Chromatin undergoes major remodeling around DNA double-strand breaks (DSB) to promote repair and DNA damage response (DDR) activation. We recently reported a high-resolution map of γH2AX around multiple breaks on the human genome, using a new cell-based DSB inducible system. In an attempt to further characterize the chromatin landscape induced around DSBs, we now report the profile of SMC3, a subunit of the cohesin complex, previously characterized as required for repair by homologous recombination. We found that recruitment of cohesin is moderate and restricted to the immediate vicinity of DSBs in human cells. In addition, we show that cohesin controls γH2AX distribution within domains. Indeed, as we reported previously for transcription, cohesin binding antagonizes γH2AX spreading. Remarkably, depletion of cohesin leads to an increase of γH2AX at cohesin-bound genes, associated with a decrease in their expression level after DSB induction. We propose that, in agreement with their function in chromosome architecture, cohesin could also help to isolate active genes from some chromatin remodelling and modifications such as the ones that occur when a DSB is detected on the genome. Genomic stability requires that deleterious events such as DNA double-strand breaks (DSBs) are precisely repaired. The natural compaction of DNA into chromatin hinders DNA accessibility and break detection. Therefore, cells respond to DSBs by triggering multiple chromatin modifications that promote accessibility and facilitate repair. We have recently developed a novel system whereby a restriction enzyme can be induced to inflict multiple DSBs across the human genome. This system permits high-resolution characterization of changes in the chromatin landscape that are induced around DSBs. While we previously reported the profile of H2AX phosphorylation (a primary event in chromatin remodelling that takes place in response to DSBs), we now provide the high resolution mapping of cohesin, a complex implicated in the 3-D organisation of chromosomes within the nucleus. Unexpectedly, we have discovered that cohesins play a role in the maintenance of gene transcription in regions where chromatin has been remodelled during the DSB response. IRradiation Induced Foci) and full activation of the DNA Damage Response (DDR) in vivo. Remarkably, γH2AX spreads across large chromatin domains surrounding DSBs, around 50 kb in yeast AsiSI) fused to the oestrogen receptor ligand binding domain. Using this system, we monitored γH2AX distribution and changes in transcription, around more than 20 DSBs located on chromosomes 1 and 6 using ChIP-chip DNA packaging into chromatin hinders detection and repair of DNA Double Strand Breaks (DSBs), and therefore DSB repair occurs simultaneously with multiple chromatin modifications, including histone acetylation, ubiquitylation and phosphorylation, as well as ATP dependant nucleosome remodelling and chromatin protein deposition or exclusion (for review Damage Induced Cohesion)  . This leImportantly, beyond its role in DSB repair and sister chromatid cohesion, another function for cohesin has recently emerged. In vertebrates, the cohesin complex accumulates at specific loci, mainly enhancer/promoters and sites bound by the CTCF insulator protein AsiSI-induced DSBs in order to both further refine its function in DSB repair and its potential impact on γH2AX spreading. Here we show that, in contrast to yeast, cohesin is only moderately recruited to AsiSI-induced DSBs in human cells and does not spread over more than 5 kb. Remarkably, cohesin binding antagonizes γH2AX accumulation within γH2AX domains. Depletion of the SCC1 cohesin subunit leads to both an increase in γH2AX and a DSB-dependent transcriptional downregulation of genes within γH2AX domains, suggesting that cohesins are, at least in part, responsible for the accurate transcriptional control observed in γH2AX domains. Finally, we also analyzed the consequences of cohesin depletion on the positions of γH2AX domain boundaries, and found that while most of these boundaries remained unaffected, at some genomic locations cohesin helped to confine γH2AX spreading.Given the multiple roles of cohesin in DSB repair, higher-order chromatin structure and transcriptional control, we decided to characterize the cohesin profile around AsiSI-ER fusion restriction enzyme (the AsiSI-ER-U20S cell line). Treatment with hydroxytamoxifen (4OHT) triggers nuclear localisation of the enzyme and induces DSBs at defined genomic loci, enabling ChIP analyses of protein recruitment at DSBs AsiSI-induced DSBs   , consistAsiSI-induced DSBs on chromosomes 1 and 6 (AsiSI sites), the SMC3 profile showed a weak increase upon 4OHT addition over a ∼5 kb region surrounding the DSB (AsiSI sites (on a 2 kb window) was significant (p<0.05)  .SceI-ER U20S cell line, in which one single DSB is induced upon 4OHT treatment. As observed on AsiSI-induced DSBs, we could detect a 4OHT-dependant increase of SCC1 at the I-SceI-induced DSB (300 bp), but not at 2.4 kb from the DSB  often coincided with peaks of SMC3 monitored before 4OHT treatment  of the establishment or maintenance of γH2AX on chromatin. Importantly, we could also detect this increase by immunofluorescence  within γH2AX domains We confirmed these data by Q-PCR on selected SMC3-bound  and two SMC3-unbound (GBP5 and GBP6) genes . TransfeAsiSI-induced γH2AX domains and active genes harbour low levels of γH2AX Gene transcription remains unaffected within AsiSI-induced γH2AX domain, we found that some domains appeared to be cohesin-independent while others showed extended spreading upon SCC1 depletion. This difference was not a consequence of elevated γH2AX within domains, since among domains that incurred a similar increase in γH2AX upon SCC1 depletion, some domains showed extended spreading   . Thus, aAccumulation of cohesin around DSBs has been proposed to enhance cohesion between sister chromatids in order to promote efficient repair by homologous recombination (HR) , in agreement with reports of γH2AX increase in irradiated, SCC1- and SMC3-depleted cells Both Pol II and Pol I transcription are down regulated in the vicinity of a DSB in an ATM-dependent manner AsiSI sites in the cell population, we also cannot exclude that other unrelated factor participate in the protection of active genes in γH2AX domains.First we observed a moderate but general increase of γH2AX levels on the cohesin-target genes encompassed in γH2AX domains after cohesin depletion, indicating that cohesins contribute to maintain reduced γH2AX level on genes. Secondly, for eight genes located in various domains, this was associated with a significant DSB-dependant transcriptional decrease. Since the effect of cohesin depletion on γH2AX levels occurred on most genes of the domains, it is likely that the trend observed on these eight genes is a general feature illustrating the role of cohesin in transcriptional maintenance, although further genome wide studies would be required to generalize our findings. It is also important to underline that both γH2AX increase on genes and DSB-dependant transcriptional decrease in cohesin depleted cells were quite moderate, and thus, while this could be to due siRNA efficiency, or to an asynchronous cleavage of This cohesin-dependant gene protection is unlikely to be a damaged–induced process since cohesin recruitment at DSB only occurs on the surrounding 2kb. Instead we favour the hypothesis that the cohesins already present on a normal, undamaged genome could protect active genes from the chromatin changes induced by DSBs, such as γH2AX which has been proposed to enhance chromatin compaction Since cohesins are known to mediate chromatin looping, they could also be involved in anchoring the chromosomal domain, within which γH2AX would spread. While we found that cohesin depletion triggers boundary expansion at some domains, we could not find a corresponding enrichment in cohesin at those positions . Thus, iIn summary, our results suggest that phosphorylation of H2AX after DSB is established on a pre-existing chromatin/chromosomal organization . While fRabbit polyclonal antibodies against SMC3 were raised using recombinant SMC3 (αSMC3-A) or an SMC3 peptide (αSMC3-B) and have been described in AsiSI-ER-U20S and I-SceI-ER-U20S cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with antibiotics, 10% FCS (Invitrogen) and 1 µg/mL puromycin at 37°C under a humidified atmosphere with 5% CO2. AsiSI-ER-T98G cells were cultured in Minimum Essential Media (MEM) GlutaMAX, supplemented with MEM Non Essential Amino Acid (NEAA), antibiotics, and 10% FCS (Invitrogen).AsiSI-ER-T98G cells was achieved by 72 hours of serum starvation (0% FBS). Cells were collected in G1 and G2 phase after 10H and 28H, respectively, of 20% FBS re-induction. Synchronisation of AsiSI-ER-U20S in G2 was achieved by an 18H R0-3306  9 µM treatment. For siRNA transfection, 5.0×106 cells were electroporated with 10 µL of 100 µM siRNA using the Cell Line Nucleofector kit V (Amaxa), according to the manufacturer instructions, and collected 48H after transfection. Sequences for siRNA are displayed Synchronization of II RT (Promega) with random hexamers. cDNAs were analyzed by Q-PCR using primers described in RNA was extracted using the RNAeasy kit (Qiagen) following manufacturer instructions. 1 µg of RNA was reverse transcribed using Im-Prom6 cells) were fractionated as reported Cell pellets   and analyzed as described in http://genome.ucsc.edu.In order to plot data with respect to transcription start sites (TSS), gene transcript positions and orientations were obtained from the refFlat table from UCSC (hg18). All genomic coordinates were from the genome assembly NCBI Build 36.1, and annotations were retrieved from the UCSC genome browser Microarray probe coordinates and data have been submitted to Array Express under accession number E-TABM-1164.AsiSI-ER-U20S cells. T4 ligase was heat inactivated at 65°C for 10 min, and DNA was fragmented by EcoRI digestion at 37°C for 2 h followed by heat inactivation at 70°C for 20 min. After a preclearing step, DNA was pulled down with streptavidin beads (Sigma) at 4°C overnight, and then washed 5 times in RIPA buffer and twice in TE. Beads were resuspended in 100 µL of water and digested with HindIII at 37°C for 4 h. After phenol/chloroform purification and precipitation, DNA was resuspended in 100 µL of water, and submitted to Q-PCR, using primers described in The full procedure for the cleavage assay has been previously described After transfection with siRNA, and 4OHT treatment for 4H, cells were fixed in PBS containing 3.7% formaldehyde for 15 min at RT, permeabilized in PBS-0.5% Triton X100 for 10 min, and blocked with 3% bovine serum albumine (BSA) for 30 min. After 2 h incubation with γH2AX antibody , cells were washed with PBS and probed for 1H with an Alexa Fluor 594 anti mouse antibody (Molecular Probes). Slides were mounted with Vectashield (Vector Laboratories), and images were acquired using a Leica microscope equipped with a charge-coupled device camera , and the MetaMorph software .Quantification of fluorescence levels was done on a least 100 nuclei using home-developed macros in ImageJ software  to normalize background, thresholds and measures.Figure S1AsiSI-ER-U20S cells without 4OHT treatment, using a mix of the two SMC3 antibodies or no antibody (mock), as indicated. SMC3 enrichment was scored by Quantitative Real Time PCR (Q-PCR) on a previously characterized cohesin binding site gapdh promoter (negative control). ChIP efficiency was calculated as % of input DNA immunoprecipitated. A representative experiment is shown.Validation of SMC3 antibodies. A, The specificity of SMC3 antibodies (α-SMC3-A or α-SMC3-B as indicated) was analyzed by western blot with HeLa nuclear extracts. B, HeLa nuclear extracts were immunoprecipitated using either a α-SMC3-A or α-SMC3-B antibodies as indicated. Flowthrough and immunoprecipitated samples were analyzed by western blot probed with α-SMC3-A or anti-SCC1 (Abcam) C, ChIP analyses were performed in (PDF)Click here for additional data file.Figure S2gapdh promoter as a negative control. ChIP efficiency was calculated as % of input DNA immunoprecipitated. A representative experiment is shown.Validation of SA1/SA2 antibodies. SA1 and SA2 antibodies were validated by western blot with HeLa nuclear extract (A), using control or SA1/SA2 siRNA transfected HeLa cells extracts (B), and in ChIP assay followed by Q-PCR (C) using a previously characterized cohesin binding site as a positive control (PDF)Click here for additional data file.Figure