Post-translational modifications of core histones affect various cellular processes, primarily through transcription. However, their relationship with the termination of transcription has remained largely unknown. In this study, we show that DNA damage-activated AKT phosphorylates threonine 45 of core histone H3 (H3-T45). By genome-wide chromatin immunoprecipitation sequencing (ChIP-seq) analysis, H3-T45 phosphorylation was distributed throughout DNA damage-responsive gene loci, particularly immediately after the transcription termination site. H3-T45 phosphorylation pattern showed close-resemblance to that of RNA polymerase II C-terminal domain (CTD) serine 2 phosphorylation, which establishes the transcription termination signal. AKT1 was more effective than AKT2 in phosphorylating H3-T45. Blocking H3-T45 phosphorylation by inhibiting AKT or through amino acid substitution limited RNA decay downstream of mRNA cleavage sites and decreased RNA polymerase II release from chromatin. Our findings suggest that AKT-mediated phosphorylation of H3-T45 regulates the processing of the 3′ end of DNA damage-activated genes to facilitate transcriptional termination. N-terminal tails of core histones are rich in basic amino acids and undergo many post-translational modifications. These modifications correlate closely with transcription . DNA damDespite these insights, however, the link between DNA damage-induced histone phosphorylation and transcriptional regulation remains poorly understood. Post-translational modifications of histone proteins in chromatin are critical in gene transcription, regulating their access to transcriptional regulators by altering the electrostatic or structural characteristics of chromatin. Acetylated histones H3 and H4 are detected in the promoter regions of actively transcribed genes. H3-K4 trimethylation peaks during transcriptional initiation, whereas di- and trimethylated H3-K36 occupies these sites throughout transcriptional elongation .Transcription in eukaryotes is mediated by RNA polymerase II and termination is a prerequisite for genomic partitioning and precise expression of neighboring genes . ParticuIn this study, we demonstrate that also known as protein kinase B (AKT) phosphorylates threonine 45 of histone H3 (H3-T45) upon DNA damage and that AKT-mediated phosphorylation of H3-T45 regulates transcriptional termination. These findings unravel an unexpected function of AKT—as a histone kinase—in the transcriptional termination of genes in response to DNA damage.HeLa, MCF7 and MCF10A cells were obtained from American Type Culture Collection (ATCC). Except for MCF10A, the cells were cultured in Dulbecco's modified Eagle's medium, supplemented with 10% (v/v) fetal bovine serum (FBS) and antibiotics. MCF10A cells were cultured in DMEM-F12 media, supplemented with 5% (v/v) FBS, L-glutamine, antibiotics, insulin , cholera toxin , hydrocortisone  and hEGF . Polyethylenimine  was used to transfect HeLa cells. MCF10A cells were electrophoresed with the Neon Transfection System (Invitrogen) per the manufacturer's instructions.Escherichia coli strain DH5α and proteins were isolated using Glutathione Sepharose™ 4B beads (Amersham Biosciences) per the manufacturer's instructions. His6 fusion proteins were expressed in the BL21DE3 strain and purified with Ni-NTA agarose (Qiagen) per the manufacturer's instructions.Mammalian expression vectors for dominant negative (DN) and myristoylated (Myr) AKT1 were purchased from Millipore. Expression vectors for wild-type (WT) and T45A H3 were generated by subcloning the corresponding polymerase chain reaction (PCR) fragment into the Platinum-A (Plat-A) cell line and MCF10A cells that stably overexpressed these clones were generated per the manufacturer's instructions . Core histone proteins were generated by subcloning the corresponding PCR fragment into pGEX4T-1 (Amersham Biosciences) or pRSETB (Invitrogen). glutathione S-transferase (GST) fusion proteins were expressed in the Anti-actin was purchased from Sigma; anti-Myc (9E10) was obtained from Covance; anti-pan H3, anti-H3  were purchased from Abcam; anti-H2A, anti-H2B, anti-H3 (phosphorylated-S10), anti-H2A/H4 (phosphorylated-S1) and anti-H2AX (phosphorylated-S139) were acquired from Upstate; anti-H2B (phosphorylated-S36) was obtained from ECM Biosciences; anti-phosphorylated RNA Pol II-S2 (3E10) and anti-phosphorylated RNA Pol II-S5 (3E8) were purchased from Millipore; anti-p53  and anti-Rpb1 were purchased from Santa Cruz; and anti-AKT1, anti-AKT2, anti-pan AKT and anti-AKT (phosphorylated-S473) were acquired from Cell Signaling.Rabbit polyclonal H3-T45 phosphorylation-specific antibody was generated from Openbiosystems (Thermofisher Scientific) by immunizing three Specific Pathogen Free (SPF) rabbits with phosphorylated H3-T45 peptide. H3 antibodies  were a generous gift from Dr Kyong-Tai Kim .Lentiviral vectors that contained the human AKT1-targeting sequences pLKO.1-sh-AKT1 #1 (TRCN0000010171), #2 (TRCN0000010174), #3 (TRCN0000039794), #4 (TRCN0000039795), and #5 (TRCN0000039793) and AKT2 pLKO.1-sh-AKT2 #1 (TRCN0000000563), #2 (TRCN0000039968), #3 (TRCN0000265834), #4 (TRCN0000265848) and #5 (TRCN0000265851) were purchased from Sigma. As a control, the pLKO.1 vector was used. Lentivirus was produced per the manufacturer's protocol using the BLOCK-iT Lentiviral RNAi expression system (Invitrogen). Twenty-four hours after lentiviral infection, infected cells were selected with puromycin (1 μg/ml) for 2 weeks and then used in the experiments. Because pLKO.1-sh-AKT1 #3 and pLKO.1-sh-AKT2 #2 were the most effective, we used these vectors in most experiments, unless specifically noted.2, 75 μM ATP and 10 μCi 32P-ATP. The reaction mixture was incubated for 2 h at 30°C and loaded onto sodium dodecyl sulphate-polyacrylamide gel electrophoresis gels. The gels were dried for 1 h at 80°C and exposed to X-ray film overnight at −80°C.Anti-Myc conjugated protein G agarose beads were used to immunoprecipitate total lysates of HEK293T cells that overexpressed DN/Myr-AKT1. Immunoprecipitated protein G agarose beads or active AKT1 (Upstate) was mixed with substrate in 20 mM MOPS (pH 7.2), 25 mM β-glycerol phosphate, 5 mM EGTA, 1 mM sodium orthovanadate, 1 mM dithiothreitol, 75 mM MgClImmunofluorescent staining was performed as described .7) were harvested and crosslinked with formaldehyde to a final concentration of 1%. The crosslinking reaction was stopped by adding glycine to a final concentration of 0.125 M. The cells were harvested and washed twice with cold phosphate buffered saline and cytosolic fractions were eliminated with buffer A . Nuclear pellets were washed and resuspended in 1× micrococcal nuclease reaction buffer  and the chromatin was digested with micrococcal nuclease (New England Biolabs). The digestion was stopped with EDTA. The remainder of the procedure was performed as described  and KRIBB (Korean Research Institute of Bioscience & Biotechnology). Briefly, DNA fragments were ligated to a pair of adaptors for sequencing on an Illumina Hiseq-2000. The ligation products were size-fractionated to obtain 200–300-bp fragments on a 2% agarose gel and PCR-amplified for 18 cycles. Each library was diluted to 8 pM for 76 cycles of single-read sequencing on the Illumina Hiseq-2000 following the manufacturer's recommended protocol.http://picard.sourceforge.net). We used the MACS v.1.4.2 tool  compared with matched control samples. Using HOMER (The sequencing reads were mapped against the human genome GRCh37/hg19) using Bowtie v.2.1.0  with def using Bong HOMER , H3-T45 ng HOMER . The aveng HOMER .® (Invitrogen). Five micrograms of total RNA was biotinylated with reaction cocktail  and vortexed for 30 min at room temperature. RNA was precipitated with glycogen, ammonium acetate and 100% ethanol at −80°C overnight. One microgram of biotinylated RNA was purified with streptavidin-coated magnetic beads, washed and reverse-transcribed using Superscript III reverse-transcriptase (Invitrogen) per the manufacturer's protocol. cDNA was treated with RNaseA (Invitrogen) at 37°C for 30 min and used as template for real-time qPCR analysis.Nascent RNA quantification was performed using the Nascent RNA capture kit (Life Technologies) per the manufacturer's recommendations. Briefly, 0.1 mM 5-ethynyluridine (EU) was added to the cell culture medium for 18 h and total RNA was extracted with TriZol® (Invitrogen) and reverse-transcribed . mRNA and antibody-bound chromatin levels by chromatin immunoprecipitation assay were quantified by real-time qPCR with the SYBR® Green qPCR Kit  on the iQ5 and CFX Connect Real-time PCR Detection System (Bio-Rad) and then normalized to actin or 1% input chromatin using the 2−ΔΔCT2-ΔΔCT calculation method. Conventional PCR was performed using EX Taq polymerase (Takara) on a MyCycler (Bio-Rad). The sequences of the primers for mRNA quantification are listed in Supplementary Table S1.Total RNA was extracted with TriZolP-values were calculated using the student's t-test calculator (http://www.physics.csbsju.edu/stats/t-test.html). A value of P < 0.05 was considered to be statistically significant. All data are representative of at least three independent experiments.Data are presented as means ± standard deviations and We attempted to identify the phosphorylation sites on core histones in response to DNA damage . Thus, MCF10A normal breast epithelial cells were treated with adriamycin and key phosphorylation sites were examined by western blot. As expected, we observed the induction of phosphorylated H2AX-S139 ,5. BasedAt the promoter region, DNA damage quickly reduced Chk1-mediated phosphorylation of H3 -T11, repressing the transcription of cell cycle-activating genes such as cyclin B1 . Phosphohttp://scansite.mit.edu), AKT was predicted to be the H3-T45 kinase with highest stringency and the amino acid sequence of H3-T45 harbors a perfectly conserved substrate recognition motif for AKT , AKT was found to interact directly with