresence of a consensus binding motif did not have an impact on whether atarget gene was repressed or activated. Interestingly, the distance betweena binding site and a transcription start site did not significantly reducedthe ability of CUX1 to regulate a target gene. Moreover, CUX1 not only wasable to regulate the next adjacent gene, but also regulated the gene locatedbeyond this one as well as the gene located further away in the oppositedirection.We expressed physiological levels of a tagged-p110 CUX1 protein and performedchromatin affinity purification followed by hybridization on ENCODE andpromoter arrays. Targets were validated by chromatin immunoprecipitation andtranscriptional regulation by CUX1 was analyzed in expression profiling andRT-qPCR assays following CUX1 knockdown or p110 CUX1 overexpression.Approximately 47% and 14% of CUX1 binding sites were respectively mappedless than 4 Kbp, or more than 40 Kbp, away from a transcription start site.More genes exhibited changes in expression following CUX1 knockdown thanp110 CUX1 overexpression. CUX1 directly activated or repressed 7.4% and 8.4%of putative targets identified on the ENCODE and promoter arraysrespectively. This proportion increased to 11.2% for targets with 2 bindingsites or more. Transcriptional repression was observed in a slightly higherproportion of target genes. The CUX1 consensus binding motif, ATCRAT, wasfound at 47.2% of the CUX1 binding sites, yet only 8.3% of the CUX1consensus motifs present on the array were bound Our results demonstrate that p110 CUX1 can activate or repress transcriptionwhen bound at a distance and can regulate more than one gene on certaingenomic loci. While these assays are stillemployed, it is clear that they cannot provide definitive evidence that atranscription factor regulates a specific gene. Additional evidence must alsoinclude chromatin immunoprecipitation assays to demonstrate "in vivo" DNA binding,and change in expression of the endogenous gene target in response to the knockdownand/or overexpression of the transcription factor.The last decade has seen significant advances in the field of transcription. Thediscovery of nuclear histone acetyltransferases (HATs) in the mid nineties hasliterally opened a new field of investigation into post-translational modificationsthat target histones and modulate the chromatin state either locally or over largegenomic loci. The multiple types of modifications that take place on specifichistone residues and the regulatory cascades that can be triggered in this mannerled investigators to propose that a "histone code" regulates gene expression in amanner reminiscent of the genetic code translating nucleic acid coding sequencesinto protein sequences ,2. In pacription. We now cription-16. AnotCUX1) has previously been called CCAAT-displacement protein(CDP), CDP/Cut and Cut-like 1 (CUTL1). CUX1 encodes two mainisoforms that exhibit different DNA binding and transcriptional properties (reviewedin [Cut homeobox 1 (iewedin ). The fuiewedin . In mid-iewedin ,20. Thisiewedin ,22. Theiewedin ,20,23-27iewedin . Itstraiewedin .Cux1 in mice causes perinatal lethality in alarge proportion of animals due to delayed lung development and associatedrespiratory failure [Homozygous inactivation of  failure . Survivi failure -30. In t failure -36.Cell failure ,37,stre failure , cell mi failure ,39-41, r failure , and den failure . Which CMany specific transcription factors are able bind to genomic sites that are far awayfrom TSS. These studies also revealed that only about up to 10% of putativetranscriptional targets showed evidence of regulation in response to changes intranscription factor concentrations -46. Whet2  using a population of Hs578tcells stably expressing moderate levels of a p110 CUX1 protein with two epitopetags at its C-terminus, p110-TagUsing a stringent false discovery rate (FDR = 0.05), 513 CUX1 bindingsites were identified on the ENCODE array Table .Mapping of CUX1 binding sites relative to transcription start sites (TSS)generated a bell-shaped curve of low height around TSS Figure A. 14.2% We compared the distribution of CUX1 binding sites with those of 3 randomlygenerated sets of binding sites, as well as those of c-Myc and E2F1 using thedata of Bieda et al., 2006  as well as purifiedchromatin from Hs578t cells expressing p110 CUX1-Tag2 were used in hybridizationon the promoter array of NimbleGen. Using a stringent false discovery rate(FDR = 0.05), 5828 CUX1 binding sites were identified on 4706 genepromoters , was foundto be present at 47.2% of the 5828 bound genomic sites , following changes in CUX1 levelsTable . 20 geneSimilar results were obtained when we analyzed the expression of putative targetsidentified on the promoter array. A total of 347 genes, 8.4% of all putativetargets for which expression profiling results could be matched, were regulatedby CUX1. 287 and 85 genes exhibited regulation in response to CUX1 knockdown orp110 CUX1 overexpression, respectively. 181 (52%) were up-regulated by CUX1while 167 (48%) were down-regulated by CUX1.We noted that CUX1 regulated 7.4% and 8.4% of putative targets from the ENCODEand the promoter arrays, respectively. We next investigated the relationshipbetween the position of a CUX1 binding site relative to a transcription startsite and the probability of a gene to be regulated in response to changes inCUX1 levels. When genes were classified according to the distance between theCUX1 binding site and the transcription start site, we did not observesignificant difference in the fraction of targets that were regulated by CUX1Figure . Again, The presence of multiple CUX1 binding sites has a modest, yet significant, impacton the probability that a gene is regulated by CUX1. CUX1 regulated 7.9%, 11.2%of genes that contain respectively one or two CUX1 binding sites, respectivelyTable .Intuitively, one would assume that a transcription factor is more likely toregulate the closest promoter. Yet, some enhancers will exhibit an effect on apromoter situated on one side, but no effect on the promoter that is on theother side on the map. This sort of selectivity between an enhancer and apromoter has been explained by the presence of boundary or insulator elements orby specific interactions between proteins bound at the enhancer and theregulated promoter. Previous studies on CUX1 have all focused on genes thatcontain a CUX1 binding site within the immediate promoter. To begin toinvestigate the rules that govern the action of CUX1, we calculated the fractionof different types of CUX1 targets that were regulated in response to changes inCUX1 levels. Three types of genes were analyzed: 1, genes that are the closestto the CUX1 binding site; 2, genes that are further away and in the otherdirection from the CUX1 binding site; 3, genes that are located further away andare separated by another gene from the CUX1 binding site. For each category, wecalculated the percentage of genes that exhibit a 1.25 or 1.5-fold change inexpression following p110 CUX1 overexpression or CUX1 knockdown. Strikingly,essentially similar fractions of genes were regulated whether they were closestto the CUX1 binding site or were located further away in the other direction, was found tobe present at 47.2% of the 5828 bound genomic sites Table  and 10. CUX1 can be purified efficiently by immunoprecipitation or affinity chromatography.Following cross-linking, however, the yield of purification is drastically reducedsuch that we need 500 million cells to perform chromatin immunoprecipitation oraffinity purification (ChIP or ChAP) for CUX1. This caveat has limited our abilityto perform ChIP-sequencing and therefore our study relied on microarrayhybridizations. While sequence coverage is admittedly smaller on microarrays, datacollected from both ENCODE and promoter arrays have enabled us to define theimportance of the CUX1 consensus binding site in the recruitment of CUX1 to genomiclocations and determine whether CUX1 can regulate genes at a distance.Our results demonstrate that p110 CUX1 can mediate transcriptional repression oractivation of specific genes when bound at variable distances from the transcriptionstart site. Although the CUX1 consensus binding motif, ATCRAT, plays a role in therecruitment of CUX1 to specific genomic sites, protein-protein interactions mustcontribute to its transcriptional activity.Hs578T is a human mammary carcinoma cell line . Previou2 (CUX1 a.a. 612–1336with protein A and CBP tags inserted at the C-terminus) (Clontech). Preparationof the retroviruses and stable cell lines was done as previously described[Retroviruses were produced by transfecting 293VSV cells with the pREV/TRE vectoreither empty or encoding p110 CUX1-Tagescribed.2 protein would be expressed at moderate level,we employed the pRevTRE retroviral vector (Clontech), which contains the minimalCMV promoter with a tetracycline responsive element. Importantly, notetracycline was added to the medium. Moreover, the Hs578T breast tumor cells donot express a tetracycline-responsive transactivator. Basal expression from thepRevTRE vector was previously shown to be very low [8 Hs578T. The cell nuclei were purified asdescribed in [2 protein was purified by theTaptag purification method with some modifications [2/DNA werewashed in wash buffer I , wash buffer II, wash buffer III  andthen TEV buffer (10 