S3AsiSI-ER-U20S cells synchronized in G2 upon RO-3306 treatment (18H at 9 µM), before and after 4OHT, and analyzed by Q-PCR. Fold enrichment at two DSBs is shown relative to the negative locus (devoid of AsiSI sites). Note that 4OHT dependant recruitment of SCC1 at DSBs in G2 is similar to the changes observed upon DSB induction in asynchronous cells Click here for additional data file.Figure S4AsiSI-ER-U20S cells, either untreated or treated with 4OHT during 4H or 14H, were subjected to ChIP analyses using SCC1 (Abcam) or γH2AX antibodies as indicated. Enrichment was scored by Q-PCR in the vicinity of an AsiSI-induced DSBs (DSB1) and normalized to the signal observed on a genomic location devoid of DSBs. A representative experiment is shown.SCC1 recruitment at DSBs does not increase over time. (PDF)Click here for additional data file.Figure S5AsiSI-ER-U20S. A, Detailed view of the SMC3/input (black) in untreated AsiSI-ER-U20S cells and SCC1/input (grey) from HeLa cells, retrieved from AsiSI-ER-U20S cells (upper panel) and the SCC1/input signal in HeLa cells (retrieved from Profile of SMC3 in (PDF)Click here for additional data file.Figure S6AsiSI sites. Paired t-test was performed on the average value of data points within a 2000 bp window centered on the site using SMC3 data from −4OHT and +4OHT samples, as in p values were logged (base 10) and box plotted to demonstrate the significance of the t-test p value calculated around AsiSI sites , distribution (scattered uniformly across both chr 1 and 6) and number (24 sites per random run) to the actual SI sites  depicted(PDF)Click here for additional data file.Figure S7AsiSI-ER U20S 4OHT-treated or untreated cells. Western blot against SMC3 and SCC1 showed that the soluble pool of cohesin is not depleted after DSB induction. B, ChIP against SCC1 was performed in I-SceI-ER U20S cells before and after 4OHT treatment. The fold enrichment relative to a negative locus was scored by Q-PCR at the immediate vicinity of the I-SceI break (300 bp), and further away (2.4 kb), as indicated. A representative experiment is shown.The lack of cohesin spreading around DSBs is not due to a limited amount of soluble cohesin. A, Soluble (chromatin unbound) and insoluble (chromatin bound) fraction were prepared from (PDF)Click here for additional data file.Figure S8γH2AX is depleted at cohesin binding sites. Regions enriched in SCC1 were identified using the algorithm detailed in (PDF)Click here for additional data file.Figure S9p value are indicated.Cohesin rich genes show a low level of γH2AX. The average Log2 (γH2AX/input) (y axis) and Log2 (cohesin/input) (x axis) were calculated over the entire length of each of the 359 genes encompassed within γH2AX domains, and plotted against each other. Results are shown for SMC3 (top panel), and SCC1 retrieved from the HeLa dataset (PDF)Click here for additional data file.Figure S10AsiSI-ER-U20S. A, Detailed view of the SCC1 distribution observed in HeLa cells retrieved from AsiSI-ER-U20S cells . ChIP-chip data are presented as Log2  on selected areas of the chromosome 1. B, The location and orientation of the 3072 genes located on chromosome 1 and 6 were used to subset data with the transcribed sequence on the right hand side. The Log2 (SCC1/input) obtained in AsiSIER-U20S cells was plotted using a 200 bp sliding window for averaging and is shown over a 20 kb window centered on the TSS.Profile of SCC1 in (PDF)Click here for additional data file.Figure S11AsiSI site (arrow), expressed as log2 and smoothed using a 500 probes sliding window. B, The average Log2 (γH2AX/input) (y axis) and Log2 (SCC1/input) (x axis) were calculated over the entire length of each of the 359 genes encompassed within γH2AX domains, and plotted against each other. The correlation coefficient and the p value are indicated.SCC1 counteracts γH2AX. A, Detailed views of the γH2AX/H2AX signal (red) and the SCC1/input signal (black) around an (PDF)Click here for additional data file.Figure S12AsiSI-ER-U20S cells were transfected by electroporation with control (CTRL) or SCC1 siRNA. 48 hours after transfection, mRNA was extracted, reverse transcribed and the amounts of SCC1 and ribosomal phosphoprotein P0 cDNAs were measured by Quantitative real time PCR. SCC1 cDNA levels are shown relative to P0 levels. The mean and standard deviation of the mean (SDOM) from 3 independent experiments are shown. B, SCC1 protein level was analyzed by western blot in Control or SCC1 siRNA transfected AsiSI-ER-U20S cells . The same blot was also probed for alpha-tubulin as a loading control (lower panel). C, The depletion of chromatin-bound SCC1 by siRNA was analyzed by ChIP in siRNA transfected AsiSI-ER-U20S cells, using an anti SCC1antibody (Abcam ab992) or no antibody. SCC1 enrichment was assessed by Q-PCR on the four regions further described in SCC1 is depleted at the RNA level, protein level, and on chromatin upon SCC1 siRNA treatment. A, (PDF)Click here for additional data file.Figure S13AsiSI sites upon SCC1 depletion. Detailed views, around selected AsiSI sites, indicated by arrows (upper and middle panels) and on two genomic regions devoid of AsiSI sites (lower panels). γH2AX enrichment over input in control (dark red) and in siRNA SCC1 (light red) transfected AsiSI-ER-U20S cells, are shown expressed as log2 and smoothed using a 500 probes sliding window. ChIP-chip analysis was performed using chromatin from AsiSI-ER-U20S cells treated with 4OHT. The average of two independent experiments is shown. Note that within domains, the γH2AX signal increases upon SCC1 depletion. Genomic coordinates (x-axis) are indicated in megabase (MB).Example γH2AX profiles around (PDF)Click here for additional data file.Figure S14AsiSI sites, in cells transfected with control or SCC1 siRNA as indicated. The box plots represent the distribution of the values obtained for the 24 AsiSI sites. The γH2AX level in SCC1 depleted cells is significantly different from the level observed in control cells. B, Same as in A except that random windows of 4 kb (left panel), and 80 kb (right panel) outside γH2AX domains were averaged.γH2AX increases around DSBs in SCC1 depleted cells. A, The average Log2 (γH2AX/input) was calculated over a 4 kb window (left panel) or an 80 kb window (right panel) surrounding (PDF)Click here for additional data file.Figure S15AsiSI-ER-U20S cells were transfected with Control (CTRL) or SCC1 siRNA for 48 hours. Untreated or 4OHT treated cells were subjected to ChIP analyses against γH2AX. Enrichment was scored by Q-PCR within 5 γH2AX domains. Distances of the primers from the DSB are indicated. Data are normalized to the signal observed on a genomic location devoid of DSBs. Representative experiments are shown.4OHT-induced γH2AX increases in SCC1 depleted cells compared to control cells. (PDF)Click here for additional data file.Figure S16AsiSI sites. Genomic DNA was extracted from siRNA transfected AsiSI-ER-U20S cells treated or not with 4OHT for 4H and assayed for cleavage at AsiSI sites. Pulled down DNA was analyzed by quantitative PCR amplification using primers close to two cleaved AsiSI sites. The mean and standard deviation of the mean (SDOM) from 3 independent experiments are shown.SCC1 depletion does not change the cleavage efficiency of (PDF)Click here for additional data file.Figure S17AsiSI-ER-U2OS cells transfected with Control (CTRL) or SCC1 siRNA for 48H were treated with 4OHT for 4H and subjected to γH2AX immunofluorescence . Images were quantified and classified based on the percentage of their nucleus covered by γH2AX staining. Data are represented as the percentage of cells falling in each of four different categories. The mean and SDOM of three independent experiments are shown. SCC1 depletion leads to an increase of γH2AX as detected by immunofluorescence. (PDF)Click here for additional data file.Figure S18AsiSI site was calculated (x-axis) and plotted against the γH2AX ratio in cells transfected with siRNA SCC1 versus siRNA CTRL(y axis). Pearson correlation and p value are indicated.γH2AX increases on SCC1 rich domains upon SCC1 depletion. The averaged SCC1 signal over an 80 kb window around each (PDF)Click here for additional data file.Figure S19p values (paired t-test) are indicated above. Note that the biggest increase of γH2AX upon SCC1 depletion occurs at TSSs. While we can see a significant increase both upstream and downstream, it is much weaker than the increase observed at the TSS.γH2AX increases at TSS upon SCC1 depletion. The average Log2 (γH2AX/input) upstream to promoters (−2000 to −1600 bp) (left panel), at promoters (−200 to +200 bp) (middle panel) and downstream of promoters (+1600 to −2000 bp) (right panel) for the 359 genes encompassed in γH2AX domains were calculated, in Control and SCC1 siRNA transfected cells as indicated. Distributions are represented as box plots. The (PDF)Click here for additional data file.Figure S20p value are indicated.γH2AX increases on SCC1 rich genes upon SCC1 depletion. For each gene encompassed in γH2AX domains, the SCC1 signal was averaged and plotted against the ratio of γH2AX in SCC1 depleted versus control cells. Pearson correlation and (PDF)Click here for additional data file.Figure S21Genes showing high changes in γH2AX between SCC1 siRNA and control cells, show higher levels of cohesin. As for (PDF)Click here for additional data file.Figure S22Detailed views of the areas analyzed by Q-PCR with SCC1 siRNA. Detailed views of the SMC3/input (black) and γH2AX/H2AX (red) ChIP-chip data, on five cohesin-bound regions (A–E) and two cohesin-unbound regions (F–G). Positions of the primer pairs used for the Q-PCR analysis presented in (PDF)Click here for additional data file.Figure S23AsiSI-ER-U20S cells were transfected with the indicated siRNAs. After 48 h, cells were treated or not with 4OHT as indicated. Total RNAs were extracted and reverse transcribed. The amount of each cDNA was measured by quantitative real-time PCR, divided by the amount of P0 cDNA and calculated relative to 1 for cells transfected with the control siRNAs and not treated with 4OHT.Expression fold changes upon 4OHT treatment and SCC1 depletion. (PDF)Click here for additional data file.Figure S24Cohesin binding does not correlate with boundary position. A, Boundaries of γH2AX domains were aligned and overlaid (right and mirror left borders are combined). Data are shown over a 200 kb window centered on domain boundaries and averaged using a 10 kb window size. Profiles are shown for γH2AX (red) and for SMC3 (black) in control cells. Note that we cannot see a specific increase of SMC3 at the boundary. B, Detailed views of right boundaries from two γH2AX domains. Signals for SMC3 (black), γH2AX in control cells (red) and γH2AX in SCC1 depleted cells (orange) are presented. The upper domain shows an extension of γH2AX upon SCC1 depletion, while the lower domain does not (grey arrows). However this is not correlated with the presence of SMC3 at the boundaries (black).(PDF)Click here for additional data file.Figure S25AsiSI-ER-U20S cells and the spatial compartmentalization observed in GM06990 cells (see arrows).Comparison of γH2AX boundaries with chromosomal domain transitions. Hi-C realized with the human lymphoblastoid cell line GM06990, led to the identification of chromosomal domains at various scales, from the nucleus scale (such as the open and closed chromatin compartments) to a megabase scale. Chromosomal domains could be easily visualized using a heatmap to depict intrachromosomal interactions (PDF)Click here for additional data file.Figure S26AsiSI domains i.e. 46 boundaries) were retrieved and averaged (left and mirror right boundaries were combined). The averaged boundary (the 0 position) correlates with a chromosomal domain transition found using Hi-C.Averaged interaction matrix around γH2AX domain boundaries. The Hi-C interaction matrix (PDF)Click here for additional data file.Table S1AsiSI sites on chromosome 1 and chromosome 6. All genomic coordinates are from the genome assembly NCBI Build 36.1. The AsiSI sites efficiently cleaved were determined thanks to our previous analysis using both the γH2AX signal and the cleavage signal List of cleaved (PDF)Click here for additional data file.Table S2List of γH2AX domain boundaries on chromosome 1 and chromosome 6 determined using the γH2AX signal in siRNA control transfected cells. All genomic coordinates are from the genome assembly NCBI Build 36.1. The boundaries positions were determined using the algorithm described in (PDF)Click here for additional data file.Table S3List of primers used in this study.(PDF)Click here for additional data file.