histones and phosphorylated H3-T45 in vitro   form of AKT1 phosphorylated histone H3, while the dominant-negative (DN) AKT1 failed to do so  Figure . FurtherGAPDH and HPRT1, were phosphorylated H3-T45-negative .H3-T45 lies in the N-terminus of the first helix of H3 and constitutes the nucleosome entry/exit point. This residue is assumed to have significant function, because it makes contact with genomic DNA –30. MCF1e Figure , with loCDKN1A locus by ChIP-qPCR recapitulated the genome-wide ChIP-seq results. Phosphorylated H3-T45 signals in CDKN1A rose significantly on induction of CDKN1A transcription by ADR, peaking 3′ of the TTS, where RNA Pol II dissociated from the chromatin , indicating that AKT, at least, is an H3-T45 kinase under DNA damage conditions. These data suggest that DNA damage-induced H3-T45 phosphorylation by AKT occurs exclusively 3′ of the TTS in actively transcribed genes, where RNA Pol II-S2 phosphorylation is maximized.CDC7, PKC-δ and DYRK1A have been reported to phosphorylate H3-T45 ,30,32. TAKT1, AKT2 and AKT3, on chromosomes 14, 19 and 1, respectively, encode distinct AKT isoforms : a long, uncleaved pre-mRNA transcript and a shorter transcript that results from cleavage at the poly(A) site. The cDNA was analyzed with primer pairs to three regions: (i) the last intronic region, amplifying pre-mRNA (not mature mRNA), (ii) the poly(A) site, to detect uncleaved pre-mRNA product and (iii) the downstream RNA after cleavage at the poly(A) site, generating a product that lacks a 5′ cap and is degraded rapidly by exonucleases   site were similar, despite the induction of pre-mRNA . However, the levels of downstream product increased in AKT inhibitor-treated cells and T45A mutant cells. The amount of product that spanned the poly(A) site was unaltered by AKT inhibition or the T45A mutation, indicating that they affect the degradation of downstream RNA products after poly(A) site cleavage but not cleavage of the mRNA proper.CDKN1A, MDM2, SMAD3 and KLF5 (Figure Next, we used a nascent RNA capture system, which quantifies transcripts precisely . With th5 Figure . Further5 Figure , demonstDNA damage induces histone phosphorylation to maintain genomic stability. For example, H2AX-S139 is phosphorylated primarily at DNA double-strand breaks and recruits damage repair complexes . Unlike Based on our data, AKT alone is unlikely to differentiate targets for transcriptional termination, because a significant amount of H3-T45 phosphorylation simply follows RNA Pol II-S2 phosphorylation Figure  and E. ACDH1 promoter with Snail1 to repress transcription (We wondered whether H3-T45 phosphorylation in other processes, such as DNA replication and apoptosis, correlates with transcriptional termination. Previous studies have suggested that H3-T45 is phosphorylated under different conditions by distinct kinases: PKC-δ under apoptotic conditions , Cdc7 ducription , a link cription ,39, H3-TAKT isoforms are highly homologous but have distinct tissue expression patterns and substrate specificity . On DNA Recent studies claim that defective termination at the 3′-end impairs splicing, enhances RNA degradation and reduces transcriptional initiation at the promoter , suggestIn summary, we have provided evidence for the connection between post-translational histone modifications and transcriptional termination for the first time. DNA damage leads to the activation of AKT, which in turn phosphorylates H3-T45, facilitating transcriptional termination for maximum transcriptional efficiency. These observations shed significant new light on the cellular response to DNA damage to regulate transcription through the termination phase.Supplementary Data are available at NAR Online.


Danio rerio) to 0.01, 0.1, and 1 mg/L BPA for 15 d. We observed a significant increase in egg production, together with a reduced rate of fertilization in fish exposed to 1 mg/L BPA, associated with significant alterations in the transcription of genes involved in reproductive function and epigenetic processes in both liver and gonad tissue at concentrations representing hotspots of environmental contamination (0.1 mg/L) and above. Of note, we observed reduced expression of DNA methyltransferase 1 (dnmt1) at environmentally relevant concentrations of BPA, along with a significant reduction in global DNA methylation, in testes and ovaries following exposure to 1 mg/L BPA. Our findings demonstrate that BPA disrupts reproductive processes in zebrafish, likely via estrogenic mechanisms, and that environmentally relevant concentrations of BPA are associated with altered transcription of key enzymes involved in DNA methylation maintenance. These findings provide evidence of the mechanisms of action of BPA in a model vertebrate and advocate for its reduction in the environment.Bisphenol A (BPA) is a commercially important high production chemical widely used in epoxy resins and polycarbonate plastics, and is ubiquitous in the environment. Previous studies demonstrated that BPA activates estrogenic signaling pathways associated with adverse effects on reproduction in vertebrates and that exposure can induce epigenetic changes. We aimed to investigate the reproductive effects of BPA in a fish model and to document its mechanisms of toxicity. We exposed breeding groups of zebrafish ( E217β-estradiol5Fc5-formylcytosine5hmC5-hydroxymethylcytosinearandrogen receptoramhanti-Müllerian hormonecyp19a1aaromataseBPAbisphenol Adnmt3DNA (cytosine-5)-methyltransferase 3dnmt1DNA methyltransferase 1esr1estrogen receptor 1esr2aestrogen receptor 2aesr2bestrogen receptor 2bERestrogen receptorGSIgonadosomatic indexHSIhepatosomatic indexhdac1histone deacetylase 1hdac3histone deacetylase 3mecp2methyl CpG binding protein 2mbd2methyl-CpG-binding domain protein 2mbd3amethyl-CpG-binding domain protein 3a5mCmethylcytosinePCAprincipal component analysisrpl8ribosomal protein L8vtg1vitellogenin 1Bisphenol A (BPA) is a commercially important high production chemical widely used in the production of epoxy resins, utilized in food and beverage packaging, dental sealants, and as a monomer component of polycarbonate plastics.BPA is moderately water soluble, entering the environment via direct discharge from BPA production and processing industries, wastewater treatment plants and leachate from landfill sites.esr1 expression in the livers of male fathead minnows  exposed for 4 d to 10 μg BPA/L, consistent with an estrogenic mode-of-action.cyp19a1a), which is responsible for the irreversible conversion of androgens into estrogens and is a key regulator of estrogen synthesis in the gonads. This enzyme was significantly upregulated in both the ovary and testis of Gobiocypris rarus exposed to 15 µg/L BPA for 7d, followed by suppression after 35 d exposure.BPA has been shown to act as an estrogen receptor (ER) agonist,Xiphophorus helleri was also reported.Adverse impacts on reproduction have been observed in several fish models. A multi-generational study in fathead minnow showed that BPA reduced gonadal growth in males and females, reduced hatching in F1 offspring of fish exposed to 640 µg/L and induced the estrogen regulated egg yolk protein, vitellogenin, a well established biomarker of xenoestrogen exposure, in the liver of male fish exposed to 640 and 1280 µg/L BPA.Evidence also exists supporting the involvement of BPA in the etiology of a range of human disease phenotypes including cardiovascular disease,Gobiocypris rarus found BPA exposure to be associated with altered DNA methylation in the 5′ flanking region of cyp19a1a (aromatase), and the effects to be time-dependent.dnmt1) in ovarian tissue has been reported, with a significant decrease in global DNA methylation.To date, few studies have investigated the potential for BPA to induce epigenetic and transcriptional changes in fish. A study in Given the extensive use and ubiquity of BPA, it is important to understand the mechanisms mediating its toxic effects and the impacts these can have on both wild populations and human health. The present study aims to investigate the effects of BPA on reproduction in the zebrafish model and identify epigenetic and transcriptional changes associated with BPA exposure. We exposed breeding groups of zebrafish to BPA for 15 d to determine if reproduction was affected by the exposure. The concentrations tested included environmentally relevant concentrations found world-wide (0.01 mg/L) and at point sources (0.1 mg/L).The mean measured concentrations of BPA in the tank water were between 100 and 139% of the nominal concentrations for all treatments, and are presented in Supporting Information Table S1.The mean mass and length of male and female fish were 460.0 ± 0.008 mg and 36.5 ± 0.02 mm, and 480.6 ± 0.01 mg and 35.7 ± 0.03 mm, respectively. There were no significant differences in size or condition factor  between treatment groups.No alterations in general feeding and swimming behavior were observed in any spawning group, with the exception of the mortality of one female in the 0.1 mg/L BPA treatment. The egg output calculations for that group were adjusted accordingly. Hepatosomatic index  in males was significantly increased in fish exposed to 1 mg/L BPA, but no effects of BPA were observed in females . There were no significant differences in the gonadosomatic index  of males or females as a result of the BPA exposure.P = 0.098). During the exposure, groups treated with 1 mg/L BPA spawned a significantly greater number of eggs per female when compared to all other treatment groups (P ≤ 0.01); this increased egg production intensified throughout the exposure period , indicating that the effects of BPA on fertilization became progressively more pronounced over the exposure period.During the 10 d pre-exposure period there were no differences in cumulative egg production between treatment groups . In females, esr2b was significantly upregulated following exposure to 0.01 mg/L BPA (P = 0.044). For genes involved in epigenetic regulation, the