mM Tris–HCl (pH8.0), 100mMNaCl, 0.1% TX-100,0.5 mM EDTA, 10% glycerol, 1 mM DTT). After TEV protease digestion,the released protein/DNA complexes were purified by affinity chromatography oncalmodulin beads in the presence of calcium and then eluted with EGTA. Afterde-crosslinking, samples were treated with RNase A and Proteinase K. Un-enrichedinput chromatin was put aside as a control.The method of chromatin affinity purification (ChAP) has previously beenvalidated , and desvery low -63.ChAPribed in , then lyications . The IgGChAP purified chromatin was amplified by the method of Ligation-Mediated PCR asdetailed previously . BrieflyLabeled samples were hybridized to either NimbleGen's HG17 ENCODE or their HG18Human Promoter Array Set high density oligonucleotide tiling array (385 kprobe format) and then washed according to the manufacturer's instructions.Arrays were scanned on an Agilent 5 μm scanner model G2505B usingcustomized scan area settings .http://www.R-project.org) or scripts written in PERL . All peaksidentified in ChAP-Chip experiments on the Encode Array and the Human PromoterArray Set are provided , grid alignment, raw signalextraction, peak identification and peak mapping were carried out using theNimblescan v8.0 software according to the company's instructions. Identifiedpeaks were considered significant with a false discovery rate (FDR) below 0.05,which is considered highly confident. Further analysis of identified bindingsites was carried our using either the R platform for statistical computing in the ENCODE regions classified by 4 criteria: L1(P < 0.0001 and 98th percentile), L2(P < 0.0001 and 95th percentile), L3(P < 0.05 and 98th percentile) and L4(P < 0.05 and 95th percentile) [We used ChIP-chip binding sites for E2F1 and c-Myc downloaded from the website ofDr. Peggy Farnham laboratory at<centile) . Based ocentile) -69 was uhttp://genome.ucsc.edu/ENCODE/). Genomic locations were comparedto those of the CUX1 binding sites using scripts written in R. UCSC Accessionnumbers of the tracks used are: wgEncodeEH000503 (GEO accessions GSM736552 andGSM736634) for the DHS data and wgEncodeEH000786 for the ChromHMM data.Data tracks were downloaded from the UCSC's Encode data portal(Genomic sequences corresponding to regions of interest (binding sites or other)were obtained using the online GALAXY platform. Scripts written in R were usedto identify the ATCRAT consensus motif within regions of interest.De Novo motif discovery was performed using the DREME (Discriminative DNA MotifDiscovery) motif discovery tools form the MEME suite of tools. Comparison withknown DNA binding motifs was performed using the TOMTOM algorithm using theJASPAR CORE database as a reference for comparison. (meme.nbcr.net/)-72.Identification of overrepresented gene functions was carried out using the onlineannotation tool DAVID. Genes that were bound by CUX1 (Targets) were comparedwith all genes present on the microarray (Background). Overrepresentation of afunction depends on the increase in the proportion of genes involved in a givenfunction between CUX1 targets and the background. The P-value is determinedusing an improved Fisher’s exact test from the DAVID software,74.For overexpression, Hs578t cells were infected with a lentiviral vectorexpressing p110 CUX1. Duplicate infections were carried out in parallel andcells were harvested after 24 hours. For CUX1 knockdown, a stable Hs578tcell line containing a doxycycline inducible shRNA was established by retroviralinfection. Doxycycline was applied to the cells for 6 days before harvestwith control cells left untreated. Knockdown experiments were carried out inbiological duplicates in parallel.Total RNA was isolated from cells using the Arcturus Picopure RNA isolation kit.2 μg of RNA was then amplified using the Arcturus RiboAmp PLUS RNAamplification kit according to the manufacturer's instructions for a singleround of amplification. Amplified mRNA (aRNA) was labeled using the ArcturusTurbo Labelling Cy5 and Cy3 kits using the manufacturer's instructions with amodification: The labeling reaction was carried out using 5 μg of aRNAin a 20 μl volume instead of 50 μl to increase the dyeincorporation rate. Labelled aRNA was hybridized to Agilent's Whole Human GenomeMicroarry (G4112F) according to the manufacturer's instructions, washed andscanned on a 5 μm Agilent scanner. Hybridizations of the biologicalduplicates of each experiment were carried out in technical duplicates using dyeswaps (Cy3 and Cy5), for a total of 4 replicates for each of the overexpressionand downregulation experiment.Raw signal and background intensities were extracted from the scanned images ofexpression arrays using the Feature Extraction software from Agilent. Raw datawas processed and normalized using the R platform and the LIMMA package. ProcessIndependent p110 CUX1 overexpression and CUX1 shRNA knockdown experiments werecarried out in Hs578t cells using retroviral vectors. 10 genes were selectedfrom each experiment and real-time PCR was used to confirm the changes inexpression seen in expression profiling.ChAP: Chromatin Affinity Purification; ChIP: Chromatin Immuno Precipitation; DHS:DNAse hypersensitivity site; ENCODE: ENCyclopedia of DNA Elements; FDR: Falsediscovery rate; TAP: Tandem affinity purification; TSS: Transcription startsite.The authors declare that they have no competing interests.AN, CV and AA conceived the study. CV, AA, RH, PLC, LL, and GB performed theexperiments. CV and AA performed data analysis. AN and CV wrote the manuscript. Allauthors read and approved the final manuscript.Distribution of 3 random sets of binding sites relative to transcriptionstart sites.Click here for fileDistribution of binding sites relative to transcription start sites for 6transcription factors.Click here for fileCUX1 Binding site peaks from ENCODE array ChAP.Click here for fileCUX1 Binding site peaks from Promoter array ChAP.Click here for fileExpression profiling results following overexpression and knockdownof CUX1.Click here for file


Bacillus subtilis. Among them, the transcriptionally regulated promoters involved in carbohydrate utilization are a research priority. Expression systems based on Bacillus promoters for xylose, maltose, and mannose utilization, as well as on the heterologous E. coli lactose promoter, have been successfully constructed. The promoter of the mtlAFD operon for utilization of mannitol is another promising candidate for its use in expression vectors. In this study, we investigated the regulation of the mtl genes in order to identify the elements needed to construct a strong mannitol inducible expression system in B. subtilis.Several vector systems have been developed to express any gene desired to be studied in mtlAFD operon (mtlAP) and mtlR (mtlRP) encoding the activator were investigated by fusion to lacZ. Identification of the mtlA Pand mtlR Ptranscription start sites revealed the σA like promoter structures. Also, the operator of mtlA Pwas determined by shortening, nucleotide exchange, and alignment of mtlA Pand mtlR Poperator regions. Deletion of the mannitol-specific PTS genes (mtlAF) resulted in mtlA Pconstitutive expression demonstrating the inhibitory effect of EIICBMtl and EIIAMtl on MtlR in the absence of mannitol. Disruption of mtlD made the cells sensitive to mannitol and glucitol. Both mtlA Pand mtlR Pwere influenced by carbon catabolite repression (CCR). However, a CcpA deficient mutant showed only a slight reduction in mtlR Pcatabolite repression. Similarly, using groE Pas a constitutive promoter, putative cre sites of mtlA Pand mtlR Pslightly reduced the promoter activity in the presence of glucose. In contrast, glucose repression of mtlA Pand mtlR Pwas completely abolished in a ΔptsG mutant and significantly reduced in a MtlR (H342D) mutant.Regulation of the promoters of mtl operon promoter (mtlAP) is a strong promoter that reached a maximum of 13,000 Miller units with lacZ as a reporter on low copy plasmids. It is tightly regulated by just one copy of the mtlR gene on the chromosome and subject to CCR. CCR can be switched off by mutations in MtlR and the glucose transporter. These properties and the low costs of the inducers, i.e. mannitol and glucitol, make the promoter ideal for designing regulated expression systems.The  Bacillus subtilis has been vastly investigated not only as the model of Gram-positive bacterium, but also as an expression platform for homologous and heterologous gene expression. Efficient secretion of enzymes such as proteases, lipases, and amylases along with its safe use and completely sequenced genome is promising for industrial applications. However, several bottlenecks exist that limit the expression potential, e.g. low level of heterologous protein production, instability of the plasmids, inclusion body formation, and production of extracellular proteases . Th. ThPmtlAe Figure . Accordid Figure . Also inmtlA Pand mtlR Pon the plasmids pKAM12 and pKAM18 in B. subtilis 3NA was investigated by cultivating the strains in LB liquid medium, as well as in Spizizen salts medium, with the addition of either mannitol, glucose, the non-PTS sugars glucitol and xylose, alone or in combination of two sugars . Therefore, further measurements were performed in LB medium due to a higher activity of the promoter and a shorter lag phase of growth. As shown in