Although transcriptional regulation of gadE in the context of acid resistance under the aerobic growth environment of Escherichia coli has been extensively studied, regulation of the operon under the physiologically relevant environment of anaerobic growth and its effect on the expression of the multidrug efflux proteins MdtEF in the operon has not been disclosed. Our previous study revealed that anaerobic induction of the operon was dependent on ArcA, the response regulator of the ArcBA two-component system, in the M9 glucose minimal medium. However, the detailed regulatory mechanism remains unknown. In this study, we showed that anaerobic activation of mdtEF was driven by the 798 bp unusually long gadE promoter. Deletion of evgA, ydeO, rpoS, and gadX which has been shown to activate the gadE expression during acid stresses under aerobic condition did not have a significant effect on the anaerobic activation of the operon. Rather, anaerobic activation of the operon was largely dependent on the global regulator ArcA and a GTPase MnmE. Under aerobic condition, transcription of gadE was repressed by the global DNA silencer H-NS in M9 minimal medium. Interestingly, under anaerobic condition, while ΔarcA almost completely abolished transcription of gadE-mdtEF, further deletion of hns in ΔarcA mutant restored the transcription of the full-length PgadE-lacZ, and P1- and P3-lacZ fusions, suggesting an antagonistic effect of ArcA on the H-NS mediated repression. Taken together, we conclude that the anaerobic activation of the gadE-mdtEF was primarily mediated by the two-component system ArcBA through antagonizing the H-NS mediated repression.The  Escherichia coli and the MexAB-OprM and MexXY-OprM systems in Pseudomonas aeruginosa, renders the bacteria a low level, intrinsic resistance to a wide range of toxic substances  antiporter GadC  of gastric acid  broth (USB) or M9 minimum medium supplemented with 0.2% glucose (USB), 0.2% casamino acids (CAA) (USB), 4 μg/ml thiamin (Sigma), 0.1 mM CaCl2, 1 mM MgCl2, 10 μg/ml ferric ammonium citrate (Sigma), and 0.5 μM (NH4)6Mo7O24 (Sigma). Without specific indication, exponential phase cells were harvested when A600 of cultures reached around 0.3. Antibiotic concentrations used for bacterial culture or colony screening were 100 μg/ml ampicillin (USB), 20 μg/ml kanamycin (USB), or 25 μg/ml chloramphenicol (USB). All other chemicals without specification were purchased from USB.The bacteria strains and plasmids used in this study are listed in Table -lacZ fusions were constructed based on the methods described previously . Primers for construction of other promoter-lacZ fusions were designed similarly and the detailed sequences are available upon request. The PCR fragments were purified using illustra GFX PCR DNA and Gel Band Purification Kit (GE) followed by NotI/HindIII (NEB) digestion, and then were ligated into NotI/HindIII (NEB) digested single copy plasmid pNN387 using Quick Ligase (NEB). Colonies were screened by chloramphenicol resistance and then verified by colony PCR and DNA sequencing .Promoterchromosomal mdtE-FLAG in various of deletion mutants was achieved by P1 phage transduction using the lysate from the strain AY0451 .Construction of 3 cells/ml) into screw-capped culture tubes filled with M9 medium, and subsequently cultured in 37°C water bath without aeration. When A600 of the cultures reached ~0.3, tetracycline (10 μg/ml) was added to terminate protein synthesis and cell growth, and the cultures were placed on ice until assayed. The assays were performed in triplicate, and results were presented as the mean in either Miller unit or the percentages relative to the activity of full length promoter-lacZ fusion in WT (wild type) strain. Error bars indicate the standard deviation.β-galactosidase activity assay was performed based on the method by Miller  and detaE. coli MG1655 was mixed with 1.25 ml ice-cold ethnol/phenol stop solution (5% water-saturated phenol pH 4.5 in ethanol) and placed on ice for 10 min before being harvested by centrifugation at 4000 g for 9 min at 4°C. After removing supernatant, the cell pellet was frozen in liquid nitrogen and stored at −80°C to aid lysis. Cells were lysed by resuspending in 800 μl TE buffer (pH 8.0) containing 1.4 μl 36 kU μl−1 lysozyme (epicenter), and then placed in 64°C water bath for 2 min. After incubation, 88 μl 3M NaOAc (pH 5.2) was added to adjust the pH and ion strength of the lysate solution. Subsequently, acid-phenol/chloroform extraction followed by ethanol precipitation was performed to obtain the total RNA following the manufacturer's instruction. To remove trace amount of genomic DNA contamination, the extracted RNA was subject to DNase I treatment using the turbo DNA Free Kit (Amibion). Absence of genomic DNA contamination was confirmed by PCR suing the prepared RNA as template. The quantity of RNA was determined using NanoDrop 2000 (Thermo Scientific).8 ml anaerobically grown  culture of 1 μg of RNA which was treated with the turbo DNA Free Kit (Amibion) as above and shown as tight, no smearing band on the agrose gel (following the instructions of the FirstChoice RLM-RACE Kit (Ambion)) was treated with the Tobacco Acid Pyrophosphatase (TAP), ligated to the 5′ RACE adapter, and reverse transcripted with reagents supplied in the FirstChoice RLM-RACE Kit (Ambion) following the manufacturer's instruction. The obtained cDNA was subsequently utilized to perform outer (primer: PgadEout: 5′-TCCAGAAATTTAATCGCTTCTTCATC-3′) and inner (primer: PgadEin: 5′-GTACTCGAGGTGATTATCTTTCAACTGCCAAAAGC-3′) PCR using Fastart Taq (Roche) DNA polymerase. The PCR products were gel band purified using PCR DNA and Gel Band Purification Kit (GE) and cloned into BamHI and XhoI sites of pPK7035  to log phase (A600 = 0.3) in M9 minimal medium supplemented with 0.2% glucose, 0.2% CAA, 4 μg/ml thiamin, 0.1 mM CaCl2, 1 mM MgCl2, 10 μg/ml ferric ammonium citrate, and 0.5 μM (NH4)6Mo7O24. Cells were harvested by centrifugation at 3800 g at 4°C for 10 min. Cell pellet was resuspended in lysis mix (BugBuster reagent (Merck) supplemented with 2.5 mg/mL lysozyme and 10 U/mL DNase I (Invitrogen)). Following the cell lysis, a small fraction of the lysis was subject to the Dc Protein Assay kit (BioRad) to measure the concentration of total proteins in the lysate. The volumes of the cell lysis loaded onto the SDS-PAGE were then determined based on the concentrations of each of the samples such that each of the lanes contains the same amount of total protein. The lysis was then heated at 55°C for 25 min prior to being subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Separated proteins were electrically  transferred to a nitrocellulose membrane (Bio-Rad) for 1.5 h. After blocking with 5% non-fat milk in 1% TBST , the membrane was incubated with monoclonal anti-FLAG antibody (Sigma), and then secondary antibody . After treatment of the membrane by the ECL Plus Western Blotting Detection Reagents , the protein bands were visualized by X-ray film development. Signals corresponding to MdtE-FLAG proteins were quantified using ImageJ  and are presented as percentages relative to the level of MdtE-FLAG in the wild type strain.in vitro phosphorylation of His6-ArcA protein was following the method described previously  to induce the overexpress of His6-ArcA. After grown for additional 2.5 h, cells were harvested by centrifugation. Cell pellet was then resuspended in 4 ml of buffer A  containing 1.3 mg/ml lysozyme, 30 g/ml DNase and RNase. Cells were then incubated at room temperature for 30 min followed by sonication to lyse the cells. Cell lysate was obtained by centrifugation at 15,000 g for 30 min and then was subjected to a 1.5-ml Ni-nitrilotriacetic acid–agarose column (Qiagen) equilibrated with buffer A. Following washing the column with 10 ml buffer A containing 10 mM imidazole, His6-ArcA was eluted with buffer A containing 50 mM imidazole. Purified His6-ArcA protein was phosphorylated by incubating the protein in TEGD buffer  supplemented with 5 mM MgCl2 and 50 mM  carbamoyl phosphate. The mixture was incubated for 90 min at 25°C, and then the phosphorylated ArcA was used immediately for DNA-binding reaction. To initiate the DNA binding reaction, various concentration of DNA probes and phosphorylated proteins were mixed and subsequently incubated at 37°C for 20 min in EMSA binding buffer . The reaction mixture was then subjected to 6% non-denaturing polyacrylamide gel electrophoresis in 0.5 × TBE buffer. The polyacrylamide gel was visualized under UV light (254–366 nm) following the staining in 0.5 × TBE buffer containing 0.5 μg/ml ethidium bromide (EB) for 10 min.Purification and subsequent MdtEF are located downstream of the gadE gene, which encodes a central regulator of the glutamic acid dependent acid resistance system in E. coli  is involved in the transcription of mdtEF under anaerobic condition. To address this, we constructed lacZ fusion of the intergenic region (denoted as PmdtE-lacZ) in the single copy plasmid pNN387 and performed β-galactosidase activity assay. It was shown that no transcription from PmdtE-lacZ was detected under either aerobic or anaerobic condition, while transcription from PgadE-lacZ constructed in the same vector was significantly induced under anaerobic condition (data not shown). These results suggested that anaerobic transcription of mdtEF was primarily initiated from the gadE promoter. Therefore we focused on the 798 bp PgadE region in the following investigation to examine its contribution to the expression of mdtEF under anaerobic condition.gadE start codon respectively, have been identified in E. coli grown in LB glucose to early stationary phase (A600 = 1.75) , -124 (T2/T4), -566 (T3), is active under anaerobic conditions. We next investigated the transcription regulatory mechanism of gadE-mdtEF from all these sites under anaerobic condition.In previous studies, three transcription starting sites, located at -124 (T1), -324/-317 (T2), and -566 (T3) relative to the gadE region in previous studies  detected from the RACE analysis might be a digestive product from the transcripts initiated from the other three sites.Following the designation of the Py Figure   of the transcription of PgadE-lacZ, and deletion of H-NS caused a moderate increase of PgadE-lacZ activity, suggesting that these regulators are not responsible for the activation of mdtEF under anaerobic condition. Interestingly, deletion of the GTPase MnmE caused a significant decrease of the transcription of gadE-mdtEF, suggesting that MnmE may also contribute to the activation of gadE-mdtEF under this condition. Since the current study focuses on the regulation of the multidrug efflux genes mdtEF present in the same operon, we next examined whether deletion of these genes caused alteration of MdtEF protein levels. We constructed chromosomal mdtE-flag in these strains (except for ΔgadE) and performed Western blot to examine the level of MdtE-FLAG. As shown in Figure mnmE, which caused a more significant reduction