most pronounced changes observed were for dnmt1, which was significantly downregulated in the livers of females exposed to 0.01 mg/L BPA (P = 0.040) and in both males and females exposed to 0.1  and 1 mg/L BPA . In addition, changes were also observed for histone deacetylase 3 (hdac3), methyl-CpG-binding domain protein 2 (mbd2) and methyl CpG binding protein 2 (mecp2) in males, and for mbd2 in females , which decreased with increasing concentrations of BPA. In addition, for anti-Müllerian hormone (amh), BPA affected gene transcription (P ≤ 0.05) and a decreasing trend across all concentrations was observed, but this was not statistically significant . Similarly to the testis, in the ovaries of exposed females, the transcript encoding esr2a was significantly downregulated following exposure to 1 mg/L BPA (P ≤ 0.001). In addition, there were similar (but non-significant) trends for other genes involved in reproductive function including esr1 and ar, which appeared to decrease with increasing exposure concentrations . Although no significant group-wise changes in dnmt1 transcription were observed in the testis . In addition, changes in mbd2 transcription were observed in the testis, with a significant increase in transcription measured in males exposed to 0.01 mg/L BPA (P = 0.020), but reduced expression in males exposed to 1 mg/L BPA . No differences in DNA methylation were seen for this region in ovaries from exposed female fish . The analysis of 11 CpG sites across the promoter of dnmt1 identified significant increases in DNA methylation for a number of sites in the liver  and the testes . Although group-wise comparisons of this region revealed no significant differences in the female ovaries .Targeted DNA methylation profiling in the promoter region of ale fish . BPA was ovaries , dnmt1 pdnmt1 transcription in the ovary and in the liver of both males and females following exposure to BPA, including at environmentally relevant concentrations in females. In association with this, we found a reduction in global DNA methylation, probably due to the decrease in dnmt1 expression. At the highest concentration tested, BPA caused reduced fertilization, potentially via estrogenic mechanisms. Together, our data provide evidence of the molecular mechanisms of action of BPA and the potential for it to cause adverse health impacts in vertebrates.Exposure to BPA resulted in a consistent downregulation of We provide evidence that BPA exposure results in an impairment of reproductive function in breeding zebrafish. These effects included an increase in the number of eggs spawned and a decrease in fertilization success in groups exposed to 1 mg/L BPA. A number of mechanisms may contribute to the observed effect of BPA on reproduction, including stimulation of estrogen responsive processes via the interaction of BPA or its metabolites with estrogen signaling pathways, as previously reported for a range of organisms.esr1 or DNA methylation across the esr1 promoter in the gonads and livers of both sexes, but a significant association between BPA concentration and decreased transcription was found for the livers of females, and a trend for reduced expression was also observed in the ovaries and testis, similar to that described previously.esr1 transcript in the testis may contribute toward the observed decline in fertilization success at this concentration.We found no evidence for significant alterations in the transcription of esr2a in both ovaries and testes, but not in the liver. Similarly, a decrease in esr2a transcription was reported in ovaries of Gobiocypris rarus exposed to 0.05 mg/L BPA for 35, d and was associated with disruption of oogenesis and the occurrence of atretic follicles.esr2a is more sensitive compared to esr1, to the natural estrogen, 17β-estradiol (E2).esr2b in the livers of males and females but not in the gonads, and, importantly, for females this effect was observed at the environmentally relevant concentration of 0.01 mg/L BPA. In parallel, BPA induced a significant increase in the transcription of the egg yolk protein, vtg1, and an increase in HSI in males, likely as a result of increased vitellogenin production in hepatocytes, indicating an association between the induction of esr2b in males and the induction of vtg1, as previously reported for fathead minnows.esr1 and esr2b in the liver, and esr2a in the gonads.BPA was found to downregulate cyp19a1a transcript in the testis of males exposed to 1 mg/L BPA, and a significant association between transcription and BPA exposure concentration. In ovaries, a decreasing trend was also observed. These findings suggest potential feedback mechanisms were activated to counteract the estrogen/androgen ratio imbalance caused by BPA, through reducing the irreversible conversion of testosterone into estrogens. Similar findings have recently been reported for the Chinese rare minnow (Gobiocypris rarus) following a long term exposure to BPA,In addition to the disruption in estrogen receptor signaling, changes in sex steroid biosynthesis may have contributed to the observed disruption of reproduction in colonies exposed to 1 mg/L BPA. We found a significant decrease in amh transcription was associated with increased BPA exposure concentrations. Similarly, in mammals, downregulation of AMH has been reported following exposure to BPA.amh promoter in the testis (CpG 1), demonstrating that exposure to BPA caused epigenetic alterations at this specific gene locus. There was also a significant correlation between the level of methylation in CpG 1 and amh transcription, and with BPA exposure concentration. This suggests that epigenetic mechanisms may be playing a role in the observed decline in amh transcript in testis tissue, which in turn could have consequences for the functioning of the testis, resulting in de-masculinization.In the testis, a decrease in amh and cyp19a1a, both expressed in these cells in the testis.Fertilization success decreased over time with the mean fertilization rate dropping from 89% on day 1 to 69% by day 15. These findings are consistent with those of Haubruge et al., who reported declines in sperm count of 40-75% in guppies exposed to 0.274 or 0.549 mg/L BPA.cyp19a1a in females indicate a disruption of the estrogen/androgen balance within ovaries and consequent alterations in sex steroid signaling pathways, putatively leading to alterations in oogenesis and oocyte quality. This hypothesis is supported by previous studies in which BPA was shown to affect oogenesis.esr2a) in the gonads of both sexes, suggesting similar mechanisms could be occurring.Changes in fertilization success may have occurred not only due to effects of BPA on spermatogenesis but also due to BPA-induced alterations in egg quality. Females exposed to 1 mg/L BPA produced an increased number of eggs, but these eggs may have lacked the quality required for fertilization success and embryo survival. Many factors contribute to egg quality, of which the hormonal environment during oogenesis is a critical one.There is now strong evidence demonstrating that BPA has the potential to induce changes in DNA methylation at both gene-specific and genome-wide levels in exposed organismsdnmt1, for all three BPA concentrations tested in ovaries of females—including at environmentally relevant concentrations—and the DNA methylation pattern in the promoter region of the dnmt1 gene was found to be significantly associated with BPA exposure concentrations for four CpG sites. The expression of dnmt1 is known to be associated with changes in global DNA methylation, and inactivation of dnmt1 has been shown to cause global demethylation of the genome.dnmt1 transcription. In contrast, previous studies in Gobiocyprisrarus, have reported global DNA hypermethylation in ovaries exposed to 0.015 mg/L BPA for 35, ddnmt1 is reported to be an important maternal transcript involved in the regulation of DNA methylation during the first stages of embryo development, particularly prior to the zygote genome activation.dnmt1 observed in ovaries of females exposed to all three concentrations of BPA could have potential consequences for the appropriate development of offspring, in addition to influencing the level of DNA methylation in the ovary of exposed females.In our study, we found a significant decrease in the expression of the DNA methylation maintenance enzyme, dnmt1 transcription was also negatively associated with BPA exposure concentrations and a significant hypermethylation of two CpG sites in the promoter region of the dnmt1 gene in fish exposed to 0.1 mg/L BPA was observed. In addition, we measured a significant decrease in global DNA methylation in the testis of fish exposed to 1 mg/L BPA, suggesting that the BPA-induced reduction in global methylation is likely to be functionally linked to the decrease in dnmt1 transcription. These data align with the reported hypomethylation of sperm associated with the presence of BPA in urine, in a study of male factory workers in China.For males, dnmt1 transcription in males and females, including at environmentally relevant concentrations, demonstrating the very significant impact of BPA on the expression of this key DNA methylation maintenance enzyme. In addition, we report significant hypermethylation of the promoter region of the dnmt1 gene in both male and female livers. Based on the positive association between the expression of this gene and global DNA methylation, it is plausible that the suppression of dnmt1 may impact on global methylation as seen in other tissues. However, this could not be measured in the liver due to technical limitations related to the amount of DNA obtained from this tissue. The fact that changes in the transcript and methylation profile for dnmt1 occur at environmentally relevant concentrations highlights the potential for BPA to cause epigenetic effects in exposed organisms within current exposure scenarios.In the liver, we observed a significant decrease in