Table mtlA Pactivity dropped at least twofold and mtlR Pactivity about threefold. This means that both promoters underlie catabolite repression.Induction and catabolite repression of s Figure . After omtlA, mtlF, mtlD, and mtlR, were replaced or disrupted by an erythromycin resistance gene in order to investigate their probable influence on mtlA Pactivity, as well as on cell growth, with mannitol or glucitol as carbon source. The mutants, i.e. KM12 (ΔmtlAF::ermC), KM13 (ΔmtlAFD::ermC), KM15 (ΔmtlR::ermC) and KM37 (mtlD::ermC) were used to inoculate minimal medium with 1% mannitol to an optical density of 0.01 (OD600) and incubated for 16 h. Very weak growth of bacteria to an OD600 of maximum 0.2 was detected with KM13, KM12, and KM15 strains; however, KM37 strictly showed no growth , mannitol or glucitol was monitored. As depicted in Figure mtl operon, the complete gut operon, namely gutRBPydjE, was replaced by the chloramphenicol resistance gene in order to investigate the probable interaction of the two degradation pathways. The gut genes were deleted in B. subtilis 3NA as well as in KM13 (ΔmtlAFD::ermC). The latter strain was used as the control in which no growth should be observed for both sugars. Growth of KM39 (ΔgutRBPydjE::cat) in minimal medium containing either 1% glucitol or 1% mannitol as the sole carbon source was strongly reduced in the case of glucitol, whereas for mannitol it was not affected. In the double mutant KM40 growth on either glucitol or mannitol was strongly reduced as expected , but also enhanced the glucose repression from 2.2-fold in the wild type to 5.6-fold in the mutant. As expected, addition of mannitol or glucitol inhibited the growth during the 1 h of induction. However, the cells grew normally when glucose was added (data not shown). In accordance with pKAM12, similar results were obtained with pKAM18 in KM12 and KM37 mutants . Finally, deletion of the mtlR encoding the activator of the operon drastically decreased the activity of the mtlA Pto about 95 M.U.  was transformed by pKAM18 harboring mtlRP. In this mutant the promoter was no longer inducible. This confirms the assumption that mtlR Pis an autoregulatory promoter. The maximum activity was reduced to 16 M.U., which was approximately the basal activity of mtlR Pin the wild type strain, and the presence of glucose led to a further reduction.Next, the ptsH-H15A mutation was transformed by pKAM12. Results of induction of the TQ432 pKAM12 by different sugars are shown in Table mtlR mutant, no induction was observed by mannitol or glucitol. Moreover, no glucose repression was observed on the basal expression level due to a slow metabolism of the glucose. These results were in line with the in vitro studies of Joyet et al. [32P).It is known that PEP-dependent phosphorylation of HPr His15) plays an essential role in the activity of PRD containing regulators such as ManR  plays an. Therefot et al.  in whichB. subtilis TQ303, which lacks the ccpA gene, a central element of the catabolite repression system, the effect of catabolite repression on mtlA Pwas investigated with plasmid pKAM12. This mutant grew slower than the wild type and showed a longer lag phase. In contrast to our expectations, the ccpA mutant demonstrated a similar or even slightly stronger catabolite repression than the wild type (3.7 versus 2.2-fold) when mannitol together with glucose were added  can be stalled when the HPr (S46~P)-CcpA binds to DNA [groESL operon (PgroE) was fused to an untranslated region of mtlR P(UTRPmtlR) , has been reported [cre site with the cre consensus sequence revealed only one mismatch  was applied [groE-UTRPmtlR was used as the negative control. The constructs PgroE-(cre)-UTRPmtlR were fused to lacZ in pSUN279.2. Transformation of B. subtilis 3NA by pKAM88 (PmtlAcre), pKAM89 (PmtlRcre), pKAM90 (acsAcre), and pKAM91 (mtlAcre) was carried out. Afterwards, the transformants were grown in LB and at the OD600 of 0.4 either glucose (0.2%), xylose (0.2%), or no sugar was added. For all 4 promoters containing a putative cre site a reduction of β-galactosidase activity was observed by the addition of glucose. For the cre site inside the mtlA it was only 1.2-fold. The cre sites of mtlA Pand mtlR Preduced the activity of groE P1.5- to 1.6-fold, whereas the cre site of acsA repressed the promoter expression 2.6-fold.In order to clarify whether the s to DNA . In this) Figure . In addireported . Comparih Figure . As a po applied ,43, whilcre sequences had weak effects on the catabolite repression of mtlA Pand mtlRP. Thus, we hypothesized that the catabolite repression in this system is mainly due to phosphorylation and dephosphorylation of one of the PRD domains of MtlR via HPr and maybe via the IICBAGlc transporter. The ptsG encoding the transporter of glucose is the first gene of an operon, followed by ptsH and ptsI [ptsG by an antibiotic resistance gene may result in the loss of expression of ptsH and ptsI encoding the HPr and Enzyme I proteins, respectively. Therefore, a two-step method was developed. In the first step ptsG was replaced by an erythromycin resistance gene. These mutants were unable to grow on mannose and other PTS sugars. In the second step, the antibiotic resistance gene was replaced by the promoter ptsGHI Pin which the selection was based on regaining the ability to grow on mannose. The resulting strain MW373 was transformed by pKAM12 and pKAM18 and incubated in the presence of different sugars . Transformation of the mtlR-H342D strain, namely KM163, by pKAM12 was followed by induction studies. As shown in table mtlAP. This phenomenon was also observed by other PTS sugars such as mannose and sucrose, although fructose showed no influence (data not shown).As shown, glucose repression was completely abolished in Δpression . Therefomtl operon and its activator was investigated by fusion of the promoter region of mtlAFD and mtlR to lacZ. Identification of transcription start sites revealed that mtlA Pand mtlR Phave the σA promoter structure. Shortening the upstream region of mtlA Pshowed that the two first base pairs of the predicted binding site by Watanabe et al. [mtlA Pactivity. In fact, these two nucleotides are not part of the highly conserved sequence found between mtlR Pand mtlA Poperator regions, and therefore might not belong to the binding site. Changing the base pairs between the predicted MtlR binding site and -35 showed a reduction in the activity of the promoter, although the inducibility of the promoter remained intact. Generally, transcriptional activators in their active conformation bind as homodimers or homomultimers to their operators [mtlA Pwith a shortened MtlR binding site. Furthermore, alignment of the operator region of mtlA Pand mtlR Pindicated that the short palindromic sequence in the operator is enclosed by conserved flanking sequences, consisting of 11 base pairs  mutant. These results were in line with recent in vitro studies on MtlR indicating that phosphate transfer from HPr (H15) to PRDII of MtlR is essential for stimulation of MtlR activity. This is similar to other PTS transcriptional activators [mtlAF) resulted in a constitutive activity of mtlA Pand mtlR Pindicating an inhibitory effect of the EIICBMtl and EIIAMtl on mtlA Pand mtlRP. MtlR belongs to the LevR-type regulator. Four such transcription activators are known in B. subtilis  [Lev [Cel [Man [Activity of tivators . On the nd MtlR) . The inhlR) [Lev ,55, EIICLev [Cel , and EIICel [Man  have alsmtlD with erythromycin resistance gene made the cells sensitive to glucitol as well as mannitol. So far, it is clear that mtlD not only plays a vital role in the consumption of mannitol, but also is necessary for assimilation of glucitol. Induction of mtlA Pin the gutRBPydjE deleted mutant showed that the gut system components, especially GutR, have no effect on mtlA Pactivity (data not shown). Northern hybridization studies, as well as induction studies, proved the ability of glucitol to induce the mtl operon [B. subtilis was observed by Chalumeau et al. [mtlA Pand mtlR Pby glucitol is likely due to the uptake of glucitol by mannitol transporter where EIICBMtl and EIIAMtl dephosphorylate the Cys419 and H599 residues of MtlR, and activates the regulator. Previously, the weak activity of mannitol 1-phosphate dehydrogenase with sorbitol 6-phosphate was detected by Horwitz and Kaplan [Mtl during transport. Afterwards, mannitol 1-phosphate dehydrogenase oxidizes glucitol 6-phosphate to fructose 6-phosphate. Therefore, deletion of the mannitol dehydrogenase should lead to an accumulation of the non-metabolized glucitol 6-phosphate and finally to the killing of the cell.The replacement of l operon . A weak u et al. . This cou et al. ,58. Thusmtl deficient mutants, catabolite repression of mtlA Pand mtlR Pwas investigated in CcpA-dependent CCR mutants. Expression of mtlA Pin the ΔccpA mutant was dramatically reduced, although the catabolite repression was increased. In fact, the ΔccpA mutant grew slower than the wild type strain due to pleiotropic roles of CcpA in the nitrogen regulation, branched chain amino acid synthesis as well as tricarboxylic acid cycle [mtlR Pwas reduced in the ΔccpA strain; however, the mtlR Pactivity was not completely relieved from CCR. Obviously, no exact interpretation was obtained by investigation of general trans elements of