of MdtE expression at translational level. Together these results confirmed that ArcA and MnmE are primarily responsible for the anaerobic activation of mdtEF.We constructed strains containing deletion of each of these genes and measured β-galactosidase activities of PgadE-mdtEF in E. coli grown in M9 glucose medium at log phase. As shown in Figure gadE-lacZ activities in wild type strain, and in ΔarcA, ΔmnmE, ΔevgA, ΔydeO, ΔgadE, ΔgadX mutants are very low (around 20 Miller units), consistent with the fact that ArcA is not active under this condition. However, in Δhns mutants, the PgadE-lacZ activities increased more than 10-fold to about 250 Miller units, suggesting H-NS strongly represses the expression of gadE-mdtEF under aerobic condition in the M9 glucose minimal medium. This result is consistent with previous findings that H-NS represses the transcription from PgadE through its interaction with various sites in the gadE promoter region  containing glucose or during stationary phase in rich medium  are involved in the transcription initiation of gadE-mdtEF, we cannot rule out the possibility that they might participate in the regulation of individual promoters contained in the complete 798 bp promoter of gadE. For example, it is possible that a certain regulator activates the transcription from one promoter region while represses transcription from another promoter, consequently cancels its effect on the transcription from the full promoter in our assay. To verify this, we examined the anaerobic transcription activity of those PgadE truncations in the deletion mutants constructed in this study except for GadX and GadW since they were indicated to be active in low pH, complex medium or stationary phase , indicating that these regulators had no effect on the transcript from the individual promoters contained in the 798 bp full promoter of gadE either under anaerobic condition.Our investigation above showed that ArcA and MnmE are the major activators responsible for the expression of gadE caused different effects on the transcription from different promoters contained in the full 798 bp PgadE region, which is dissimilar from that observed in the case of ΔevgA, ΔydeO, and Δhns. As shown in Figure gadE deletion had no significant effect on the transcription from the lacZ fusions of the full length, P3, P3+P2/P4, as well as P3+P2/P4+P1, deletion of gadE caused almost complete abolishment of transcription from the P2/P4+P1 and the P1 promoters. This observation combined with the results above which showed that individual P2/P4 was not active under anaerobic conditions suggested that GadE autoactivates the transcription of gadE-mdtEF from its P1 promoter. This result is consistent with a previous truncation study performed under aerobic condition, where deletion of gadE caused decrease of transcription from P2/P4+P1 and P1 promoters  or mnmE (gray bars) caused decrease of transcription from the full length and all the truncation mutants with variable promoter lengths, indicating ArcA and MnmE might affect transcription from multiple promoters, especially the P3 and P1 regions, since P2/P4 and P individual promoters were not active under this condition as shown above. Considering that the activation of the P1 promoter is dependent on GadE, it is possible that ArcA and MnmE primarily activate the P3 promoter.Since ArcA and MnmE were identified as transcription activators of the y Figure . SurprisgadE is through its direct binding on the gadE promoter region, we performed EMSA to measure the direct binding of ArcA-P (phosphorylated His6-ArcA) to the P1 and P3 promoters. As shown in Figure gadE promoter encompassed in the individual P1 and P3 promoters and activates the expression of gadE under anaerobic condition. Genome wide CHIP-chip (Chromatin immunoprecipitation with microarray) and CHIP-seq study which determines all the ArcA binding sites across the E. coli K-12 MG1655 genome under anaerobic conditions in glucose minimum media also identified two broad ArcA binding peaks in the gadE promoter. Furthermore, the presence of the ArcA peaks correlated with the presence of a strong σ70 peak comparing with the very low σ70 peak on this region under aerobic condition , further confirming the ArcA dependent activation of the gadE-mdtEF operon through its direct binding of multiple sites on the gadE promoter.To examine whether the ArcA dependent activation of PgadE-mdtEF is largely repressed by the DNA silencer H-NS under aerobic conditions in M9 glucose medium, whereas under anaerobic condition in the same medium its expression is activated primarily in an ArcA dependent manner. This led us to ask how the H-NS mediated repression is replaced by the ArcA dependent activation during the transition of the facultative bacterium E. coli from its aerobic to anaerobic growth. To answer this question, we first examined which individual promoter region is responsible for the H-NS mediated repression under aerobic condition. As shown in Figure gadE, deletion of hns caused de-repression of transcription from the P1 and P3 promoters, but no effect on the transcription from the P2/P4 and P promoters, indicating that H-NS represses the transcription of PgadE from both the P1 and P3 promoters under aerobic condition in the M9 glucose medium. This pattern is similar to that of how ArcA achieves its activation of PgadE under anaerobic condition which is also through its interaction with the P3 and P1 promoters.Our results above showed that the expression of gadE-mdtEF during the transition from aerobic to anaerobic growth. One possibility is that H-NS is inactive under anaerobic conditions and it disassembles from the gadE promoter, allowing ArcA which is active under this condition to bind the promoter of gadE and initiate gene transcription. Alternatively, under anaerobic conditions H-NS can still bind to the gadE promoter, however, its binding is antagonized by the activated ArcA, resulting in the recruitment of RNAP and transcript initiation of the operon under this condition. To differentiate these two possibilities, we constructed ΔarcA Δhns double deletion and examined transcription of PgadE-lacZ in this strain. If H-NS is inactive under anaerobic condition and is disassembled from the gadE promoter, then transcription of PgadE-lacZ in ΔarcA Δhns should be similar to that of ΔarcA alone. However, if H-NS was not inactivated, rather, it can still bind to the promoter but its binding was antagonized by ArcA, then its binding to the gadE promoter should be resumed when ArcA is absent and an increase of PgadE-lacZ activity should be observed in Δhns ΔarcA than ΔarcA single deletion strain. As shown in Figure gadE-lacZ in ΔarcA Δhns double deletion did display significantly higher activity than that in ΔarcA strain, suggesting that the anaerobically up-regulated gadE-mdtEF expression was through the antagonization of H-NS mediated repression by ArcA. Western blot analysis of chromosomal MdtE-FLAG showed the same pattern as that of transcription assay . To address this, we tested the effect of ΔmnmE on the transcription of two ArcA dependent promoters, PlpdA and the mutated PicdA. Transcription of lpdA gene has been shown to be mediated by the repression of ArcA under anaerobic conditions. In the case of icdA, it contains two known promoters, PI and PII, in which PI is the primary promoter and is repressed only by ArcA. In the mutated PicdA-lacZ, transcription from PII was eliminated by removal of its −10 element such that transcription repression of this PicdA-lacZ is exclusively ArcA dependent . If MnmE affects either the production or phosphorylation of ArcA (which affects the regulatory activity of ArcA), ΔmnmE should cause full or partial de-repression of these two promoters under anaerobic condition. As shown in Figure icdA-lacZ and PlpdA-lacZ fusions were significantly repressed by ArcA under anaerobic condition, since ΔarcA caused de-repression of their transcription under anaerobic condition in M9 glucose medium, the same condition used in this study. However, ΔmnmE had little or no effect on the transcription of these two promoters, suggesting that MnmE does not affect the production or phosphorylation of ArcA in vivo. Therefore, the observed MnmE mediated anaerobic activation of gadE-mdtEF was not due to its potential effects on ArcA.As shown in Figure E. coli, transcriptional regulation of the gadE-mdtEF operon under various of growth phases and conditions has been extensively investigated. While previous studies largely focused on its relevance in bacterial acid resistance under aerobic growth environment, the current study aimed to dissect the up-regulation of the operon under the physiologically relevant environment of anaerobic growth and its effect on the production of the multidrug efflux protein MdtEF.Owing to the important roles of GadE and MdtEF in acid resistance and drug efflux respectively in gadE expression in the context of acid resistance have been disclosed. These include the EvgA-YdeO-GadE feed forward regulatory loop which is effective in minimal glucose medium under aerobic condition  was observed, deletion of rpoS or gadE did not have any effect on the transcription of PgadE-lacZ in this growth phase (data not shown), whereas ΔarcA still caused significant decrease of the gadE-mdtEF expression in the same growth phase. These results suggested that anaerobic expression of the gadE-mdtEF operon is primarily regulated by ArcA in both log phase and stationary phase under anaerobic condition. It is noted that previously a putative ArcA binding site located at the P2 region was proposed ” as the template and the motif was proposed as the ArcA binding site by Lynch and Lin , significant accumulation of the indole nitrosative by-product occurred when pH dropped to 5.8 or below  in the presence of the acrAB genes, suggesting that the MdtEF pump exhibits at least certain degree of substrate specificity even in the presence of the housekeeping pump AcrAB. It is highly likely that MdtEF also expels other metabolic by-products accumulated during acid stresses. If that is the case, there are probably more extensive relevance between MdtEF mediated drug efflux and the physiology of E. coli during acid resistance. This relevance probably can be disclosed through metabolic profiling by comparing the total metabolites in ΔmdtEF and WT strain grown during acid stresses.This raises the interesting question of whether the housekeeping pump AcrAB is also involved in the nitrosative and/or acid resistance by expelling metabolic by-products under anaerobic conditions. Although the broad substrate specificity of AcrAB-TolC was established in terms of its capability of exporting antibiotics, we speculate that the same degree of broad substrate specificity may not apply in terms of its capability of exporting physiological substances, since it has been well recognized that expression of certain efflux pump genes other than the housekeeping AcrAB pump is activated by specific environmental or