dnmt1 transcription may be causing demethylation of specific areas of the genome or within specific cell types, but this may not be detectable by a global measurement of DNA methylation, including all cell types simultaneously. This may explain why dnmt1 transcription appears to be more sensitive to BPA exposure compared to global methylation measurements.It is important to note that global DNA methylation in this study, measured using the LUMA assay, provides only an estimate of the total DNA methylation across all areas of the genome and all cell types in a given tissue. Decreased mbd2 was significantly altered following exposure to BPA in both male testis and female livers. mbd2 belongs to a family of nuclear proteins capable of binding specifically to methylated DNA, and may also function to repress transcription from methylated gene promoters.mecp2 transcription in male livers, a gene involved in transcriptional repression by associating with methylated CpG dinucleotides where it silences transcription by recruiting histone deacetylases, resulting in chromatin remodeling.hdac3 transcription was also observed. These findings suggest that BPA is not only interacting with the processes linked to DNA methylation, but also has the potential to disrupt processes linked to chromatin structure and potentially impact on gene function via these mechanisms.The transcript profile for Despite the advances in our understanding of the epigenetic and transcriptional consequences of BPA in a model vertebrate, there are some limitations to the methodologies used: the locus-specific DNA methylation measurements conducted were based on the sodium bisulphite treatment of genomic DNA and, therefore, cannot distinguish between DNA modifications such as 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and methylcytosine (5mC), which have unknown functional significance.dnmt1, which occurred in conjunction with decreases in global DNA methylation. Of note, some changes were observed after exposure to environmentally relevant concentrations of BPA (0.01 mg/L), corresponding to current exposure scenarios for both humans and wildlife. These findings provide evidence of the adverse effects of BPA in a model vertebrate and advocate for BPA's replacement within consumer products and its reduction in the environment.Overall, we have found evidence that BPA caused significant disruption to reproduction in breeding zebrafish exposed to 1 mg/L BPA, likely via estrogenic mechanisms. The potential for BPA to cause disruption of reproduction shown here raises concerns for its toxicity when organisms are exposed to BPA in environments affected by other stressors, including other environmental endocrine disruptors with similar mechanistic pathways that may act additively to cause reproductive disruption. Importantly, BPA also caused significant alterations in the transcription of a number of genes involved in epigenetic regulation in both liver and gonad tissue, most notably on All chemicals were obtained from Sigma-Aldrich, UK, unless stated otherwise.22H2O, 9.4 mg/L NaHCO3, 50 mg/L MgSO47H2O, 2.5 mg/L KCl, 50 mg/L Tropic Marin Sea Salt), aerated, and heated to 28°C in a reservoir, before it was supplied to each aquarium using a flow-through system. Tanks were aerated and supplied with a flow rate of 48 L/day of water.Artemia nauplii once daily  and TetraMin tropical flake food  twice daily, to satiation.Wild type WIK strain adult zebrafish (originating from a stock population at the University of Exeter) were maintained according to the conditions reported in Paull et al.The selected 15 groups that showed consistent breeding and behavioral patterns during the initial acclimation period were subjected to a 10 d pre-exposure period, followed by a 15 d exposure period. Reproductive data for the 10 d pre-exposure period were collected to ensure that all breeding groups were reproducing consistently and there were no differences between reproductive measurements for any of the breeding groups prior to the chemical exposure period. Three independent replicate breeding groups were assigned at random to each treatment. A flow-through system was used to dose the tanks for 15 d with three concentrations of BPA  using ethanol (0.0005%) as a solvent. An absolute control receiving water alone and a solvent control receiving the same concentration of ethanol as the chemical exposures were also included.On day one of the exposure period, tanks were spiked with the appropriate amount of BPA to achieve the required exposure concentrations. Flow rates were monitored daily to ensure the chemical concentrations remained consistent and dosing stocks were replaced every day. Water samples from each tank were collected on days 5, 10, and 15 of the exposure, and were stored at −20°C until chemical analysis.The effects of BPA on reproduction were determined by measuring the egg production and fertilization success of individual groups. Eggs were collected each morning approximately one hour post-fertilization (hpf), washed and transferred to petri dishes for analysis. The numbers of fertilized and unfertilized eggs were determined by visual inspection for each treatment using a dissection microscope .3. The gonads and livers were dissected and weighed, and the gonadosomatic index (GSI) = gonad weight (mg)/[total weight (mg)- gonad weight (mg)] × 100 and hepatosomatic index (HSI) = liver weight (mg)/[total weight (mg)- liver weight (mg)] × 100 were calculated. Gonads and livers were collected, snap frozen in liquid nitrogen and stored at −80°C until analysis for transcript profiling and DNA methylation.On day 15 of the exposure period, all fish were sacrificed humanely using a lethal dose of benzocaine followed by destruction of the brain, in accordance with UK Home Office regulations. The wet weight and fork length were recorded, and the condition factor for each fish was calculated (k) = [weight (g) × 100]/[fork length (cm)]2) between the mean Ct and the logarithm of the cDNA dilution was > 0.99 in each case, and efficiencies were between 1.86-2.24. The primer sequences, annealing temperatures, PCR product sizes and PCR efficiencies for each primer pair are shown in Supporting Information Table S2.Transcript profiling of genes encoding epigenetic regulatory proteins and genes involved in reproductive function was conducted using real-time quantitative PCR (RT-QPCR) as previously described.rpl8), which has been shown to have stable expression in the livers and gonads following exposures to estrogens in another cyprinid fish species.RNA and DNA were extracted together from the livers and gonads of 8 male and 8 female fish from each treatment group using the AllPrep DNA/RNA Micro Qiagen Kit  according to the manufacturer's instructions, which allows for extraction of both RNA and DNA from the same tissue sample. NanoDrop ND-1000 Spectrophotometer  was used to assess RNA and DNA purity and concentration. RNA was treated with DNase I (Qiagen) to remove any potential DNA contamination. cDNA was synthesized from 2 µg of total RNA using random hexamers  and M-MLV reverse transcriptase , according to manufacturer's instructions. cDNA was then diluted 1:2 and RT-QPCR was performed in duplicate using an iCycler iQ Real-time Detection System  and SYBR Green chemistry, as previously described.esr1, amh, and dnmt1 were obtained from Ensembl  esr1 (ENSDARG00000004111) has 3 known transcripts  and 2 transcription start sites (TSSs). The dnmt1 gene (ENSDARG00000030756) also has 2 TSSs and 3 transcripts . amh (ENSDARG00000014357) has one transcript  and one TSS . Target sites within the promoter sequences were chosen based on their proximity to the TSSs and estrogen-responsive elements (EREs), identified using JASPAR,DNA sequence data for the promoter regions of Template preparation and pyrosequencing was carried out as described by Tost and Gut (2007) The LUMA assay was performed as described by Karimi et al. (2006) using DNA extracted from gonad samples from 8 individual fish per treatment.For analysis of the concentrations of BPA in the exposure water, methanol, acetonitrile and water, both HPLC and LC-MS grade, HiPerSolv CHROMANORM®, were purchased from VWR Int. One mL of each water sample was added to a glass vial and mixed with 1 mL of HPLC-grade acetonitrile. Before LC-MS/MS analysis, aliquots were vortexed and diluted in a mixture of acetonitrile and water (1:3 v/v). Analyses were performed using a Surveyor MS Pump Plus HPLC pump with an HTC PAL autosampler coupled to a TSQ Vantage triple quadrupole mass spectrometer equipped with heated electrospray (HESI II) source . Chromatographic separation was achieved using a reversed-phase, 3 µm particle size, C18 Hypersil GOLD column 50 mm × 2.1 mm i.d. . Analytes were separated using a linear gradient of water and methanol. The initial conditions for the gradient consisted of 10% methanol, which was increased to 100% in 4.5 min and maintained for 1 min before returning to the initial 10% methanol. The flow rate was 500 µL/min. The temperature of the autosampler was set at 8°C, and the column was kept at a room temperature. The HESI probe was operating in the negative mode and an ion-spray voltage of −4.0 kV was applied. The heated capillary temperature was set at 275°C and the vaporizer temperature was 60°C. Nitrogen was employed as sheath and auxiliary gas at a pressure of 30 and 5 arbitrary units, respectively. The argon CID gas was used at a pressure of 1.5 mTorr and the optimum collision energy (CE) for each transition was selected. Quantification of BPA was performed using two characteristic multiple reaction monitoring (MRM) transitions of precursor ion 227.1→ 212.1 (CE: 20 V) and 227.1 → 133.1 (CE: 28 V).2) was calculated using linear modeling for fertilization rates. Linear mixed effects models were generated using the lme4 package Statistical analyses were carried out using R (version 3.0.2).P ≤ 0.05, post-hoc testing was carried out using the pairwise