CCR. Therefore, we focused on the cis element of CCR, which is the cre site of mtlA Pand mtlRP. The influence of cre sites from mtlA, PmtlR, acsAP, and mtlA were compared by using the constitutive promoter of groESL operon. In the presence of glucose the cre sites of mtlA Pand mtlR Psignificantly repressed the expression of lacZ from PgroE, whereas the cre site located inside the mtlA gene had almost no effect. Therefore, it seems that two weak cre sites, located in the regulator and operon promoter work together to repress the mannitol genes, whereas in the similar B. subtilis mannose system, a strong cre site in the promoter of the regulatory gene (manRP) is the only cis element for CcpA-dependent CCR [mtl operon representing the CcpA-dependent CCR in mannitol PTS [In addition to id cycle ,61. In cdent CCR . These ritol PTS ,63.ptsG was disrupted [ptsG was deleted from the genome in a way that ptsGHI Pwas fused to ptsHI. As expected, activity of the mtlA Pand mtlR Pin the ΔptsG mutant was identical in the presence and absence of glucose. The reason why we did not see any effect of the cre/CcpA system in the presence of glucose can be explained by the fact that HPr kinase is only active in the presence of glycolytic intermediates. Due to the deletion of ptsG no glucose is taken up by the EIICBAGlc. From the second glucose transporter (GlcU) it is known that this gene (glcU) is expressed 3 h after sporulation under the control of σG [glcP (glucose/mannose:H+ symporter), a third glucose permease depends on glucose 6-phosphate produced by glucose PTS [cre/CcpA dependent CCR. Since CcpA-dependent CCR was completely abolished only in ΔptsG and ptsH-H15A mutants, CcpA dependent CCR might not play the main role in the mannitol regulation. The strong effect of glucose might be explained by a hierarchy in the affinity of HPr (H15~P) to EIICBAGlc versus EIIAMtl and PRDII domain of MtlR. Alternatively, EIICBAGlc might also inactivate MtlR directly by dephosphorylation of PRDII domain. Mutation of the histidine 342 to aspartate located in PRDII domain of MtlR supported the assumption of dephosphorylation of MtlR by glucose PTS components. In this mutant addition of PTS sugars such as glucose, mannose, and sucrose (except fructose) increased the activity of the promoter. This could be due to dephosphorylation of the EIIA and EIIB domains of MtlAF/or MtlR by the specific transporters of glucose, mannose, and sucrose: However, further experiments are necessary to clarify this effect. Overall, catabolite repression in mtl system is mainly functional at posttranslational level by PtsG and at the transcription initiation level by CcpA dependent CCR, which switch off the expression of mannitol uptake system.So far, it is shown that the glucose repression is abolished when isrupted ,65. In ool of σG ,67. Finacose PTS ; therefomtl operon, as well as its regulator, resulted in construction of a highly inducible expression system in B. subtilis based on mannitol as a cheap inducer. Activity of mtlA Pand mtlR Pin several mutants revealed that this system is mainly regulated by the phosphorylation and dephosphorylation state of the specific regulator. This accelerates the further manipulation of the strain to enhance the expression system.Characterization and optimization of the promoter region of The authors declare that they have no competing interests.ptsG mutant and wrote the corresponding section in Materials. JA supervised and coordinated the project. KMH wrote the paper which was later revised and corrected by JA. All authors approved the final version of the manuscript.KMH designed and performed the experiments and analyzed the data. MW constructed the


In eukaryotic cells, transgene expression levels may be limited by an unfavourable chromatin structure at the integration site. Epigenetic regulators are DNA sequences which may protect transgenes from such position effect. We evaluated different epigenetic regulators for their ability to protect transgene expression at telomeres, which are commonly associated to low or inconsistent expression because of their repressive chromatin environment. Although to variable extents, matrix attachment regions (MARs), ubiquitous chromatin opening element (UCOE) and the chicken cHS4 insulator acted as barrier elements, protecting a telomeric-distal transgene from silencing. MARs also increased the probability of silent gene reactivation in time-course experiments. Additionally, all MARs improved the level of expression in non-silenced cells, unlike other elements. MARs were associated to histone marks usually linked to actively expressed genes, especially acetylation of histone H3 and H4, suggesting that they may prevent the spread of silencing chromatin by imposing acetylation marks on nearby nucleosomes. Alternatively, an UCOE was found to act by preventing deposition of repressive chromatin marks. We conclude that epigenetic DNA elements used to enhance and stabilize transgene expression all have specific epigenetic signature that might be at the basis of their mode of action. In the nucleus of eukaryotic cells, linear chromosomal DNA associates with histones and other proteins to form chromatin. Besides packaging DNA into a smaller volume, chromatin also serves as a mechanism to control DNA expression and replication . NumerouTelomeres are regions of highly repetitive DNA that protect the ends of linear chromosomes from DNA repair or recombination . GeneralThe subtelomeric DNA is heavily methylated at CpG dinucleotides, and inactivation of DNA methyltransferases from mouse embryonic stem cells results in telomeric instability . Similarloci. This phenomenon, termed the telomere position effect (TPE), occurs in both lower and higher eukaryotes  and scaffold attachment regions are A + T rich DNA of variable length that were originally found to bind the nuclear matrix or scaffold, a poorly understood protein structure within the cell nucleus . They arSTabilizing Anti-Repressor (STAR) elements are genetic elements proposed to counteract chromatin-associated repression effects, thereby leading to high and stable expression of transgenes . They welocus, a genomic region encompassing the promoters of the heterogeneous nuclear ribonucleoprotein A2/B1 (HNRPA2B1) and of the chromobox protein homologue 3 (CBX3) housekeeping genes.Ubiquitous chromatin opening elements (UCOEs) are regulatory elements derived from promoter-containing CpG islands of ubiquitously expressed housekeeping genes . It was In this study, we used a dual-reporter system to evaluate epigenetic regulatory elements for their ability to protect transgenes expression and to modify the chromatin structure when placed in the prevailing heterochromatic environment of telomeres. Two fluorescent protein-coding reporter genes were placed in a telomeric-proximal or telomeric-distal position relative to a regulatory element, and the strength of various epigenetic regulators was assessed in terms of barrier activity, anti-silencing effect and general transcriptional activation capability. We show that epigenetic regulators such as MAR, UCOE and the chicken cHS4 insulator resulted in an increased number of cells expressing the telomeric distal reporter gene, whereas the telomeric-proximal reporter gene was usually highly repressed, implying a barrier effect, while some of the MAR elements showed an additional anti-silencing activity. Chromatin immunoprecipitation (ChIP) on monoclonal cell populations revealed that epigenetic regulators act differently on the chromatin structure. The positive effects on transgene expression of MAR elements may be due to the enrichment of both histones H3 and H4 acetylation in their proximity, whereas UCOE may prevent the deposition of heterochromatic histone marks such as H3K9me3, H4K20me3 and H3K27me3. Overall, we conclude that the MAR and UCOE strong chromatin boundary as well as the MAR anti-silencing activity may result from the deposition of distinct chromatin structures.de novo telomere formation at the genomic integration site, a MCS for the insertion of DNA elements, and two distinct reporter genes, DsRed and eBFP2 being respectively at a telomeric proximal and telomeric distal position relative to the MCS . The UCOE corresponds to the BspEi-Esp3I fragment of the HNRPA2B1-CBX3 locus (Supplementary Data).A novel dual reporter system for the study of epigenetic regulators at human telomeres was used. Construction of this new system, termed pSTE-TR-RB, started with the introduction of a new multiple cloning site (MCS) into pBS-Puro, a BlueScript vector containing a puromycin resistance cassette. DsRed and eBFP2 (enhanced blue fluorescent protein 2) coding sequences under the control of minimal CMV (mCMV) promoter were polymerase chain reaction (PCR) amplified from pCMV-DsRed Express (Clontech) and pEBFP2  respectively. These sequences, together with telomeric repeats from pGE2min plasmid , were cl the MCS A. All DN the MCS ,34, as w the MCS . The mulX3 locus , while tX3 locus . Neutral2 in DMEM:F12 medium (Gibco) supplemented with 10% foetal bovine serum (Gibco). Low passage HeLa cells were transfected with linearized plasmids using FuGENE6 according to the manufacturer’s instruction (Roche). Selection was carried out with 2.5 μg/ml puromycin (Sigma) for 15 days, after which selection was stopped and cells were grown for