physiological conditions Piddock, . We recegadE at exponential growth in LB medium with glucose (LBG) or stationary growth of E. coli in unbuffered LBG under aerobic condition has been reported  suggested that MnmE does not affect the production and/or activity of ArcA under anaerobic conditions, and thus the observed reduction of gadE-mdtEF expression in ΔmnmE strain was not due to its potential effect on ArcA.MnmE is a GTP binding protein that is involved in tRNA modification . This observation is not in agreement with a previous finding which suggested that MnmE dependent gadE expression was induced only in the presence of glucose. Thus, although several studies support the notion that the GTPase MnmE participates in the transcriptional activation of the gadE-mdtEF operon under certain growth conditions and phases, how exactly MnmE is involved in the process is still ambiguous. This is largely due to the fact that the exact biological function of MnmE itself is still elusive. Obviously, further studies of MnmE protein is needed before we can reveal its role in the up-regulation of gadE-mdtEF under the physiologically relevant conditions.Studies also indicated that MnmE dependent conditional expression of gadE-mdtEF promoter is highly conserved among the pathogenic E. coli strains, including pathogenic strains O157:H7, 2369, RS128 and CFT073 and Shigella (96–99% identity), and GadE has been demonstrated to be indispensable for the survival of E. coli O157::H7 in a simulated gastric environment  activates the expression of the gadE-mdtEF operon, it is conceivable that synergetic activation of the system by both regulatory circuits may occur during the transition of E. coli to human host, particularly the passage of the gastrointestinal tract, thus greatly facilitate the adaptation and survival of this neutrophilic bacterium in its host niches.It is noteworthy that the 798 bp sensory integration region of the The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Our understanding of genomic regulation was revolutionized by the discovery that the genome is pervasively transcribed, giving rise to thousands of mostly uncharacterized non-coding ribonucleic acids (ncRNAs). Long, ncRNAs (lncRNAs) have thus emerged as a novel class of functional RNAs that impinge on gene regulation by a broad spectrum of mechanisms such as the recruitment of epigenetic modifier proteins, control of mRNA decay and DNA sequestration of transcription factors. We review those lncRNAs that are implicated in differentiation and homeostasis of metabolic tissues and present novel concepts on how lncRNAs might act on energy and glucose homeostasis. Finally, the control of circadian rhythm by lncRNAs is an emerging principles of lncRNA-mediated gene regulation. This paradigm was fundamentally called into question by results obtained from whole-transcriptome sequencing efforts  over thnsortium . Althougnsortium , recent nsortium . One lognsortium , to Drososophila , plants osophila  and humaosophila . Given tosophila . It was osophila  and goesXIST . Interestingly, lncRNAs on one hand exhibit many similarities with protein-coding transcripts: As true for mRNAs, lncRNAs are transcribed by RNA-polymerase (Pol) II  II , spliced(Pol) II , are par(Pol) II  and even(Pol) II . Further(Pol) II . It is n(Pol) II . On the (Pol) II . Further(Pol) II .Currently, intensive research efforts are underway to better understand the molecular basis of gene regulation by lncRNAs. To date, four major paradigms have emerged on how lncRNA impinge on gene regulation:cis- or trans. Indeed, early insights into lncRNA-based gene regulation have revealed the recruitment of the inhibitory polycomb repressive complex (PRC) 2 and the activating Trithrorax/MLL chromatin modifiers to specific genomic loci by the lncRNAs HOTAIR , which control the expression of neighboring protein-coding genes. Elegant follow-up studies using chromosome conformation capture (3C) technology revealed that the co-activator complex Mediator is involved in tethering eRNAs to their gene targets. Hence, lncRNAs regulate the three-dimensional (3D) structure of chromosomes via Mediator-dependent chromosome looping  that acts as BACE1 ceRNA and concomitantly increases Bace1 mRNA stability and leads to augmented deposition of Aβ-plaques in Alzheimer’s disease  and are required for AR-dependent gene transcription. In androgen-refractory prostate cancer, PRNBR1 and PCGEM1 are robustly expressed and are implicated in the ligand-independent activation of AR signaling  into changes in gene expression. NFkB activation induces the transcription of a specific subset of lncRNAs, apart from the induction of classical inflammatory protein-coding genes. Among this subset of TNF-regulated lncRNAs, a lncRNA termed Lethe is recruited to the NFkB effector subunit RelA in an inducible fashion and inhibits RelA from DNA-binding and target gene activation  1alpha transcription factors. This HIF1a-linc-p21 circuit controls the hypoxia-evoked increases of the glycolytic “Warburg effect” in tumor cells . Key to the understanding of whole-body metabolism are the pleiotropic effects of the anabolic master regulator insulin which simultaneously controls peripheral as well as central-nervous system-related aspects of metabolism . ResistaCRNDE ] points H19 is involved in the intergenerational transmission of diabetes mellitus [gestational diabetes mellitus (GDM)] and the GDM-associated impairments of islet infrastructure and function . Concomitantly, RNAi-mediated SRA loss-of-function interfered with in vitro differentiation of 3T3-L1 preadipocytes  serves as storage organ for excess nutrients, brown adipose tissue (BAT) dissipates the proton gradient across mitochondrial membranes to generate heat via the BAT-intrinsic uncoupling protein 1 UCP1; . The accipocytes . In a seipocytes  of whichAlu elements located within the 3′UTR of an SMD target and an Alu site localized within a class of lncRNAs called 1/2sbsRNAs (1/2-STAU1-binding site RNAs) triggered SMD , a condition of reduced terminal differentiation of myoblasts. Reinstating DMD-associated downregulation of linc-MD1 expression via lentiviral delivery led to improved maturation of DMD myoblasts. In a study published by Braveheart (Bvht) was demonstrated to be required for differentiation of mesodermal progenitors toward mature cardiomyocytes via interaction with PRC2 epigenetic modifiers. This report for the first time implicated a tissue-specific lncRNA in maintaining cell fate during mammalian organogenesis.The differentiation of skeletal muscle cells (myogenesis) is regulated by a complex, yet well understood, evolutionarily conserved circuitry of protein-coding genes which control the timely growth, morphogenesis, and terminal maturation of muscle progenitors , a CNS-controlled genetic disorder circadian rhythm with an associated dysregulation of metabolism and the development of obesity, was shown to be influenced by a PWS-associated lncRNA called 116G. After splicing, a lncRNA consisting of the remnants of 116G (termed 116HG) bound to the transcriptional activator RBBP5 and ensures a physiological circadian rhythm in the brain. Mice deficient for 116HG exhibit metabolic disorders due to the dysregulation of diurnally expressed circadian genes like Clock, Cry1, and Per2 in the CNS  such as miRNAs can be packaged into exosomes and released after binding to recipient cells, thus constituting a novel and intriguing way for ncRNAs to regulate systemic aspects of metabolism . Similarin vivo -mediated inhibition of disease-associated lncRNAs  effectively improves degenerative diseases like myotonic dystrophy type 1 (DM1) in mice  efficiently silence the expression of ncRNAs such as miRNAs and are generally well tolerated in vivo . These ain vivo  and adopin vivo  with unpc health . In conte models ] showcas in mice .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


CHD5 is down-regulated in several cancers, including leukemia and is responsible for tumor generation and progression. However, the mechanism of CHD5 down-regulation in leukemia is largely unknown. In this study, quantitative reverse-transcriptase polymerase chain reaction and western blotting analyses revealed that CHD5 was down-regulated in human leukemia cell lines and samples. Luciferase reporter assays showed that most of the baseline regulatory activity was localized from 500 to 200 bp upstream of the transcription start site. Bisulfite DNA sequencing of the identified regulatory element revealed that the CHD5 promoter was hypermethylated in human leukemia cells and samples. Thus, CHD5 expression was inversely correlated with promoter DNA methylation in these samples. Treatment with DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (DAC) activates CHD5 expression in human leukemia cell lines. In vitro luciferase reporter assays demonstrated that methylation of the CHD5 promoter repressed its promoter activity. Furthermore, a chromatin immunoprecipitation assay combined with qualitative PCR identified activating protein 2 (AP2) as a potential transcription factor involved in CHD5 expression and indicated that treatment with DAC increases the recruitment of AP2 to the CHD5 promoter. In vitro transcription-factor activity studies showed that AP2 over-expression was able to activate CHD5 promoter activity. Our findings indicate that repression of CHD5 gene expression in human leukemia is mediated in part by DNA methylation of its promoter.Chromodomain helicase DNA binding protein 5 (CHD5) was previously proposed to function as a potent tumor suppressor by acting as a master regulator of a tumor-suppressive network.  