multiple comparisons of means method with false discovery rate P value adjustment. Where the Kruskal-Wallis test was used, post-hoc testing was carried out using the Wilcoxon signed rank test accounting for repeated measures within the data sets. P values of ≤ 0.05 were considered to be significant. All data are presented as mean ± SEM.In order to determine the effects of BPA on the reproductive and molecular endpoints measured, statistical comparisons were performed between the solvent control and the groups exposed to BPA, and comparisons between the water control and the solvent control were also conducted to confirm that no significant differences occurred as a result of the presence of the solvent. Comparisons between treatments were performed using one-way analysis of variance (ANOVA) and Kruskal-Wallis tests. Where ANOVA analysis found a For transcript profiles, data points classified as outliers (using Chauvenet's criterion) and data points for which the expression was below the assay detection limit were excluded from analysis. Where amplification was detected in more than 70% of individuals, data were represented as fold-change relative to the expression in the water control group and groups were then compared using one-way ANOVA and Kruskal-Wallis tests with post-hoc tests as described previously. Where amplification was detected in less than 70% of individuals, data were represented as the proportion of individuals for which the target genes were detected, and analysis was conducted using a binomial generalized linear model. In the gonadal data sets, PCA was also performed using the prcomp function to identify the main trends in gene expression.dnmt1 transcription, as above. The relationship between BPA concentration and transcript expression or methylation was also determined using regression analysis, calculated using linear modeling.In order to determine if there were associations between the methylation levels for specific loci in the promoter regions of genes of interest and their transcription, correlation analysis was conducted. Where data was normally distributed Pearson correlation was used, and where data did not meet the assumptions of parametric testing, Spearman correlation analysis was performed. Correlation analyses were also conducted to determine the relationship between global methylation and All graphs were plotted using untransformed data for ease of interpretation, and were created using the R packages ggplot2,


Streptomyces coelicolor is well studied, little is still known about the role small noncoding RNAs (sRNAs) play in regulating gene expression in this organism. Here, we report the identification of a second target of the sRNA scr5239, an sRNA highly conserved in streptomycetes. The 159 nt long sRNA binds its target, the mRNA of the cobalamin independent methionine synthase metE (SCO0985), at the 5’ end of its open reading frame thereby repressing translation. We show that a high methionine level induces expression of scr5239 itself. This leads, in a negative feedback loop, to the repression of methionine biosynthesis. In contrast to the first reported target of this sRNA, the agarase dagA, this interaction seems to be conserved in a wide number of streptomycetes.While transcriptional regulation of the primary and secondary metabolism of the model organism  Escherichia coli and Salmonella typhimurium . The pH was adjusted to 7.2 with NaOH and the volume was increased to 90% of the final volume. After autoclaving the C, N and P sources were added  was described previously  and further divided by the amount of protein used to calculate specific Gus activity [A415 mg-1 min-1], which was used herein to represent Gus units (GU). All assays were conducted in triplicate and repeated at least once. Data are presented as the mean +/- standard deviation.108 pre-germinated spores of S. coelicolor strains were inoculated in 200 ml TSB medium and grown at 28°C under continuous shaking. After 3 days, 50 ml samples were harvested, washed twice in Evans medium and resuspended in Evans medium containing amino acids as indicated. The strains were incubated as before for another 24 h, harvested and washed twice in 1x ZAP . Cell disruption was done as described for the GUS measurements only in 800 μl 1x ZAP. Fifty μg of crude extract were separated on a 20 x 20 cm 8% PAA SDS gel [2O overnight. The next day, the gel was washed three times for 20 s in 50% ethanol. Next, it was incubated in solution 1 (0.5% Na2S2O3) for one min followed by three washing steps in ddH2O for 20 s each. After 20 min incubation in solution 2 , the gel again was washed three times for 20 s in ddH2O and finally developed in solution 3 . Once the protein bands were clearly visible, the reaction was stopped by addition of acetic acid to a final concentration of ~ 10%.10 SDS gel  overnighProtein bands showing a differential expression pattern were excised using a clean scalpel. Protein identification was done in the mass-spec facility of the Institute of Pharmaceutical Chemistry, Goethe University Frankfurt, Germany.Streptomyces genomes. However the only validated target of scr5239, the agarase dagA is present merely in S. coelicolor. This led us to the hypothesis, that scr5239 had to have further—possibly more conserved—target mRNAs.The sRNA scr5239 can be found in approximately two-thirds of the available sequenced S. coelicolor wild type cells grown in minimal medium supplemented with glutamine or glutamate. These two amino acids have a contrary effect; glutamine leads to a repression, glutamate to an induction of scr5239 expression  as a cofactor to catalyse the ive mRNA . By comp [et al.  predicteS. coelicolor grown on solid medium done in our lab supports this prediction. It places the metE transcription start point at 1,037,861 nt, 382 nt upstream of the GTG start codon  [In the next step, we tested whether scr5239 affects n region . Using Rn region  for sequ the ORF . On the metE mRNA we performed mobility shift assays with scr5239 and the metE mRNA. To omit possible interference of the B12 riboswitch during in vitro transcription or during refolding of the RNA after its purification, we used a 215 nt fragment of the metE mRNA (M1) covering the nucleotides-155 to +60. The mobility shift assay was performed using radiolabeled scr5239 RNA mixed with rising amounts of M1 RNA. metE 5’UTR including the riboswitch. For that we performed another gel retardation assay using the complete metE 5’UTR spanning the nucleotides-382 to +60 , where the sRNA was replaced by a kanamycin resistance cassette . As a control we cloned a shorter fragment covering the same 5’UTR part but only the first six nucleotides of the metE ORF thereby omitting the scr5239 binding site , ArgR (arginine) and NdgR  [metH and the methylenetetrahydrofolate reductase metF that catalyzes the second to last step of methionine synthesis. In this study, Kim et al. used the DNA sequences of the presumed promoter regions of the respective genes as bait and screened for NdgR binding. The metE probe, however, failed to capture any NdgR even though they used the whole intergenic region upstream of metE as bait. Kim et al. identified two possible binding motifs for NdgR within the probes that were captured by the protein. However, both of them are absent in the intergenic region upstream of the metE gene. This implies, that the two enzymes MetE and MetH are regulated by different means although they perform the same reaction: MetH expression is dependent on the presence of the transcriptional activator NdgR. The expression of MetE, on the other hand, depends both, on the absence of coenzyme B12 and of the sRNA scr5239.Little is known about the regulation of amino acid metabolism in hionine) ,33,34. NmetE. If coenzyme B12 is available the B12 dependent methionine synthase MetH is expressed while transcription of metE is terminated by the B12 riboswitch in its 5’UTR. If B12 is scarce, transcription of the metE mRNA is permitted and MetE is expressed. It is still unknown, if and how MetH expression is downregulated under these conditions. NdgR activates its transcription upon nutrient downshift [ownshift , but themetE mRNA thereby reducing methionine synthesis.Expression of MetE leads to an increased methionine level, which—by a yet unknown mechanism—induces the expression of scr5239. The sRNA then represses the translation of the metE is regulated by a B12 riboswitch in its 5’UTR in most bacteria. This allows the cell to make the decision if coenzyme B12 is available and if therefore the expression of a cobalamin-independent methionine synthase is necessary. Some bacteria, like Bacillus clausii, additionally have a SAM riboswitch in tandem to the B12 riboswitch in their metE 5’UTR. Also the expression of metH and metK is controlled by a SAM riboswitch in this organism. This setup generates a feedback inhibition where high SAM levels directly repress the expression of these three enzymes [metE , therefore we propose that here the conserved sRNA scr5239 takes over its regulatory function: to give a feedback about the cells methionine (and SAM) supply.The S-methylation of homocysteine is the final step in methionine biosynthesis. While there are several alternative ways leading to homocysteine, this last step is highly conserved. The two methionine synthases MetH and MetE can be found in parallel in a large number of bacteria, although there are species that carry one but not the other . The tra enzymes . StreptoB. subtilis, the glmS riboswitch probably represents the simplest solution. The ligand binding pocket of the aptamer domain not only binds intracellular Glucosamine-6-phosphate as an activating agent. It also binds Glucose-6-phosphate, which acts as an inhibitor of ribozyme activity. The riboswitch, thus, integrates metabolite information to sense the overall metabolic state of the cell [add gene from Vibrio vulnificus. This translational regulatory element senses temperature and ligand concentration to confer efficient regulation over the range of physiologically relevant temperatures, allowing gene expression control to adapt to the differing temperatures experienced in the free-living and in the infectious environments [B. clausii, two different riboswitches respond independently to SAM and vitamin B12, respectively. This system’s output fits a Boolean NOR gate, because transcription is terminated in the presence of only one or both ligands [Listeria monocytogenes which control the expression of the virulence regulator PrfA act as sRNA themselves [Enterococcus faecalis. This riboswitch can lead to premature termination of the sRNA-target interaction therefore regulating the sRNA function. Obviously there are plenty of possibilities to perform multiple input integration, however to our knowledge; metE is the first example of an sRNA regulating gene expression in combination with a riboswitch.Nature has come up with diverse solutions to tackle the problem of multiple signal input. In the cell . A seconronments . As alre ligands . In the  ligands  or, evenemselves . On the What is the connection of the regulation of methionine synthesis to the glutamine/glutamate complex? Glutamine acts as nitrogen storage in the cell. Feeding the cells with glutamine shifts the balance towards nitrogen surplus and leads to repression of scr5239 expression . It is nS1 FigScr5239 signal was normalized to the 5S rRNA loading control.