at least 2 weeks without selection pressure.HeLa Tet-off cells (Clontech) were cultivated at 37°C, 5.5% COExpression of reporter genes in transfected HeLa cells was measured by a CyAn ADP cell analyzer (Dako) and analysed with FlowJo software (Treestar). Sorting of cells for the establishment of monoclonal populations was done with a FACSAriaII (BD Biosciences) at the Flow Cytometry Core Facility of the Swiss Institute of Technology of Lausanne .HeLa cells were cross-linked in 1% formaldehyde for 7 min and lysed in the presence of protease inhibitor cocktail . Chromatin was then sonicated on ice until sheared to an average size of 400 bp, diluted in ChIP dilution buffer  and pre-cleared for 90 min in the presence of rProtein A sepharose beads . The pre-cleared chromatin was immunoprecipitated overnight at 4°C. Antibodies against acetylated histone H3 (06-599), acetylated histone H4 (06-866) and trimethylated histone H3K27 (07-449) were obtained from Millipore, whereas antibodies against trimethylated histone H3K4 (ab8580), monomethylated H2BK5 (ab12929), monomethylated H3R2 (ab15584), trimethylated histone H3K9 (ab8898) and trimethylated histone H4K20 (ab9053) were purchased from Abcam. Anti GFP antibody was used as negative control (Roche). The Immunoprecipitated complexes were recovered by incubation with 12 μl of rProtein A sepharose beads followed by four washes with immunoprecipitation buffer . Beads were re-suspended in elution buffer  and reverse cross-linked overnight at 65°C. Precipitated DNA was recovered using the Qiagen PCR purification kit.Eff corresponds to the primer pair efficiency calculated using LinRegPCR software (The quantification of ChIP DNA recovery was performed by quantitative PCR using a LightCycler 480 instrument with SYBR Green-reagent (both from Roche). For the amplification of eBFP2 and DsRed, a combination of a gene-specific forward primer with a common reverse primer for both DsRed and eBFP2 on the mCMV promoter was used. Primer sequences were as follows: eBFP2 forward primer 5′-CAGCTCCTCGCCCTTGCTCA-3′, DsRed forward primer 5′-CGCACCTTGAAGCGCATGAA-3′, mCMV promoter reverse primer 5′-AGAGCTGGTTTAGTGAACCGTCAGATC-3′. The relative positions of these amplicons are shown in software .Supplementary Data). In addition to epigenetic regulators, neutral sequences of various lengths were also cloned as spacer controls, as the strength of telomeric silencing in HeLa cells was found to partly depend on the distance from the telomeres , similarly to what was observed in yeast , suggesting an anti-silencing effect for these elements when placed at a telomeric location.Analysis of polyclonal populations of stably transfected cells revealed that most of the DNA elements were able to increase significantly the number of cells expressing at least one of the two reporter genes when compared to a plasmid containing a neutral control sequence of similar length . In stabSupplementary Figure S1, pSTE-RB-HS4 and pSTE-RB-S4 constructs). Overall, these results showed a significant barrier activity for MAR and UCOEs, as well as for the previously known cHS4 insulator. In addition, a transcriptional activating effect was observed for MARs and, to a lesser extent, for STAR 40. Interestingly, the AT-rich core sequence of MAR 1-68 and MAR X-29 were unable to mediate a barrier or transcriptional effect, implying that other elements are required to mediate the MAR effect, in agreement with previous bioinformatics-based modelling of MAR elements , together with their respective spacer controls (1 kb and 3 kb CTRLs), were sorted into monoclonal populations and grown without selection pressure. Clones displaying homogeneous and relatively low expression of DsRed were retained, in order to exclude cells with potential non-telomeric integration of multiple plasmid copies. The mean fluorescence of eBFP2 and DsRed was assessed using 12 clones generated for each of the DNA element and spacer control by flow cytometry, to evaluate the expression variability between distinct clones A and B. Starting from the stably transfected polyclonal populations, non-expressing cells were also sorted and grown as monoclonal populations. Although no expression could be detected by fluorescence-activated cell sorting (FACS), PCR amplification on genomic DNA extracted from some silenced cloned confirmed that the reporter genes were still integrated into the genome (data not shown), implying a strong silencing effect rather than a loss of the integrated vector. Among these silent clonal populations, several were able to spontaneously recover eBFP2-expressing cells after 2 weeks of culture without selection C. The nuIn order to shed light on the mechanism of action of the epigenetic regulatory elements used in this study, the epigenetic landscape on the reporter genes promoter was assessed by ChIP. Different histone modifications were investigated, including open chromatin marks such as histone 3 and histone 4 acetylation, trimethylated Histone H3 on lysine 4 (H3K4me3), monomethylated H2B on lysine 5 (H2BK5me1) and monomethylated H3 on arginine 2 (H3R2me1), as well as repressive/heterochromatic marks such as trimethylated H3K27 (H3K27me3), trimethylated H3K9 (H3K9me3) and trimethylated H4K20 (H4K20me3).Supplementary Figure S2), and it was shown to correlate well with the mRNA levels of the transgenes . Fluorescence in-situ hybridization (FISH) analysis was performed on metaphasic chromosomal spreads using probes consisting of the telomeric repeats and of the dual reporter system without DNA elements nor telomeric repeats (pSTE-RB), which confirmed a single integration site at telomeric/subtelomeric position for all analysed clones . The copy number of integrated transgenes per genome was also estimated by qPCR on total cellular DNA, yielding values indicative of single copy integration (data not shown). For every genetic element, enrichment values from ChIP experiments performed on the two clones were averaged, to minimize possible clone-specific effects, and they were expressed as fold increase over the values obtained from the controls with spacer DNA of similar size, as obtained from three independent ChIP experiments performed with at least two different chromatin preparations . At the chromatin level, the human MAR 1-68 and X-29 and the murine MAR S4 appeared to be the most active DNA elements in terms of augmenting the chromatin marks associated with a permissive chromatin structure . The MAR X-29 promoted deposition of active chromatin marks on both reporter genes, with an increase in the acetylation of histone H3 (AcH3) and H4 (AcH4) and the trimethylation of H3K4 compared with the spacer control. In addition, it reduced the presence of heterochromatic histone marks on the telomere proximal gene (H3K9me3 and H4K20me3). MAR S4 acted mainly on the telomere proximal gene, increasing the deposition of H3K4me3 and acetylated H3 and H4, as well as decreasing the level of all three repressive/heterochromatic marks. MAR 1-68 acted mostly on the telomeric distal gene, which is the gene whose expression could be activated from a silent state . Expression of eBFP2 in the presence of MAR 1-68 was found to be accompanied with increased levels of euchromatic marks, such as AcH4, H2BK5me1 and H3R2me1, as well as with increased levels of H3K9me3, a modification often associated with heterochromatin. In presence of the chicken lysozyme MAR, little differences were detected for active and inactive chromatin modifications, possibly reflecting the fact that it was found to be a relatively weaker MAR element when compared to the human and murine MARs telomeric regions such as H3K9me3, H4K20me3, H2BK5me1 or H3K2me1 on the promoter of the telomeric-proximal reporter gene, as expected from the ine MARs . Nonetheine MARs .Supplementary Figure S2). The cHS4 insulator resulted in lower levels of heterochromatic marks on the telomeric-proximal reporter gene, which, together with the absence of effects on euchromatic marks, is consistent with the barrier function proposed for this element. Presence of STAR 40 led to a reduction of both eu- and heterochromatic marks, which might explain the lack of strong effects on gene expression. Overall, ChIP results showed epigenetic landscapes which varied from element to element, with a tendency for MARs to promote the deposition of active histone marks, whereas the UCOE prevented deposition of repressive modifications, as summarized in The analysed UCOE was also particularly active at the chromatin level. Besides a strong depletion of all repressive marks—with H3K9me3 and H3K27me3 levels below or close to the background obtained with immunoprecipitation controls—UCOE strongly promoted acetylation of H3 and H3K4 trimethylation over the promoter of the telomere-proximal DsRed gene . Yet, thThe eukaryotic genome is functionally compartmentalized into defined chromatin domains that are thought to contribute to the regulation of different biological processes including gene transcription. Integration of a transgene in the host genome may result in inconsistent expression, as elicited by the particular chromatin environment of the integration site. Epigenetic regulators are DNA sequences which may minimize this position effect, possibly leading to a more consistent expression of the transgene . Here, wloci. MARs greatly increased the