Previous studies report that tumors and cell lines commonly harbor deletions in genes that encode proteins protecting against malignancy. For example, a deletion in human chromosome 1p36, first reported for a neuroblastoma in 1977 CHD5, CHD3 and CHD4 also have plant homeodomain (PHD) motifs Based on sequence similarity with members of the chromodomain superfamily of proteins, the human chromodomain helicase DNA binding protein 5 (CHD5) is believed to function as a chromatin remodeling protein CHD5 in cancer, the mouse Chd5 has been identified as a tumor suppressor.CHD3 and CHD4 are components of the nucleosome remodeling complex; an assembly of proteins that remodels chromatin by sliding nucleosomes out of the way, thereby providing polymerase with the access needed to activate gene expression. Other than this homology between CHD5 and previously described chromatin remodeling proteins, little functional data existed for this protein. Further supporting the role of CHD5 expression has been shown to be epigenetically silenced by promoter hypermethylation in a variety of human cancers, including neuroblastomas CHD5 mutations have been implicated in head and neck squamous cell carcinoma CHD5 expression through transcriptional repression CHD5 expression is also a favorable predictor of survival following anticancer therapy CHD5 might be important for preventing cancer. Loss of Chd5 enhances proliferation; whereas Chd5 gain compromises proliferation CHD5 expression inhibited proliferation and tumor growth in neuroblastoma CHD5 facilitates expression of a tumor-suppressive network that includes p16 and p19, encoded by the cyclin-dependent kinase inhibitor 2A (Cdkn2a) locus CHD5 to bind unmodified histone 3 (H3) is essential for tumor suppression CHD5 PHD domain abrogated CHD5–H3 interaction and consequently compromised proliferation inhibition, inducing differentiation and suppressing tumorigenesis Cdkn2a, and other genes encoding proteins implicated in chromatin dynamics and cancer-associated pathways. These data taken together strongly suggest that CHD5 modulates the expression of multiple genes regulating pathways involved in tumorigenic processes In addition to regulating processes that are fundamental for cancer prevention, CHD5. Although this does not support CHD5 deficiency as a causal event in these cancers, the finding that mice heterozygous for the 4.3-Mb interval encompassing Chd5 are prone to spontaneous lymphoma CHD5 is deleted in lymphoid cancers in mice provide evidence that CHD5 deficiency plays a key role in these hematopoietic malignancies Deletions of regions including Chd5 in a mouse model of chromosomally unstable lymphoma showed that CHD5 is one of only three genes mapping to the minimally common region of deletion that they discovered in human T-cell acute lymphoblastic leukemia/lymphoma CHD5 in leukemia, we hypothesize that CHD5 is inactivated in leukemia and sought to elucidate the mechanism of inactivation. Here we demonstrate the following: CHD5 was down-regulated in human leukemia cell lines and samples, the important regulatory region of the CHD5 gene is localized 500–200 bp upstream of the transcription start site (TSS), hypermethylation was observed in these important regulatory elements, and the hypermethylation of the CHD5 promoter repressed transcriptional activity. Furthermore, the activating protein 2 (AP2) binding site was identified as a region strongly regulating CHD5 expression. Our findings indicate that repression of CHD5 gene expression in human leukemia is mediated in part by hypermethylation of the identified CHD5 regulatory element.Given the pivotal role of Human leukemia cell lines K-562, HL-60, KG-1a and Jurkat were purchased from American Type Culture Collection  and were maintained in RPMI 1640 medium  supplemented with 10% fetal bovine serum (Gibco). Bone marrow obtained from 12 healthy donors, 50 acute lymphoblastic leukemia (ALL) patients, 50 AML patients and 50 chronic CML patients  was donaTotal RNA was prepared using Trizol reagent  according to the manufacturer’s instructions. RT-PCR was performed on 0.5 mg total RNA using a PrimeScript RT Reagent Kit with gDNA Eraser . qPCR was performed using an ABI 7500 Real-Time PCR system and a SYBR Premix Ex TaqTM II kit (Takara). Gene expression of CHD5 was normalized to β-actin and all samples were analyzed in triplicate. Primers for qRT-PCR are shown in , Hercules, USA) and 50−100 µg protein was analyzed by using standard procedures, with antibodies specific for CHD5  and β-actin . Immunoreactive bands were visualized by chemiluminescent detection . β-Actin was included as a loading control. All extracts were prepared in duplicate, and at least three independent experiments were conducted.Cells were lysed in Laemmli buffer and protein concentrations were determined using Bio-Rad protein assay kits  and MethPrimer to identify CpG islands, and corresponding primers were used for BGS. Genomic DNA was isolated using the genomic DNA isolation kit . DNA (1 µg) was converted with EpiTect Bisulfite Kit (Qiagen). BGS primers [5′-GTTGTTTTGAAGATTTTGTTTT -3′ and 5′-CTAATTACTATAACAACCCCATCCC-3'] were used to amplify the CpG island located at 560–240 bp upstream of the TSS. The PCR products were purified using agarose gel electrophoresis and the Qiagen purification kit (Qiagen), and were cloned into the PMD18-T Vector (Takara) for sequencing. A minimum of eight colonies were selected for sequencing. Methylation data from BGS were analyzed using BiQ Analyzer software to generate lollipop diagrams (which list the percentage of methylation at each CpG) and to calculate the efficiency of bisulfite conversion. Analysis of non-CpG cytosines indicated the efficiency of bisulfite conversion was ∼99%.For dose–response experiments, K-562 and Jurkat cells were treated with DNA methyltransferase inhibitor 5-aza-2-deoxycytidine  at 0, 0.5, 2.5, 5, 10, and 20 µmol/L for 4 days, and HL-60 and KG-1a cells at 0, 0.1, 0.5, 1, 2.5, and 5 µmol/L. The medium was exchanged every other day with the same concentration of DAC. All experiments were performed in duplicate and repeated twice.http://genome.ucsc.edu/cgi-bin/hgGateway). The promoter was analyzed for transcription factor binding sites (TBSs) using WWW Promoter Scan The location and putative domains of the CHD5 prompter were predicted using the UCSC Genome Browser , a promoterless luciferase expression vector, to produce the pGL4.10-CHD5-2000 to −1 recombinant vector. A set of reporter constructs containing truncated CHD5 promoter sequences generated by progressively deleting 200 or 100 bp from the 5' or 3' end were PCR amplified from pGL4.10-CHD5-P-WT and self-ligated. Constructs pGL4.10-CHD5-2000 to −370 and pGL4.10-CHD5-356 to −1, in which the AP2 binding site (located at –369 to –357 bp from the TSS) is deleted, were PCR amplified from pGL4.10-CHD5-2000 to −1. A 321 bp region located at –560 to –240 was PCR amplified from pGL4.10-CHD5-2000 to −1 and the fragments were methylated in vitro with SssI, HpaII and HhaI methylases (New England Biolabs), or no enzyme (mock), according to the manufacturer’s instructions. SssI methylates all 5′-CpG-3′ sites, HpaII methylates only the CpG within the sequence 5′-CCGG-3′, and HhaI methylates only the CpG within the sequence 5′-GCGC-3′. Methylation efficiency was confirmed using HhaI, HpaII, and McrBC digestion (NEB). Methylated or mock-methylated fragments were ligated back into pGL4.10 vector to produce reporter constructs (pGL4.1-CHD5-P-CR for mock-methylated fragment). Primers used to generate these reporter constructs are listed in The CHD5 promoters was calculated by normalizing to Renilla luciferase activity and compared to normalized Luc luciferase activity from the pGL4.10-Basic vector. The experiment was repeated twice to confirm the reproducibility of results.Cells were cultured in 24-well plates. Twenty-four hours after seeding, medium was changed and cells were co-transfected with pGL4.10 vector or one of the recombinant pGL4.10 vectors and pGL4.74 (Promega), or co-transfected with pGL4.74 vector, expression vector for human AP-2 (Addgene Plasmid 12100) 5'-GCGGGGGCGGTGTTTCCT-3') and ChIP-CHD5-P-R (5'-CAGGGGGAGCCGTCGAGG-3') were used for the region containing the AP2 binding site (–369 to –357). The –468 to –360 region was amplified to determine by qPCR. Samples were run in triplicate, and data were normalized to 10% input DNA amplifications, after subtraction of signals obtained from antibody isotype control. ChIP was repeated twice to confirm the reproducibility of results.Chromatin immunoprecipitation (ChIP) analysis was performed using the Pierce® Agarose ChIP Kit . Chromatin was precipitated with an AP2α antibody . The abundance of specific DNA sequences in the immunoprecipitates was determined by qPCR. ChIP-qPCR primers ChIP-CHD5-P-F . P<0.001 was considered statistically significant unless otherwise indicated.Comparisons between two groups were performed by paired sample CHD5 expression was down-regulated in leukemia, we initially examined expression level of CHD5 mRNA and protein in leukemia cell lines. K-562, HL-60 and Jurkat cells had markedly lower CHD5 expression relative to normal mononuclear cells (NMCs). However, CHD5 expression in KG-1a cells was only slightly lower . The median expression in ALL, AML and CML was 18.20 , 14.75  and 13.79 , respectively , the frequency of 1p36 deletion in leukemia samples is less than 5%, and some of these 1p36 deletions do not include the CHD5 gene sequence using UCSC Genome Browser software indicated that the putative CHD5 promoter is located 2000 bp upstream of the TSS and includes a CpG island , KG-1a (99.4% to 50.02%), HL-60 (97.4% to 58.46%) and Jurkat cells (76.9% to 41.8%) treated with DAC  was methylated. Median methylation densities in ALL, AML, CML and NMCs were 38.5% , 22.9% , 24.5%  and 7.2% , respectively  into a luciferase reporter construct. We methylated the cloned insert using SssI methylase (methylation of 39 CpGs), HpaII methyltransferase (methylation of 6 CpGs) or HhaI methylase (methylation of 8 CpGs). Furthermore, the 30th and 31st CpG sites were methylated by SssI methylase and HpaII methyltransferase, but not by HhaI methylase. Proper methylation of inserts was confirmed by digestion with the restriction enzymes McrBC, HpaII and HhaI. CHD5 promoter activity was tested by transfection of the luciferase construct containing methylated CHD5 promoter. Methylation with SssI, HpaII or HhaI methylases repressed CHD5 promoter activity in K-562. Repression was methylation dose dependent in that SssI, which methylates all CpG sites, exhibited the greatest repression, HpaII, which methylates 6 CpG sites, exhibited moderate repression, and HhaI, which methylates 6 or 8 CpG sites, exhibited the least repression of CHD5 promoter activity  CHD5 is down-regulated in human leukemia cell lines as well as clinical samples. This observation suggests that CHD5 could be used as a candidate gene for developing a biomarker panel for hematopoietic malignancies.In this study, we found that CHD5 promoter was strongly methylated in the –780 to –450 region Click here for additional data file.Figure S2The graphical representation of the methylated region. The methylated region that Zhao et al identified at −560 to −240 is somewhat proximal to the region that Fujita et al have identified at −780 to −450. The DNA sequence of −780 to −450 marked by underline was partially overlapped with DNA sequence of −560 to −240 marked by box. These CpG sites is marked by green shading. The sequence of all CpG sites located at −560 to −240 marked by sequential number.(TIF)Click here for additional data file.Figure S3AP2 expression in K-56 cell lines treated with DAC. K-562 cells were treated with DNA methyltransferase inhibitor DAC at the indicated concentrations. AP2 expression was determined by qRT-PCR (A) and western blotting (B). β-Actin was detected as an internal control. All data are presented as mean ± SD.(TIF)Click here for additional data file.Figure S4Visualization of chromosomal aberrations of K-562 and HL-60 cell lines. The SKY/M-FISH for K-562 (A) and HL-60 (B) cell lines were quoted from SKY/M-FISH and CGH Database at NCBI (NCI and NCBI SKY/M-FISH and CGH Database (2001), http://www.ncbi.nlm.nih.gov/sky/skyweb.cgi).(TIF)Click here for additional data file.Table S1Patient samples.(DOC)Click here for additional data file.