(TIF)Click here for additional data file.S2 FigS. coelicolor M145 grown on solid medium throughout one developmental cycle. The differentially coloured bars represent the sequencing reads from each time point. The transcription start point of metE was identified at position 1,037,861 nt. Transcription termination by the B12 riboswitch occurs at a terminator structure at 1,038,093 nt (155 nt upstream of the metE start codon).RNA was prepared at five different time points from (TIF)Click here for additional data file.S3 FigUnder native conditions the M1 RNA forms at least three different conformations (see first lane). Yet all of them seem to bind scr5239.(TIF)Click here for additional data file.S4 FigmetE fragment M1 covering-155 to + 60 nt of the mRNA. FL: full-length 5’UTR of metE covering-382 to +60 nt of the mRNA. CE: 1 μg crude extract of S. coelicolor wild type. The full-length 5’UTR of metE is bound by scr5239 in a similar amount as the M1 fragment. Crude extract was added with the aim to increase binding efficiency by supplementing necessary protein(s). This did, however, in this setup not lead to an improved binding.M1: (TIF)Click here for additional data file.S5 Figa) Overview of the deletion strategy. The scr5239 gene including its promoter was replaced with a kanamycin resistance cassette by homologous recombination (-84 to +159 nt). The upstream gene SCO5238 has a putative terminator structure 20 nt upstream of the beginning of the deletion. The downstream gene SCO5239 gives rise to a leaderless transcript starting with the first nt of the start codon (unpublished data). (b) Northern blot validating the successful deletion of scr5239. The deletion was also confirmed by colony PCR of the mutant and subsequent sequencing of the PCR product (not shown).((TIF)Click here for additional data file.S6 Fig16S rRNA was used as a loading control.(TIF)Click here for additional data file.S7 Fig(a) RNAseq results of the metH gene  Cloned reporter fusion of metH to gusA used to measure a possible regulation by scr5239. The whole 5’UTR including the first 30 nt of the metH ORF where used. T represents the artificial terminator 3’ of the gusA ORF that ensures efficient transcription termination. (c) RNAhybrid predicted one possible binding site for scr5239 at-27 to-53 of the metH 5’UTR. The conserved core motif used for binding metE, however, is only partially present  Reporter gene measurement of the metH fusion protein. Deletion of scr5239 does not affect the expression of MetH:GusA.gene see . The met(TIF)Click here for additional data file.S8 Fig(a) Sequence alignment of scr5239 from eight Streptomyces species. The nucleotides used for binding metE in S. coelicolor are indicated in blue (top lane). Nucleotides predicted to be involved in metE binding in the other Streptomycetes are also indicated in blue. The highly conserved core binding motif is boxed in green. (b) Sequence alignment of the metE coding region. Colour coding as in (a). The top four sequences of scr5239 and metE are identical in their binding sites. For a detailed view of binding site variations see (TIF)Click here for additional data file.S9 FigmetE in S. coelicolor (sco), S. griseus (sgr), S. venezuelae (sve), S. bingchenggensis (sbi), and S. hygroscopicus (shy). Mutations in the sequences of the sRNA or metE are given in red, the start codon in bold.Predicted binding mode of scr5239 to (TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


Mollicutes) are insect-transmissible and plant-pathogenic bacteria that multiply intracellularly in both plants and insects through host switching. Our previous study revealed that phytoplasmal sigma factor rpoD of OY-M strain (rpoDOY) could be a key regulator of host switching, because the expression level of rpoDOY was higher in insect hosts than in plant hosts. In this study, we developed an in vitro transcription assay system to identify RpoDOY-dependent genes and the consensus promoter elements. The assay revealed that RpoDOY regulated some housekeeping, virulence, and host–phytoplasma interaction genes of OY-M strain. The upstream region of the transcription start sites of these genes contained conserved –35 and –10 promoter sequences, which were similar to the typical bacterial RpoD-dependent promoter elements, while the –35 promoter elements were variable. In addition, we searched putative RpoD-dependent genes based on these promoter elements on the whole genome sequence of phytoplasmas using in silico tools. The phytoplasmal RpoD seems to mediate the transcription of not only many housekeeping genes as the principal sigma factor, but also the virulence- and host-phytoplasma interaction-related genes exhibiting host-specific expression patterns. These results indicate that more complex mechanisms exist than previously thought regarding gene regulation enabling phytoplasmas to switch hosts.Phytoplasmas (class,  Escherichia coli is maintained at a constant level under various growth conditionsIn the regulation of bacterial gene expression, the initiation of transcription, mediated by a DNA-dependent RNA polymerase (RNAP) holoenzyme, plays an important role as the first step in the regulation process. The RNAP holoenzyme consists of a core enzyme (subunit composition α2ββ'ω) with catalytic activity of RNA polymerization, and an additional subunit known as a sigma factor involved in promoter recognition and DNA meltingMollicutes, genus ‘Candidatus Phytoplasma spp.’) infect hundreds of plant species and cause devastating yield losses in various crops worldwide57tengu and phyl1, phytoplasma genes encoding a secreted protein, are more highly expressed in plant hosts than insect hosts, and respectively induce phytoplasma-specific symptoms in plants such as witches’ broom and phyllody9Ca. P. asteris’ onion yellows strain (OY-M) are differentially expressed when grown in plant versus insect hostsPhytoplasmas  sigma factor subfamily and is present in each of the phytoplasma genomes as a multi-copy gene, with the exception of ‘Ca. P. mali’; these FliAs encode proteins containing only region 2, or regions 2 and 4  is unclear. To date, only limited studies have addressed the gene regulatory mechanisms in phytoplasmas due to the difficulties associated with their genetic engineering. All five genome-sequenced strains of phytoplasma have two types of sigma factors, RpoD and FliA12131415gene see . Phytoplgene see  online. nd 4 see  online12rpoD  and the others are categorized as fliA ( fliAOY). Despite the role of RpoD in E. coli and many other bacteria in regulating housekeeping genes17rpoD of OY-M (rpoDOY) is 4.0 times higher in insect hosts than in plant hosts11OY protein is unknown. These data raise doubts as to whether RpoDOY functions as the principal sigma factor. A few studies have approached this issue using ex vivo or in silico methods. A recent study using an E. coli-based ex vivo reporter assay (EcERA) system that evaluates the interaction between phytoplasmal promoters and sigma factors based on the luciferase reporter activity in E. coli cells showed that RpoDOY activates the promoter regions (approximately 400 bp) of several housekeeping genes highly expressed in insect hostsOY has the potential to function as the principal sigma factor and plays a major role in infected insect hosts. However, due to possible indirect effects of cognate transcription factors in living E. coli cells, avoiding false positives and negatives in this EcERA system was difficult. Thus, this system does not appear to be suitable for further research to identify promoter elements recognized by RpoDOY. In another study, bioinformatic prediction using E. coli RpoD-dependent promoter elements was performed to identify phytoplasma RpoD-dependent promoters. However, obtaining precise estimates using this approach is difficult, since phytoplasma genomes, as well as E. coli RpoD-dependent promoter elements, are strongly AT-biased4OY to determine its function.The genome of OY-M contains 13 copies of putative sigma factor genes, one of which is categorized as OY in phytoplasma gene expression in detail, we developed an in vitro transcription assay of phytoplasma genes and analyzed their promoter activity using this assay. The consensus RpoDOY-dependent promoter elements were identified for the first time. The genome-wide promoter prediction using this newly identified essential promoter elements revealed at least 88 genes that were regulated by RpoDOY, which was involved in the transcription of not only many housekeeping genes, but also