proportion of cells expressing a telomeric-distal reporter gene, while they less frequently prevented the silencing of a telomeric-proximal transgene, suggesting a prominent barrier activity for this class of epigenetic regulators. In addition, MAR elements could reactivate expression from silent clones at a high frequency, and thus possess anti-silencing effects. Finally, MARs also increased the transcription rate of the telomere-distal gene, as assessed from the fluorescence and mRNA levels, indicating that they can also act as transcriptional activators. These conclusions are further supported by the finding that the MARs locally promoted modifications of the chromatin to adopt a structure more permissive for gene expression. In particular, acetylation of histone H4 was enriched in proximity of all four MAR elements tested, while further histone H3 acetylation was promoted by the two potent MARs X-29 and S4. The presence of acetylated nucleosomes may result from the recruitment of histone acetyltransferases, as proposed for other MAR elements in human osteosarcoma U2OS cell line (We show that MARs can act to protect gene expression at telomeric ell line . Interesell line . This suell line  and partell line . This coell line ,34.de novo telomere formation at the integration site. Indeed, the increased transgene expression resulting by the insertion of MARs is in part also due to a higher number of integrated copies (The effects of some MARs on transgene expression was limited in this study by the single copy integration upon d copies ,48. Thisd copies ,34. Neved copies . Additiod copies , and we d copies .loci, the UCOE could increase the number of cells expressing the telomeric distal reporter gene, as indicative of protection against silencing effects by a barrier effect, but it did not increase fluorescence levels as would result from higher transcription rates. Interestingly, the chromatin environment in proximity to the UCOE was poor in histone modifications, particularly in heterochromatic repressive marks. One possible explanation is that UCOE are nucleosome-poor regions, in agreement with the findings that CpG islands are relatively nucleosome-deficient (locus (locus was accompanied by active histone modification marks such H3K4me3 and acetylation of both histones H3 and H4, together with low repressive marks such as H3K27me3 (loci may thus explain the lack of silent gene activation.UCOEs are CpG island-containing DNA sequences derived from the promoter regions of housekeeping genes , and theeficient  or that t (locus . ConcernH3K27me3 . The lacet al. (loci showed that the barrier activity of cHS4 was accompanied by acetylation of nearby histones (The chicken beta-globin HS4 element is a well-known insulator that was shown to protect transgene expression from TPE when the transgene was insulated by two copies of cHS4 . Here weet al. . Previouhistones ,55. Howehistones . Our datloci.STAR elements were proposed to prevent the deposition of heterochromatin-related proteins such as heterochromatin protein 1 . At teloOver 10 years ago, it was proposed that histone modifications are not isolated chromatin marks, but that they constitute together a histone code, as represented by the combinatorial patterns of histone modifications that function in concert . These pSupplementary Data are available at NAR Online, including [59–71].Swiss Commission for Technology and Innovation and from Selexis SA, and by the University of Lausanne. Funding for open access charge: University of Lausanne.Conflict of interest statement. N.M. is a co-founder and own shares of Selexis SA, a company that uses proprietary technology to generate therapeutic-producing CHO cell lines. Other authors have no competing interest to declare.


Rhodobacter sphaeroides RpoHI and RpoHII using genome-scale data derived from gene expression profiling and the direct interactions of each protein with DNA in vivo. We found that the RpoHI and RpoHII regulons have both distinct and overlapping gene sets. We predicted DNA sequence elements that dictate promoter recognition specificity by each RpoH paralog. We found that several bases in the highly conserved TTG in the −35 element are important for activity with both RpoH homologs; that the T-9 position, which is over-represented in the RpoHI promoter sequence logo, is critical for RpoHI–dependent transcription; and that several bases in the predicted −10 element were important for activity with either RpoHII or both RpoH homologs. Genes that are transcribed by both RpoHI and RpoHII are predicted to encode for functions involved in general cell maintenance. The functions specific to the RpoHI regulon are associated with a classic heat shock response, while those specific to RpoHII are associated with the response to the reactive oxygen species, singlet oxygen. We propose that a gene duplication event followed by changes in promoter recognition by RpoHI and RpoHII allowed convergence of the transcriptional responses to heat and singlet oxygen stress in R. sphaeroides and possibly other bacteria.Cells often mount transcriptional responses and activate specific sets of genes in response to stress-inducing signals such as heat or reactive oxygen species. Transcription factors in the RpoH family of bacterial alternative σ factors usually control gene expression during a heat shock response. Interestingly, several α-proteobacteria possess two or more paralogs of RpoH, suggesting some functional distinction. We investigated the target promoters of  Rhodobacter sphaeroides and several other α-proteobacteria possess two or more paralogs of RpoH, suggesting some functional distinction. Our findings predict that a gene duplication event followed by changes in DNA recognition by RpoHI and RpoHII allowed convergence of the transcriptional responses to heat and singlet oxygen stress in R. sphaeroides and possibly other bacteria. Our approach and findings should interest those studying the evolution of transcription factors or the signal transduction pathways that control stress responses.An important property of living systems is their ability to survive under conditions of stress such as increased temperature or the presence of reactive oxygen species. Central to the function of these stress responses are transcription factors that activate specific sets of genes needed for this response. Despite the central role of stress responses across all forms of life, the processes driving their organization and evolution across organisms are poorly understood. This paper uses genomic, computational, and mutational analyses to dissect stress responses controlled by two proteins that are each members of the RpoH family of alternative σ factors. RpoH family members usually control gene expression during a heat shock response. However, the photosynthetic bacterium  Escherichia coli uses the alternative σ factor σ32 to increase synthesis of gene products involved in protein homeostasis or membrane integrity E. coli σ32 are conserved across virtually all proteobacteria. This so-called RpoH family of alternative σ factors is characterized by a conserved amino acid sequence (the “RpoH box”) that is involved in RNA polymerase interactions Transcriptional responses to stress are critical to cell growth and survival. In bacteria, stress responses are often controlled by alternative σ factors that direct RNA polymerase to transcribe promoters different from those recognized by the primary σ factor Brucella melitensisSinorhizobium melilotiBradyrhizobium japonicumRhizobium eltiRhodobacter sphaeroidesE. coli ΔrpoH mutants, suggesting that these proteins can functionally interact with RNA polymerase and recognize similar promoter elements Rhizobium elti, the Δ1rpoH mutant was sensitive to heat and oxidative stress while the Δ2rpoH mutant was sensitive to osmotic stress While bacteria often possess many alternative σ factors, they usually possess only one member of the RpoH family. However, several α-proteobacteria, including R. sphaeroides RpoHI or RpoHII can complement the temperature sensitive phenotype of an E. coli ΔrpoH mutant; that singly mutant R. sphaeroides strains lacking either IrpoH or IIrpoH are able to mount a heat shock response; and that RNA polymerase containing either RpoHI or RpoHII can initiate transcription from a common set of promoters in vitroI and RpoHII have some overlapping functions in R. sphaeroides. On the other hand, in vitro transcription assays identified promoters that were selectively transcribed by either RpoHI or RpoHIIIIrpoH is under direct transcriptional control of RpoE, a Group IV alternative σ factor that acts as the master regulator of the response of R. sphaeroides to singlet oxygen stress IIrpoH mutant is more sensitive to singlet oxygen stress than the wild-type strain I and RpoHII also have distinct functions in R. sphaeroides. Finally, global protein profiles of R. sphaeroides mutants lacking IrpoH, IIrpoH, or both genes, suggested that RpoHI and RpoHII have distinct and overlapping regulons I and RpoHII is still unknown because past studies have been unable to distinguish direct from indirect effects on gene expression or identify all the direct targets for either of these σ factors.Previous work demonstrated that either I and RpoHII regulons using a combination of expression microarrays, chromatin