E. coli σ70 has the highest concentration and affinity for the core RNA polymerase. The E. coli protein Rsd is regarded as an anti-sigma factor, inhibiting σ70-dependent transcription at the onset of stationary growth. Although binding of Rsd to σ70 has been shown and numerous structural studies on Rsd have been performed the detailed mechanism of action is still unknown.Among the seven different sigma factors in in vitro and in vivo. Cross-linking and affinity binding revealed that Rsd is able to interact with σ70, with the core enzyme of RNA polymerase and is able to form dimers in solution. Unexpectedly, we find that Rsd does also interact with σ38, the stationary phase-specific sigma factor. This interaction was further corroborated by gel retardation and footprinting studies with different promoter fragments and σ38- or σ70-containing RNA polymerase in presence of Rsd. Under competitive in vitro transcription conditions, in presence of both sigma factors, a selective inhibition of σ70-dependent transcription was prevailing, however. Analysis of rsd expression revealed that the nucleoid-associated proteins H-NS and FIS, StpA and LRP bind to the regulatory region of the rsd promoters. Furthermore, the major promoter P2 was shown to be down-regulated in vivo by RpoS, the stationary phase-specific sigma factor and the transcription factor DksA, while induction of the stringent control enhanced rsd promoter activity. Most notably, the dam-dependent methylation of a cluster of GATC sites turned out to be important for efficient rsd transcription.We have performed studies to unravel the function and regulation of Rsd expression The results contribute to a better understanding of the intricate mechanism of Rsd-mediated sigma factor specificity changes during stationary phase. The regulation of synthesis often depends on the presence of small regulators, such as ppGpp or on mRNA stabilizing or destabilizing components. The stability or turnover of mRNAs for some σ factors is for instance affected by specific nucleases. In other cases inactive pre-sequences of σ factors are synthesized, which are activated by proteases, when needed. A common regulatory mechanism changing the activity of σ factors involves the action of proteins, which bind to σ factors, thereby inhibiting their association with the RNA polymerase core enzyme. These proteins are generally termed anti-sigma factors and notable examples are found for many different σ factors, including the housekeeping factor σ70Reprogramming the specificity of transcription during the change from exponential to stationary growth or under conditions of environmental stress is an essential feature of bacterial physiology. It requires that a large number of genes involved in growth and macromolecular synthesis are no longer expressed at high yield, while genes supporting maintenance and genetic stability, which are silent under exponential growth must be preferentially synthesized under conditions of generally shrinking resources 70, responsible for exponential growth, and σ38, responsible for stationary phase-specific transcription, was not easily comprehensible, because the concentration of σ70 exceeds that of σ38 at all growth phases. Moreover, the affinity of σ70 for core RNA polymerase is notably higher than that of σ3870, such as the non-coding 6S RNA, which selectively inhibits σ70 RNA polymerase holoenzyme (Eσ70) regulator of sigma D), which is considered to act as an anti-sigma factor for σ7070-specificity is not absolute 70 has been documented and analysed in detail, specific interaction was also shown to occur for instance between Rsd and the RNA polymerase core enzyme, suggesting that Rsd does not only sequester σ70 but might also affect the core enzyme of RNA polymerase 70 sequestering when cells enter stationary phase of growth 70.Originally, the change in σ factor specificity between σIn order to elucidate details of the proposed anti-sigma factor mechanisms of Rsd, and to dissect the molecular steps, which ultimately lead to a shift in transcriptional specificity, we studied the potential interactions of Rsd with components of the transcription machinery.rsd gene to unravel its implication in stationary phase adaptation. Previous characterization of the rsd gene expression has demonstrated that its intracellular level increases during the transition from exponential growth to stationary phase rsd gene in vitro and in vivo.For a complete understanding of the involvement of Rsd in stationary phase adaptation it is also important to learn more about the regulation of Rsd expression itself. Hence, we were interested to characterize details of the transcriptional regulation of the The bacterial strains and plasmids used in this study are listed in Supplementary E. coli RNA polymerase core and holoenzyme (Eσ70) as well as the sigma factors σ70 and σ38 were isolated as described previously 4SO4 precipitation and stored in 10 mM Tris-HCl, pH 8.0; 200 mM NaCl; 10 mM MgCl2; 25% (v/v) glycerol; 0.2 mM EDTA, pH 8.0; 0.1 mM DTT; 0.2 mM PMSF; 0.1 µM leupeptin; 0.1 µM pepstatin A at −20°C. The DNA binding proteins H-NS, LRP, FIS and StpA were purified as described The 70, σ38 or Rsd, respectively. Samples were cooled on ice before 0.02% (v/v) glutaraldehyde was added. The reaction mixtures were then incubated at room temperature for another 30 minutes. The reaction was stopped by the addition of 5 µl 200 mM HEPES, pH 7.4; 20% (v/v) glycerol; 0.4 mM EDTA, pH 8.0; 0.4 mM DTT and 3 µl β-mercaptoethanol. Samples were denatured at 96°C for 90 seconds and analyzed on a SDS-gel. Protein bands were visualized by Coomassie staining.For cross-linking 3 µg Rsd protein was incubated for 30 minutes in a total volume of 15 µl 50 mM HEPES, pH 7.4; 5% (v/v) glycerol; 0.1 mM EDTA, pH 8.0; 0.1 mM DTT and 3 µg of either σRsd protein was dialysed against 50 mM HEPES, pH 7.4; 200 mM NaCl; 5% (v/v) glycerol; 0.2 mM PMSF for 2 hours at 4°C. A 9 fold excess of Biotinyl-N-hydroxy-succinimide was added to the reaction mixture in a total volume of 200 µl. Samples were incubated over night at 4°C. The biotinylation reaction was stopped by an excess of glycine and samples were dialysed against 50 mM HEPES, pH 7.4; 200 mM NaCl; 25% (v/v) glycerol; 0.2 mM PMSF and stored at −20°C. The efficiency of biotinylation was verified by SDS gel electrophoresis.70, σ38 and native Rsd, respectively. Binding was allowed for 1 hour in 30 µl of TGED buffer  glycerol; 0.1 mM EDTA, pH 8.0; 0.1 mM DTT) on ice. 100 µl of Streptavidine MicroBeads (Miltenyi Biotec) together with 70 µl TGED buffer were added and the mixture was incubated at room temperature for 5 minutes before it was placed on the column. The MicroBead column was washed four times with 100 µl TGED buffer to remove unspecific bound proteins. Specifically bound proteins were eluted with 150 µl of 50 mM Tris-HCl, pH 6.8; 50 mM DTT; 10% (v/v) glycerol; 1% (w/v) SDS. The flow-through and fractions from washing and elution were collected and precipitated with 4 fold volume of acetone for 2 h at −20°C. Precipitated samples were resuspended in 15 µl Aqua dest. and analyzed on SDS containing polyacrylamide gels. Protein bands were stained with Coomassie brilliant blue prior to silver staining.Biotinylated-Rsd protein was incubated with RNA polymerase core and holoenzyme at molar ratios of 1∶1 or 1∶3 for σrrnB P1 fragment was obtained by cleavage of the plasmid pUC18-1 rrnB P1 start site). The bolA-fragment containing the coding strand  was obtained by cleavage of pUC18-bolA with EcoRI and BsaAI. The bolA fragment for the analysis of the non-coding strand  was obtained by cleavage of pUC18-bolA with HincII and XbaI. For modification with KMnO4 a DNA-fragment containing only the bolA P1 promoter (position -58 to 49 relative to the start site) was prepared by cleavage of pUC18-bolA1 with EcoRI and BsaAI. A 471 bp rsd-up-fragment, containing the rsd promoters and the upstream region, was obtained by cleavage of the vector pUC18-rsd-up with EcoRI and HincII. Additionally, a 288 bp long rsd-fragment containing the tandem-promoter rsd P1 and P2 was obtained by cleavage of the plasmid pUC18-rsd-up with BssHII and HincII. The 195 bp long up-fragment containing the upstream region without the rsd promoter was prepared by digestion of pUC18-rsd-up with the restriction enzymes SmaI and BssHII. A DNA fragment containing the fic promoter was generated by PCR from chromosomal DNA (E. coli K12 MG1655) with the primers: # fic2 5′-CTGGCCTGAAAATTACGAT-3′ and # fic-up 5′-GTTGCCGATAAGATTTCC-3′. The fragment was cleaved with BanI resulting in a 256 bp long fragment . Methylated DNA-fragments were derived from plasmids isolated from the E. coli strain XL-1 non-methylated plasmids were isolated from the dam deficient strain JM110, respectively. Methylation of the GATC-sites was verified by digestion with the methylation sensitive enzyme MboI. For binding and footprinting experiments DNA fragments were purified by agarose gel electrophoresis and end-labelled by Klenow (Promega) polymerase and incorporation of [α-32P]-dNTP.The ImageJ 1.42q was used.Limited DNase I cleavage of free and protein bound DNA was performed as described previously 4 was performed as described previously Aqua dest. and lyophylization, were separated on 10% (w/v) denaturing polyacrylamid gels and visualized by autoradiography.Modification of DNA by KMnOIn vitro transcription reactions were performed in 50 mM Tris-acetate, pH 8.0; 10 mM Mg-acetate; 1 mM DTT; 0.1 mM EDTA, pH 8.0; 10 µg/ml BSA acetylated; 160 mM potassium-glutamate in the presence of 5 nM pSH666-1 plasmid together with 30 nM of each σ-factor, when indicated, and 65 µM ATP, GTP, UTP, 5 µM CTP and 133 nM [α-32P]-CTP. The reaction was initiated by addition of 20 nM holoenzyme together with the indicated Rsd concentration. New rounds of initiation were stopped after 10 minutes at 30°C by the addition of 6 µl chase-solution . After another 10 minutes at 30°C reactions were stopped with 10 µl stop-solution  and the samples were precipitated with ethanol, re-dissolved in 20 µl formamide-buffer and separated on denaturing polyacrylamide gels. Transcription products were visualized by autoradiography.RNA was extracted from cells grown in YT-media at 37°C to either logarithmic or early stationary phase. Cells were cooled rapidly to 0°C and concentrated by centrifugation. Lysis and extraction of total RNA was performed as described previously 32P]-labelled desoxyoligonucleotide 5′-GCTCGGCGGATTTGTCCT-3′ complementary to position 160 to 142 of the rsd-mRNA generated by the vector prsd-up-cat. A second oligonucleotide 5′-TCAGCAGAGCGCAGATACCA-3′ complementary to position 28 to 9 of the RNA1 was used as an internal standard. Both oligonucleotides had been labelled at the 5′-end with [γ-32P] and T4-polynucleotidekinase . The primer extension reaction was performed with the AMV reverse transcriptase  as described previously For primer extension analysis 5 µg total RNA was incubated with 0.5 pmol 5′ [70 subunit of RNA polymerase has been characterized intensively, the interaction with other RNA polymerase components is less clear. We therefore analyzed direct protein-protein interaction between Rsd and the different RNA polymerase sigma subunits σ70 and σ38 by glutaraldehyde cross-linking.While binding of Rsd to the σ38 while for σ70 some aggregates can be observed following glutaraldehyde treatment. The existence of heterologous dimers between σ70 and Rsd are reflected by a cross-link band in the upper part of the gel (lane 9). While this interaction was expected the surprising formation of weak band(s) representing similar cross-link products between σ38 and Rsd is visible above the σ38 band in lane 11. In the case of σ70 and σ38 incubated together with Rsd and glutaraldehyde, the band intensities of free sigma factors and free Rsd are also noticeable reduced. We took this as a first indication that, in addition to the well established interaction of Rsd with σ70, the protein has also the potential to interact with σ38.As a first approach to study possible interactions between Rsd and different components of the transcription apparatus glutaraldehyde cross-linking experiments were applied. Mixtures of the proteins of interest were incubated either alone or in the presence of Rsd with glutaraldehyde. An example of such an experiment is shown in 38 dimer complexes. As alternative method we performed affinity-binding assays employing magnetic beads and biotinylated Rsd  and holoenzymes Eσ70 (α2ββ′ ωσ70). As can be seen in the upper left panel of 70), no protein that was retained from the affinity column could be detected .The 70 and Eσ38 with their corresponding promoters by gel retardation and footprinting experiments. Initial gel retardation studies with labeled promoter fragments revealed that increasing concentrations of Rsd inhibited binding of both holoenzymes to the appropriate promoters in a similar way (data not shown). To investigate potential structural rearrangements of the RNA polymerase promoter complexes in presence of Rsd we performed DNase I and KMnO4 footprint experiments. A typical DNase I footprinting result for Eσ70 RNA polymerase and the rrnB P1 promoter is shown in rrnB P1 promoter DNA was analyzed at increasing concentrations of Eσ70 RNA polymerase holoenzyme. Polymerase binding protects DNA from cleavage resulting in a well-established footprint pattern consistent with previously published rrnB P1 footprints rrnB P1 promoter, and a hyperreactive site at −37 becomes intensified (lanes 2 to 7). In the right panel the RNA polymerase concentration was set 100 nM and the samples were supplemented with increasing concentrations of Rsd. The addition of higher amounts of Rsd reduces the intensity of the footprint pattern, resulting in almost a total disappearance of the footprint and also the retardation pattern at the highest Rsd concentrations  harbours the σ70-dependent promoters rrnB P1, tac, the RNA1 promoter as well as the weak bolA P2 promoter. The latter normally does not give rise to measurable transcripts under the conditions tested. In addition, the template vector contains