virulence- and host-phytoplasma interaction-related genes. We discuss the mechanism of transcription regulation associated with host switching between plants and insects based on the function of RpoDOY.In this study, to elucidate the role of RpoDOY, we investigated the TSSs of the 16S ribosomal RNA (rrn) B gene of OY-M, as rrn is often transcribed from RpoD-dependent promoter elements in many bacteria212223rrnB was performed using total RNA from phytoplasma-infected plants, resulting in the detection of three TSSs located 91, 254, and 444 bp upstream of rrnB  and purified recombinant RpoDOY  and their putative promoter regions, was used as a template for transcription reactions. As shown in OY-dependent manner. The sizes of these transcripts corresponded to the expected sizes of transcripts initiated from P2 (254 bp upstream of rrnB) and P3 (444 bp upstream of rrnB), respectively. No transcripts corresponding to that transcribed from P1 (91 bp upstream of rrnB), which was expected to be 375 nt, were detected. These results indicate that RpoDOY is compatible with the heterologous E. coli RNAP to initiate transcription as the RNAPEc–RpoDOY holoenzyme. Our results also demonstrated that the RNAP holoenzyme with RpoDOY recognizes the rrnB promoter elements upstream of P2 and P3, but not P1.To identify which of these putative rrnB, we focused on the putative P2 promoter and performed in vitro transcription assays. We introduced a series of double-base substitutions to GG into the putative P2 –35 and –10 promoter elements of a DNA fragment containing –400 to –1 of rrnB, named PrrnB_P2  also resulted in a reduction in promoter activity to 6–22% compared with the intact PrrnB_P2 , but not in the putative P1 promoter elements (–35 5′-TTCACA-3′ and –10 5′-TAATCT-3′).To define the core promoter elements of PrrnB_P2 . In the , 50S ribosomal subunit protein L13 (rplM), 30S ribosomal subunit protein S4 (rpsD), and RNA polymerase sigma70 factor (rpoD) genes] for in vitro transcription as templates . In addition, the upstream region of the molecular chaperone gene (ibpA) was used as a template (PibpA) because a previous study suggested that it is regulated by RpoD in an AT-rich bacterium ‘Ca. Blochmannia floridanus’ibpA is reported to be regulated by an alternative heat shock sigma factor RpoH31in vitro transcription assays revealed that RNAPEc–RpoDOY produced specific transcripts from the four templates , but did not produce a transcript from PrpoD  containing upstream regions of genes associated with virulence or host–phytoplasma interactions: PAM157 (putative secreted protein), PAM289 (adhesin-like protein)PAM486 (putative secreted protein), tengu (secreted and virulence-related protein)amp (insect transmissibility-related protein)PAM157 and PAM289 were highly expressed in insect hosts and PAM486 and tengu were highly expressed in plant hosts9amp was expressed at comparable levels in both plant and insect hostsin vitro transcription assays revealed that RNAPEc–RpoDOY produced specific transcripts from all templates, PPAM157, PPAM289, PPAM486, Ptengu, and Pamp  using total RNA from phytoplasma-infected plants or insects. As a result, we mapped the 5′-ends of ibpA, infC, rplM, rpsD, PAM157, PAM289, PAM486, tengu, and amp transcripts at 170, 415, 166, 301, 177, 222, 52, 153, and 81 nt upstream, respectively, of their start codons , we searched putative RpoDOY-dependent genes that possess these promoter elements on the whole genome sequence of the OY-M phytoplasmaOY-dependent promoter elements located within a 500-bp upstream region of either ATG initiation codons or the 5′-ends of mature tRNA and rRNA of OY-M genes, which could mediate the transcription of at least 88 genes , ‘Ca. P. australianse’ (PAa), and ‘Ca. P. mali’ strain AT (ATP)]. Given that RpoD homologous proteins of these phytoplasmas contain highly conserved motifs, regions 2 and 4, which are responsible for the recognition of the –10 and –35 promoter elements, respectively  for promoter prediction. In total, 68, 71, and 100 putative RpoD-dependent genes were found in AYWB, PAa, and ATP, respectively. Of the 88 putative RpoDOY-dependent genes in OY-M, 34 genes (39%) were also predicted to be RpoD-dependent in at least one of the other phytoplasma strains. Various housekeeping genes such as ribosomal RNA genes, ribosomal protein subunit genes, and tRNAs, were found in common among the putative RpoD-dependent genes of all three phytoplasmas  that have only one TSS with –10 and –35 promoter elements resembling the RpoDOY-dependent promoters of rrnB (P2 and P3)  . Moreoven assays . These rrrnB, PAM289, and tengu) were common between the in vitro transcription assay in this study and another in vivo promoter assayrrnB and PAM289 were recognized by RNAPEc-RpoDOY in our in vitro transcription assay  were foundOY-dependent promoter elements identified in this study were consistent with previous observations of other bacteria.Our activity . In contactivity ,c. Moreovariable . In agreOY-dependent promoter possessed other sequence features common in bacteria, such as a 5′-TnTG-3′ positioned around –17 to –14 (“the extended –10 region”) and an A-rich region positioned around –42 to –39  of RpoD and the α-subunit of RNAP, respectively42B. subtilis and E. coli38C. trachomatis, nucleotide substitutions at positions 4 and 5 bp upstream of TSSs, where no conserved motif has been identified, had negative effects on promoter activityin vitro transcription assay using RpoDOY revealed that nucleotide substitutions at positions –14 and –13 (mt11), and –6 and –5 (mt15), slightly decreased promoter activity  conserved in phytoplasmas is one of the candidate transcription regulators. In some bacteria, histone-like proteins act as a transcriptional repressor by binding to DNA44OY-dependent genes, which leads to their specific expression in plant hosts. Second, non-coding RNAs may play roles as gene regulatory factors. Recent studies have revealed that non-coding RNAs such as riboswitches and small RNAs regulate their gene expression in many bacteria46in silico promoter analysis, many promoter elements that positioned intergenic regions far from the ATG initiation codon or within the coding regions of genes, implying the existence of many non-coding RNAs in phytoplasmas.How RpoDMollicutes, mycoplasmas and spiroplasmas possess rpoD as a single sigma factor gene in their genomes4951In class in silico analysis detected 88 RpoDOY-dependent genes in the OY-M genome. Some of these genes, such as znuA, rRNA-16S, and tRNA-Glu, were known as the first genes of operons that could be regulated by the principal sigma factor in other bacteria212254OY-dependent operons, RpoDOY is likely to regulate more than 88 genes identified by our in silico analysis, even though additional studies are needed to determine whether each mRNA of putative RpoDOY-dependent genes is monocistronic or polycistronic.Our Streptomyces griseus, the principal sigma factor hrdB is not controlled by itself, but by the alternative sigma factor ShbA, which is known as an ECF sigma factorOY as the principal sigma factor, PrpoD was not controlled by RNAPEc–RpoDOY  that can constitute a set of operons containing rpoDOY from the list of putative RpoDOY-dependent genes  was isolated in Saga Prefecture, JapanChrysanthemum coronarium) using the leafhopper vector insect Macrosteles striifronsThe ‘http://ai.stanford.edu/~xsliu/BioProspector/. The resulting consensus promoter sequence was illustrated based on multiple sequence alignments using the WebLogo toolhttp://weblogo.berkeley.edu/.To identify the 5′-end of mRNA in OY-M, 5′ RACE analysis was performed using the 5′ RACE System for Rapid Amplification of cDNA Ends (Life Technologies Inc.) according to the manufacturer’s protocol. Polymerase chain reaction (PCR) amplification was accomplished using Taq DNA polymerase (TaKaRa Bio Inc.), a nested gene-specific primer (RACE1 or RACE2) that anneals to a site located within the cDNA molecule see  online, OY, the full-length rpoD gene of OY-M was PCR-amplified using a primer pair rpoD_kpnF (5′-GGG GTA CCA TGG AAT TCG ATA ACA TAA TCA AAA -3′) and rpoD_salR (5′-CGA CGT CGA CTT ATT TGT GGT TGT GGT ACA AAC TTT TT-3′). The amplified DNA fragments were digested with KpnI and SalI, followed by cloning into pColdI (TaKaRa Bio Inc.) digested with the same enzymes. The resulting plasmid was subsequently transformed into E. coli strain BL21-CodonPlus™ (DE3)-RIL cells (Stratagene). The transformed E. coli was precultured at 37 °C in lysogeny broth (LB) medium containing ampicillin (50 μg/ml). The overnight culture (2 ml) was added to 100 ml LB medium containing ampicillin (50 μg/ml). After incubation for 1 h at 37 °C and subsequent incubation for 30 min at 15 °C, protein expression was induced by the addition of 0.1 mM isopropyl-β-D-thiogalactopyranoside (IPTG). Cells were cultured at 15 °C for 24 h following induction.To construct pCold_His-RpoDOY was purified as previously describedE. coli from pCold_His-RpoDOY. A DNA-dependent RNA polymerase (RNAP) holoenzyme containing RpoDOY (RNAPEc-RpoDOY) was made by adding 20 ng of purified RpoDOY to 1 U of E. coli RNAP core enzyme (Epicenter) in buffer  overnight at 4 °C. The reconstituted complex was verified by NativePAGE™ gel electrophoresis  using genome sequence files obtained from the NCBI genome database (http://www.ncbi.nlm.nih.gov/genome/ ). Whole genome sequences were searched for promoter sequence patterns using ‘dna-pattern’ of RSA-tools (http://rsat.ulb.ac.be/index.html). The resulting sequences were matched to each reference genome sequence, and the genes to which the resulting sequences were matched within 500 bp upstream of either ATG or 5´-end of mature rRNA and tRNA are listed in Promoter predictions were made in the genomes of ‘How to cite this article: Miura, C. et al. Functional characterization of the principal sigma factor RpoD of phytoplasmas via an in vitro transcription assay. Sci. Rep.5, 11893; doi: 10.1038/srep11893 (2015).