immunoprecipitation and computational methods which have been previously been shown to predict correctly direct targets for other alternative σ factors or DNA binding proteins I and RpoHII regulons function in protein repair or turnover, membrane maintenance, and DNA repair. Genes specific to the RpoHI regulon encode other proteins involved in protein maintenance and DNA repair, whereas genes specific to the RpoHII regulon include proteins involved in maintaining the oxidation-reduction state of the cytoplasmic thiol pool. We used information on the members of each regulon to generate and test hypotheses about DNA sequences that determine promoter specificity of these two RpoH homologs. The observed properties of these two R. sphaeroides RpoH homologs illustrate how duplication of an alternative σ factor and subsequent changes in promoter recognition could have allowed convergence of transcriptional responses to separate signals. In the case of R. sphaeroides, we predict that these events allowed convergence of the transcriptional responses to heat shock and singlet oxygen stresses to be under control of these two RpoH paralogs.In this study, we characterized the RpoHI and RpoHII regulons, we monitored transcript levels and protein-DNA interactions in R. sphaeroides strains ectopically expressing either RpoHI or RpoHII. To generate these strains, we constructed low copy plasmids carrying IrpoH or IIrpoH under the control of an IPTG-inducible promoter R. sphaeroides mutant strains lacking IrpoHIIrpoHI and RpoHII contained each protein at levels comparable to those following either heat shock or singlet oxygen stress  perature . RpoHI lncreased . Levels response . The abuI or RpoHII activity, we compared mRNA levels of cells expressing RpoHI or RpoHII ectopically to those of control cells lacking either IrpoH or IIrpoH. We selected differentially expressed genes with a significance level set for a false discovery rate ≤5% and that displayed at least 1.5-fold higher transcript levels in cells expressing either RpoH family member. This analysis revealed that transcripts from 241 and 186 genes were increased by expression of RpoHI and RpoHII, respectively . From the ChIP-chip analysis we identified 812 and 1353 genomic regions enriched after immunoprecipitation with antibodies against RpoHI and RpoHII, respectively, using a significance level set for a false discovery rate ≤5%. Because the signal from a single σ factor binding site extends on average over a 1 kb region, some enriched regions may contain multiple binding sites. To increase the resolution of the putative RpoHI and RpoHII binding sites, we identified the modes of the ChIP-chip signal distributions within each enriched region. This adjustment increased the number of putative binding sites for RpoHI and RpoHII to 1085 and 1765, respectively.We recognize that some of these differentially-expressed transcripts might be not be direct targets for RpoHpe RpoHI . In addiI or RpoHII regulons. Included in this list of potential regulon members were genes that are predicted to be co-transcribed using a previous computational analysis of R. sphaeroides operon organization (http://www.microbesonline.org/operons/) I or RpoHII are 1120 and 1616, respectively . The conserved sequence elements we identified mapped to putative promoter elements that were within 100 bp of the coordinates of the modes of the distributions of the ChIP-chip signal. Thus, the predictions of these searches identified conserved sequence elements that were in agreement with the experimental data. In addition, even though we analyzed the individual RpoHI and RpoHII regulons independently for these motifs, the sequence alignment algorithm converged to the same sequence elements for promoters that were predicted to be recognized by both RpoHI and RpoHII. This result is not surprising given that both σ factors have similar amino acid sequences in their DNA recognition regions and are thus expected to recognize similar promoter sequences. However, this observation supports the hypothesis that RpoHI and RpoHII recognize common promoter sequences in their respective target genes as opposed to distinct promoters.To overcome the limited resolution of the ChIP-chip experiment and predict determinants of promoter specificity for RpoHI or RpoHII and one group that is bound and transcribed by both σ factors. As noted above, some promoters appear to be bound by RpoHI or RpoHII without inducing detectable changes in transcript levels. We aligned these promoters separately to determine if they possessed unique characteristics, but no significant differences were detected (data not shown).To predict specificity sequence determinants for each RpoH paralog, the putative distinct and overlapping promoter sequences were sorted into three groups according to the expression profiling and ChIP-chip data sets and converted into sequence logos . The seqI and RpoHIII-dependent promoters, a cytosine is overrepresented at position −37 and a thymine is overrepresented at position −9. In the logo for RpoHII-dependent promoters, cytosine and thymine are overrepresented at positions −14 and −13, respectively.The conservation of a TTG motif in the −35 region in all three logos is consistent with the importance of this triplet in a previous analysis of at least one promoter known to be recognized by both RpoHI and RpoHII-specific promoter logos allowed us to identify significant differences in the promoter sequences that may be used to adjust promoter selectivity and strength for RpoHI or RpoHII. In addition, the predicted sequence elements for RpoHI or RpoHII promoters are not mutually exclusive. Rather, it appears that promoter specificities for RpoHI or RpoHII are distributed along a gradient using a combination of specific bases at various positions of the −35 or −10 promoter elements.Overall, the comparison between RpoHlacZ reporter gene and integrated these into the genome of a R. sphaeroides ΔIrpoH ΔIIrpoH mutant R. sphaeroides reporter strains ectopically expressing either RpoHI or RpoHII  I- and RpoHII-specific promoters. In the −10 region of the groE promoter, substitutions of the cytosine at position −14 for an adenine or guanine, the cytosine at position −13 for an adenine, or substitution of the thymine at position −11 for a cytosine, each reduced RpoHI-dependent promoter activity. In addition, a substitution of the adenine at position −12 for a cytosine or changing the thymine at position −9 for any other base reduced RpoHI-dependent activity by >90%. These observations are consistent with the conservation of a thymine at position −9 of the derived RpoHI promoter logo . Instead, we generated a small set of point mutations in the P1 promoter of the R. sphaeroides cycA promoter .We were unable to test activity of promoter  which wacycA P1 (G-36T) increased its transcription by both RpoHI and RpoHII  I and RpoHII is part of a general stress response that is common to the heat shock, singlet oxygen and possibly other uncharacterized signals in R. sphaeroidesR. sphaeroides and R. elti strains lacking both RpoHI and RpoHII are more sensitive to several conditions than strains lacking only one of these proteins This work revealed a surprisingly extensive overlap of the RpoHntegrity . These tI and RpoHII, it is also important to note that this set of genes may be larger than the one we characterized because some promoters known to be transcribed by both σ factors were only marginally affected by ectopic expression of either RpoHI or RpoHII. For example, the RSP_2310 (groES) promoter was shown to be transcribed by both RpoHI and RpoHII in previous in vitro experiments I and RpoHII, but did not meet all the criteria of our analysis. Thus, the groES promoter, like other promoters, may be subject to complex regulation in vivo.In considering the scope of functions that are regulated by both RpoHI or RpoHII  that serves as the master regulator of the singlet oxygen stress response R. sphaeroides ΔIIrpoH mutant is more sensitive to singlet oxygen than a wild-type or ΔIrpoH strain II-specific regulon might be expected to play an important role in the response to singlet oxygen stress. Among the genes in the RpoHII-specific regulon are others predicted to function in maintaining membrane integrity and performing DNA repair, both potential targets for damage by singlet oxygen. However, the RpoHII–specific regulon contains fewer genes encoding functions related to protein homeostasis than found in the RpoHI regulon . We have computational and experimental observations that can explain some aspects of promoter selectivity by RpoHI and RpoHII. For example, our experiments identify T-9 and other positions in the −10 element as potential candidates in this discrimination, as one or more substitutions have larger effects on activity with individual RpoHI homologs. Mutation of T-9 to any other base reduced RpoHI-driven expression of GroE promoter by more than 90%, and this same effect was observed using an authentic RpoHI promoter from R. sphaeroides. Importantly, changing the −9 position of an RpoHIIR. sphaeroides promoter to T permitted expression by RpoHI. Together, these data suggest that T-9 is either required for or significantly enhances expression of RpoHI promoters, but is likely to be less important for expression of RpoHII promoters, as there is