the σ38-dependent bolA P1 promoter. All promoters give rise to transcripts of a defined length due to the tandem rrnB terminators T1T2, which have been cloned at defined distances downstream of the respective promoters. Transcription reactions directed only by the Eσ70 holoenzyme resulted in major products for the tac promoter followed by the rrnB P1 and the RNA1 promoters. When the Eσ70 holoenzyme was used as the only polymerase no transcripts could be detected for the bolA promoters . Consistent with the expected specificity transcription with only the Eσ38 holoenzyme yielded a double band characteristic for the bolA P1 promoter . At the same time, the amounts of transcripts derived from the σ70-specific promoters are significantly lower, although the transcript from the tac promoter is still the strongest product under these conditions  the decreasing activity of the σ70-dependent promoters is compensated by a notable increase of the bolA P1 transcript . There are 5 GATC sequences within a short DNA stretch (350 bp) upstream and between the rsd P1 and P2 promoters . The presence or absence of the methylation at GATC sites was verified by restriction analysis with MboI. NAP binding was then compared to the methylated and non-methylated DNA fragments. No notable qualitative or quantitative differences in binding of the four proteins H-NS, StpA, LRP and FIS could be detected, however, suggesting that the methyl groups do not affect the specific binding of the NAPs to the rsd promoter region (data not shown).Inspection of the DNA sequence flanking the two romoters . Dam met70 and Eσ38 might be affected by Dam-specific methylation. Again, we used non-methylated and methylated DNA templates containing both rsd promoters P1 and P2 for gel retardation. Binding was performed with the two holoenzymes Eσ70 and Eσ38, separately and in combination. Moreover, binding of the different holoenzymes was analyzed in the presence and absence of Rsd. Results are shown in 70 holoenzyme binds exclusively to the σ70-dependent rsd P2 promoter  but very likely by altering sigma factor competition at the rsd promoters through the activity of Rsd. In summary, the results demonstrate a contribution of DNA methylation on the regulation of rsd expression. By changing the RNA polymerase distribution between the σ70- and σ38-dependent rsd promoters Rsd is itself involved in the altered expression (autoregulation). We conclude that the DNA methylation state very likely has an effect on the Rsd responsiveness of certain promoters. The observation that transcription of rsd appears to increase, when DNA is methylated, might also suggest a link between the Rsd expression and the cell cycle.When the methylated DNA template was used binding of the different RNA polymerase holoenzymes was generally stronger . The spenhibited . The resrsd P2 promoter has the typical characteristics of gearbox promoters, whose activity often correlates inversely with the growth rate in vivo promoter activities at different growth rates. To this aim we used a vector-based expression system (prsd-up-cat), where the rsd promoters and their upstream regulatory region was fused to a promoter-less cat gene and transcript levels of transformed MG1655 cells were determined by primer extension analysis. The amount of the plasmid-encoded RNA1 transcript served as internal control. Total RNA was extracted at exponential and stationary phase from cells grown in different media with growth rates of µ = 0.35, µ = 0.95 and µ = 2.7 and subjected to primer extension analysis. The P2-derived products represented the predominant fraction of the rsd transcripts. The results obtained were fully consistent with the gearbox nature of the P2 promoter reported earlier rsd expression is only marginal at the tested conditions.Since the rsd promoter-derived transcripts from wild-type strains and strains with mutations in the genes for a number of selected regulators, which had been transformed with the rsd promoter vector prsd-up-cat.To analyze the effects of a variety of cellular growth rate regulators we employed the same primer extension analysis described above and compared the amount of relA and dksA genes were selected to analyse effects of the stringent control and growth rate regulator ppGpp. Both effectors are known to act as synergistic regulators rsd gene served to test for possible autoregulation. An rpoS mutant was selected to determine the effect of the stationary phase master regulator. Mutants in the NAP genes hns, fis, lrp, stpA were chosen to verify the in vitro binding results. A mutant with a defect in the ssrS gene encoding the regulatory 6S RNA was selected for its functional homology to rsd. Finally, we selected a strain with the dam mutation to verify the difference in RNA polymerase binding observed with methylated and non-methylated DNA in vitro  did not result in an altered rsd mRNA level. This is of special interest because 6S RNA itself is a regulator, considered to facilitate the switch in specificity between exponential and stationary growth transcription by interfering with the Eσ70 holoenzyme rsd transcription is not altered in the ssrS mutant contrasts with earlier studies performed under long-time stationary growth, where the rsd P2 promoter was found to be negatively affected by 6S RNA Since the amounts of transcripts derived from the analysis . In addirelA, dksA, rsd, rpoS, hns, fis and dam) are summarized in dam+/- strains is presented in relA-deficient strain , the master regulator for stationary phase expression, is a dramatic increase in P2 activity during the stationary phase  were unsuccessful so far (data not shown).In the above study we show that Rsd is able to interact with RNA polymerase core enzyme and can form dimers. This is consistent with earlier reports 70 but can also associate with the stationary phase-specific sigma factor σ38in vitro. This was demonstrated by cross-linking and independently by affinity binding. Whether this interaction is of functional importance was further analyzed by transcription studies, which showed that Rsd was able to inhibit open complex formation of both holoenzymes, Eσ70 and Eσ38, at their appropriate promoters. It should be noted that this was the case when the analyses were performed in vitro under non-competitive conditions. When the effect of Rsd was studied under more complex conditions, allowing competition between different sigma factors and promoter selection by RNA polymerase, it turned out that the presence of Rsd now changed transcription specificity in favour of Eσ38 holoenzyme and σ38- dependent promoters. The results indicate that the mode of action of Rsd is not governed by a strong affinity preference for the principal sigma factor σ70 but apparently involves a balanced change in concentration and affinity of additional interacting partners, involving the accessibility of competing promoters and σ factor competition. This is particularly reflected in the different results Rsd provokes in open complex formation experiments with single promoters and single RNA polymerases, presented in Unexpectedly, we found evidence that the anti-sigma factor Rsd does not only interact with σ70 for AsiA and Rsd one could speculate that Rsd may also exert functions other than sequestering the primary sigma factor σ70. In line with this our binding and footprinting studies are not fully consistent with the simple conception that Rsd fulfils its anti-sigma factor function only by reducing the active fraction of the respective sigma factor through binding. The mechanism how Rsd contributes to the shift in sigma factor usage and promoter selection is obviously more complex. It likely involves transient interactions of Rsd with RNA polymerase core, changes in the concentration of available reaction partners, including σ70 and σ38, but also putative Rsd dimer formation. Hence, the outcome of Rsd-dependent regulation is determined by an orchestrated interplay of numerous components involved in the transcription reaction.Generally, in a simplistic view, anti-sigma factors bind to sigma and preclude the formation of transcription-competent RNA polymerase holoenzymes. A notable exception is AsiA, which acts both as anti-sigma factor and, together with the regulator MotA, as co-activator 70 is not absolute the molecular reasons for the specificity difference between σ70 and σ38 to bind to RNA polymerase core and to select their cognate promoters cannot easily be discerned because both sigma factors share high structural homology. Numerous studies to understand the specificity difference of σ70 and σ38 have revealed only some clues to explain the divergent functions of these rather homologous sigma factors 38-dependent promoters do not share good −35 sequence conservation, consistent with the minor role of this recognition element for stationary phase-specific promoters. Generally, σ38 and σ70-specific promoter sequences exhibit only limited structural differences. A conserved cytidin at position −13 is considered as presumably the most pronounced difference and appears to be a hallmark for σ38-dependent promoters 38 and σ70.In much the same way as the selectivity of Rsd for σ70 it was concluded that the binding of Rsd sterically not only interferes with the interaction of σ70 and the −35 promoter recognition element but also with the β flap, indicating that both the recognition of core polymerase as well as certain promoter structures may be influenced by Rsd 70 have been identified in subregion 4.2. However, within the major recognition helix of this domain there are only three amino acids , which differ between σ70 and σ38 among the positions identified in direct interaction with Rsd 70 and σ38. It has been shown that Rsd can interact simultaneously with σ70 regions 4 and 2, likely forming an extensive interface 38, and a low degree of amino acid conservation in subregion 3.2, are involved in core binding and may provide potential sides for a differential specificity to recognize Rsd. Unfortunately this region is not present in the crystal structure from which the contact sites have been derived. It is feasible, however, that binding of Rsd to these regions will interfere with RNA polymerase core binding. In summary, the high similarity between crucial domains of both sigma factors may explain the observations made in this study that under non-competitive conditions Rsd can recognize both, σ70 and σ38  resulted in the expected characteristic KMnO4 modification signal at positions -10T and -11T (marked on the right) indicative for a transcriptionally active open promoter complex [Supplementary Reference S1 in rrnB P1 promoter are given at the left margin. An A+G sequencing reaction of the promoter DNA was separated in lane 1 (S).(TIFF)Click here for additional data file.Figure S24 footprint analysis of Eσ38∼KMnObolA1 promoter complexes: effect of increasing Rsd concentrations. Complexes of Eσ38 holoenzyme with the bolA P1 promoter fragment were cleaved after KMnO4 treatment. The analysis of the coding strand is shown in (a). In lanes 1 to 6 increasing polymerase concentrations were applied: lane 1, no polymerase, lanes 2 to 6: 5, 10, 20, 50 nM and 100 nM Eσ38 holoenzyme, respectively. Samples in lanes 7 to 12 contained 50 nM Eσ38 each and 0, 0.5, 1, 2, 4 and 8 µM Rsd, respectively. In Lane 13 an A+G sequencing reaction of the promoter fragment was separated. With increasing RNA polymerase concentration KMnO4-sensitive positions, indicating the presence of open complexes , became visible. Nucleotide positions relative to the transcription start site of the bolA P1 promoter are given at the right margin. (b) Gel shift analysis of aliquots from the samples used for the KMnO4 footprint reaction shown in (a) prior to the modification. Lane numbers correspond to those shown in (a). The positions for the free DNA and the Eσ38∼bolA1 promoter complex are given on the left margin.(TIFF)Click here for additional data file.Figure S338∼Effect of Rsd on Eσfic promoter complex formation. (a) Binding of the RNA polymerase Eσ38 holoenzyme (200 nM) to a DNA fragment harbouring the σ38-dependent fic promoter was analyzed by gel retardation. Complex formation was challenged by increasing concentrations of Rsd. In lane 1 the free DNA is shown. Lane 2 represents the complex in the absence of Rsd. In lane 3 to 6 increasing Rsd concentrations of 1 µM (lane 3), 2 µM (lane 4), 4 µM (lane 5) and 8 µM (lane 6) were present. (b) Diagram showing the quantitative evaluation of the data from (a) indicating the remaining amounts of complex as a function of the Rsd concentration.(TIFF)Click here for additional data file.Figure S4Sequence of the rsd promoter region. The sequence given corresponds to the rsd-up fragment presented in rsd P2 transcription start site. Promoter core elements (−10 and −35 regions) are boxed and highlighted with bold letters. Transcription start sites of the rsd P1 and P2 promoters are marked by bold-type capital letters. The Rsd translation initiation codon is shown in bold-type and boxed. The five GATC sites are bold-type and underlined. NAP binding sites and the respective colour code are taken from (TIFF)Click here for additional data file.Figure S5rsd P1 promoter activity in different strain background during stationary phase of growth. For the rsd P1 promoter notable amounts of transcript were only obtained during stationary phase of growth. The diagram depicts the relative amounts of rsd P1-derived transcripts normalized to the RNA 1. Compared are the transcripts from strains with defects in relA, dksA, rsd, rpoS, hns, fis and dam relative to the transcripts from the respective wild-type strains, normalized to 1. Shown is one representative experiment out of two to three with similar results.(TIFF)Click here for additional data file.Table S1Bacterial strains and plasmids used in this study.(DOC)Click here for additional data file.Table S2Quantitative evaluation of RNA polymerase complexes formed with methylated and non-methylated rsd promoter DNA.(DOC)Click here for additional data file.Text S1Supplementary References.(DOC)Click here for additional data file.