only weak conservation of -9T in RpoHII promoters. Our data also predict that other bases, which are overrepresented in the RpoHII promoters, could be critical for expression by that σ factor. As is the case with E. coli σ32 there are likely to be specificity determinants that lie outside the canonical −35 and −10 elements in vivo and in vitro experiments with a larger suite of mutant promoters and a library of mutant RpoH proteins are needed to better define the determinants of promoter selectivity by RpoHI and RpoHII.Our experiments provide definitive evidence that some promoters are transcribed either exclusively or predominantly by RpoHR. sphaeroides, RpoHI controls functions that are necessary for maintenance of protein homeostasis and membrane integrity after temperature increase and other cytoplasmic stress, similar to the well-characterized role of E. coli σ32 in the heat shock response R. sphaeroides, some RpoHI-regulated functions are also useful for survival in the presence of other forms of stress because these target genes also contain promoters that are recognized by RpoHII. We propose that the duplication of an ancestral RpoH protein to create a second homolog of this alterative σ factor provided R. sphaeroides the opportunity to connect stress response functions to another stimulus. In this model, IIrpoH was placed under the control of the master regulator of the singlet oxygen stress response and the two RpoH proteins evolved to recognize somewhat different but compatible promoter elements to assure the optimal regulation of distinct but overlapping stress regulons. As a result of these events, the transcriptional responses of R. sphaeroides to heat shock and singlet oxygen stress were separable but allowed to converge and contain a common set of functions. It will be interesting to identify and examine other examples of such convergence across bacteria and other organisms that possess multiple homologs of RpoH or other transcription factors.In conclusion, our results suggest that, at least in E. coli strains were grown in Luria-Bertani medium R. sphaeroides strains were grown at 30°C in Sistrom's succinate-based medium E. coli DH5α was used as a plasmid host, and E. coli S17-1 was used as a donor for plasmid conjugation into R. sphaeroides. The media were supplemented with kanamycin (25 µg/ml), ampicillin (100 mg/ml), chloramphenicol (30 mg/ml), spectinomycin (50 mg/ml), tetracycline (10 mg/ml for E. coli and 1 mg/ml for R. sphaeroides), trimethoprim (30 µg/ml), or 0.1% of L-(+)-arabinose when required. Unless noted, all reagents were used according to the manufacturer's specifications. The list of bacterial strains and plasmids used in this study are summarized in I or RpoHII were constructed by separately cloning the IrpoH or IIrpoH genes downstream of the IPTG-inducible promoter in pIND4 IrpoH or IIrpoH were amplified from R. sphaeroides 2.4.1 genomic DNA using oligonucleotides containing BsrDI and BglII restriction sites . These resulting fragments were digested with BsrDI and BglII and ligated into pIND4 that was digested with BseRI and BglII to create pYSD40 (IrpoH) and pYSD41 (IIrpoH), respectively. The pYSD42 plasmid expressing the FLAG-tagged version of RpoHI was constructed following the same procedure but with an oligonucleotide primer containing a sequence encoding for three consecutive copies of the FLAG epitope (DYKDDDDK) at the N-terminus (RSP_2410_3FLAG_BsrDI GTAGCAATGCATGGACTACAAGGACCACGACGGCGACTACAAGGACCACGACATCGACTACAAGGACGACGACGACAAGAGCACTTACACCAGCCTTCCCGCTC). pYSD40, pYSD41, and pYSD42 were conjugated into R. sphaeroides ΔIrpoHR. sphaeroides ΔIIrpoH respectively.Plasmids for ectopically expressing RpoHI and RpoHII after heat shock, exponential phase aerobic cultures  of wild type R. sphaeroides strain 2.4.1 grown at 30°C, were transferred to a 42°C warm bath with samples collected before heat treatment and at 10 min time intervals after heat shock, up to 60 min. To assess induction resulting from singlet oxygen stress, similarly grown wild type cells were treated with 1 µM methylene blue and exposed to 10 W/m2 incandescent light with samples collected before treatment and at 10 min time intervals after treatment, up to 60 min. Exponentially growing aerobic cultures of R. sphaeroides ΔIrpoH and ΔIIrpoH mutants carrying the pYSD40 or pYSD42 plasmids respectively, were treated with 100 µM IPTG for one generation and harvested. All cell samples were resuspended in 3 M urea containing 1× protease inhibitor cocktail  and sonicated. Samples were centrifuged to remove debris and total protein concentration of the samples determined with a protein assay kit following the manufacturer protocol . An equal amount of total protein for each sample was loaded onto a NuPAGE acrylamide gel  and run in 1× 4-morpholineethanesulfonic acid running buffer at 150 V for ∼90 min. Proteins were transferred to Invitrolon PVDF membranes , which were subsequently incubated for 1 hr in 1× Tris-buffered saline, 0.1% Triton-X, and 5% milk protein. The membranes were incubated with rabbit polyclonal antibodies raised against either RpoHI, RpoHII or PrrA. Horseradish-peroxidase-conjugated goat anti-Rabbit IgG antibody  was used as secondary antibody for detection with Super Signal West Dura extended duration substrate .To monitor levels of RpoH2, 69% N2, 1% CO2) until they reached early exponential phase (OD at 600 nm of 0.15). At this point IPTG  was added to a final concentration of 100 µM to induce gene expression from the pIND4 derivatives. After 3 hours incubation (OD at 600 nm of 0.30), 44 ml of cell culture were collected and 6 ml of 5% v/v phenol in ethanol was immediately added. Cells were collected by centrifugation at 6,000 g and frozen at −80°C until sample preparation. RNA extraction, cDNA synthesis, labeling, and hybridization were performed as previously described on Genechip Custom Express microarrays  http://www.r-project.org/) http://www.ncbi.nlm.nih.gov/projects/geo/query/acc.cgi?acc=GSE39806).Triplicate 500 ml cultures were grown aerobically with bubbling  . RpoHII was immunoprecipitated with anti-R. sphaeroides RpoHII rabbit serum. Labeled DNA was hybridized on a custom-made tiling microarray, synthesized by NimbleGen , covering the genome of R. sphaeroides 2.4.1 limma package in the R environment) I or RpoHII were identified using CMARRT with a false-discovery rate ≤0.05 http://www.ncbi.nlm.nih.gov/projects/geo/query/acc.cgi?acc=GSE39806).Cells were harvested at mid-exponential growth (OD at 600 nm of 0.30) from the same cell cultures used for the expression microarray experiment to prepare samples for a ChIP-chip assay R. sphaeroides 2.4.1 were obtained from VIMSS MicrobesOnline (http://www.microbesonline.org/operons/) I and RpoHII were discovered using Bioprospector http://weblogo.berkeley.edu/) DNA sequences were manipulated using custom Python scripts. Operon structure predictions for in vivo activity of RpoHI and RpoHII at target promoters, β-galactosidase assays were conducted with R. sphaeroides ΔIrpoH ΔIIrpoH mutant strains containing individual reporter gene fusions. To construct this set of reporter strains ∼350 base pair regions upstream of putative target genes: RSP_1173, RSP_1408, RSP_1531, RSP_2314, RSP_2389, RSP_3274, RSP_1207 and RSP_2617, were amplified from genomic DNA using sequence specific primers, with NcoI and XbaI restriction sites at the ends of the upstream and downstream primers respectively. The amplified DNA fragments were purified, digested with NcoI-XbaI and then cloned in a pSUP202 suicide vector containing a promoterless lacZ gene (pYSD51). These Tcr plasmids were then conjugated into an R. sphaeroides ΔIrpoH ΔIIrpoH mutant lacZ fusions integrated in the genome. pYSD40, pYSD41 or pIND4 (empty vector) were then conjugated into each of these reporter strains. Exponential phase cultures of these strains, grown by shaking 10 mL in 125 mL conical flasks, were then treated with 100 µM IPTG for one generation and samples analyzed for β-galactosidase activity. β-galactosidase assays were performed as previously described To assay the I and RpoHII promoter specificity, β-galactosidase assays were conducted in R. sphaeroides tester strains containing reporter gene fusions of the cycA (RSP_0296) P1 promoter with a variety of point mutations (see Results). These reporter strains were constructed as described above, with individual point mutations being generated by overlap extension PCR I or RpoHII) was subtracted from the measured LacZ activity for each mutant promoter.To test bases that contribute to RpoHE. coli CAG57102 mutant strain, the promoter library, and the β-galactosidase assay used to test the activity of R. sphaeroides RpoHIin vivo on mutant promoters were described previously R. sphaeroides RpoHI the E. coli rpoH gene of pSAKT32 IR. sphaeroides rpoH gene. At least triplicate assays for β-galactosidase activity were performed on all strains.The construction of the Figure S1I versus FLAG-RpoHI dependent change in gene transcription levels.Scatter plot of RpoH(TIF)Click here for additional data file.Table S1I and RpoHII target genes.RpoH(XLS)Click here for additional data file.Table S2I and RpoHII target genes.Predicted promoter sequences of RpoH(XLS)Click here for additional data file.Table S3Bacterial strains and plasmids.(XLS)Click here for additional data file.