Long non-coding RNAs (lncRNAs) are transcripts without protein-coding potential but having a pivotal role in numerous biological functions. Long non-coding RNAs act as regulators at different levels of gene expression including chromatin organization, transcriptional regulation, and post-transcriptional control. Misregulation of lncRNAs expression has been found to be associated to cancer and other human disorders. Here, we review the different types of lncRNAs, their mechanisms of action on genome formatting and expression and emphasized on the multifaceted action of the H19 lncRNA. The advent of DNA tilling arrays and deep sequencing technologies has revealed that a much larger part of the genome is transcribed into RNAs than previously assumed. It is estimated that up to 70% of the genome is transcribed but only 2% of the human genome codes for proteins  and RNAsNon-coding RNAs include the well-known ribosomal (r) RNAs, ribozymes, transfer (t) RNAs, small nuclear (sn) RNAs, telomere-associated RNAs , as well as a plethora of far less characterized RNAs. Based on their size, these ncRNAs are subdivided into two groups: small ncRNAs (<200 nt) and long ncRNAs [lncRNA (>200 nt)]. Small ncRNAs, such as microRNAs (miRs), small interfering RNAs (siRNAs), or PIWI-interacting RNAs (piRNAs) received much attention and were shown to mainly act as negative regulators of gene expression. In contrast, lncRNAs represent a more functionally diverse class of transcripts. LncRNAs are found in a large diversity of animals species , but alsMajority of lncRNAs are 5′ capped, 3′ polyadenylated, multi-exonic and are subjected to transcriptional regulation as coding mRNAs . Some ofWhile only a limited number of lncRNAs has been studied, numerous evidences indicate that lncRNAs interact with a plethora of proteins. Furthermore, homologous Watson–Crick base pairing provides an efficient way by which lncRNAs may selectively interact with other nucleic acid species. It is believed that lncRNAs are involved in a diversity of cellular functions through gene expression regulation at different levels including chromatin organization, transcriptional regulation, and post-transcriptional mRNA processing .To complicate matters further, Figure 1).The ability of lncRNAs to recognize complementary sequences allows the regulation of mRNA processing at various steps, including degradation, splicing, translation, or transport (Base pairing between defined regions of the human β-site APP-cleaving enzyme 1 (BACE1) transcript and its antisense lncRNA BACE1-AS induces the mRNA stabilization and consequently the increase in BACE1 protein abundance . SimilarThe translational process may also be modulated positively or negatively by lncRNA–mRNA pairing. For example, the antisense lncRNA ULCH-AS1  enhances ULCH mRNA translation , whereasThe lncRNA MALAT1 (metastasis associated lung adenocarcinoma transcript 1) regulates pre-mRNA alternative splicing by modulating active serine/arginine splicing factors levels . In thisMeg3/Gtl2 contains in its last intron the evolutionarily conserved microRNA miR-770 whereas Meg8 transcripts have the intron-encoded miR-341, miR-1188, and miR-370. Similarly, miR-155 is processed from sequences present in BIC lncRNA that accumulates in lymphoma cells.Some lncRNAs act on post-transcriptional regulation through the modulation of the microRNA (miR) pathways. MiRs, a large class of small ncRNA, function by annealing to complementary sites in the coding sequences or 3′-untranslated regions (UTRs) of target mRNAs where they favor the recruitment of protein factors that impair translation and/or promote transcript degradation leading to a decrease in protein abundance . SpecifiFigure 2). The dihydrofolate reductase (DHFR) gene contains a major and a minor promoter. The minor promoter gives rise to a lncRNA that forms a stable triplex lncRNA-DNA association at the major DHFR promoter and interacts with the general transcription factor II B (TFIIB) leading to the dissociation of the transcriptional preinitiation complex at this major promoter and then reducing DHFR expression (A number of evidences indicate that lncRNAs can act at the level of transcription either negatively or positively through a variety of molecular mechanisms (pression .Other lncRNAs act as decoys to negatively control transcription by titrating transcription factors away from their cognate promoters. The lncRNA PANDAR (promoter of CDKN1A antisense DNA damage activated RNA) is induced in a TP53-dependent manner and inhibits apoptotic gene expression to favor cell-cycle arrest through direct interaction with, and sequestration of NFYA, a transcription factor controlling the apoptotic program upon DNA damage . SimilarRather than acting as molecular decoys, lncRNA could modulate transcription by recruiting factors at target gene promoters or acting as transcription factor co-activators. For example, a lncRNA produced at the 5′ regulatory region of the cyclin D1 (CCND1) gene in response to genotoxic stress tethers and modulates the activity of the RNA-binding protein TLS (translocated in liposarcoma) which in turn inhibits the activity of the histone acetyltransferases CBP (CREB binding protein) and EP300, leading to CCND1 transcriptional repression . The lncOther lncRNAs regulate transcription by controlling transcription factor trafficking. As such, the lncRNA NRON  interacts with importin-beta family members to inhibit nuclear translocation of the inactive dephosphorylated nuclear factor of activated T cells (NFAT) trans-activator .Figure 3).LncRNAs have been implicated in the control of gene expression through the recruitment of epigenetic modifiers at specific genomic loci. In eukaryotic chromatin, epigenetic regulation is conveyed by covalent modifications of DNA , modifications of histone tails , and the incorporation of various histone variants. These modifications locally change chromatin organization and regulate gene expression without changes in the DNA sequence. A number of evidences indicate that lncRNAs, acting as guides targeting enzymes involved in chromatin modifications, are part of this picture (The lncRNA HOTAIR (HOX transcript antisense RNA) is transcribed from the HOXC locus and targets Polycomb Repressive Complex 2 (PRC2) to silence distantly located genes, including genes at the HOXD locus and 100s of other genes on various chromosomes . Componecis  which is produced by the INK4B/ARF/INK4A locus binds specifically two Polycomb proteins, CBX7 (PRC1) and SUZ12 (PRC2). Disruption of interaction with both PRC1 and PRC2 proteins impacts the transcriptional repression at the INK4B locus in cis . As anotcis . Thus, scis , are belcis .cis, with an efficacy that correlates with the proximity of the HOXA genes relative to the HOTTIP transcriptional unit. At the target genes, knockdown of HOTTIP results in the loss of activating H3K4me3 and H3K4me2 epigenetic marks, together with the decreases in occupancy of the MLL1 protein complex responsible for the establishment of these histone modifications. Furthermore, chromosome conformation capture carbon copy (C5) assays revealed abundant long-range looping interactions, bridging the transcribed target HOXA genes into proximity of the HOTTIP transcriptional unit. Thus, the mechanism by which the lncRNA HOTTIP controls HOXA expression relies on its potential to guide the histone methyltransferase MLL1 at target HOXA gene promoters, and on the formation of chromatin loops that connect distantly expressed HOXA genes to HOTTIP transcripts.In contrast, the lncRNA HOTTIP  mediates transcriptional activation by controlling chromatin modification and organization . HOTTIP A role of lncRNAs in chromatin loop formation has also been described for the lncRNA CCAT1-L . Indeed,Thus, lncRNAs, through the recruitment of chromatin modifiers and/or the induction of chromatin loops will modulate the chromatin conformation and will format the genome in a particular configuration. This lncRNA-mediated genome formatting emerges as a crucial and fundamental mechanism by which lncRNA may act on gene expression programs.As discussed above, lncRNAs can regulate genome expression through different molecular mechanisms. However, several lncRNAs use multiple strategies that, in combination, may be required for their biological function. The action of the lncRNA H19 on gene expression illustrates the complexity of the combinatorial mechanisms of regulation achieved by a single lncRNA. H19 was the first lncRNA discovered . FurtherFigure 4). The ICR (imprinting control region), located 2 kbp upstream of the H19 promoter, controls the monoallelic expression of H19 and IGF2 by insulating communication between the 3′ enhancers and the IGF2 promoter. The chromatin insulator property of the H19/IGF2 ICR is regulated by the insulator CTCF (CCTC-binding factor), which binds specifically to the unmethylated maternal allele. On the paternal allele, the ICR methylation does not allow CTCF binding and leads to IGF2 expression . H19 has been shown to guide chromatin modifying enzymes to specific loci. In particular, To complicate matters further, H19 lncRNA mechanisms of action appear to be extremely diverse, acting at various levels . Since lncRNAs are regulating a different cellular pathways, growing evidences suggest that they could play a role in a large number of other human disorders including metabolic diseases, neurodegenerative and psychiatric disorders, cardiovascular and immune dysfunctions  designed to perfectly match exact stretches of nucleotides, guarantee a high degree of specificity leading to lncRNA degradation. The power of the siRNA approach is illustrated by the success of a number of preclinical studies where siRNAs targeted mRNAs . Similarse model  and the se model . Similartastasis .In this context, further exploration in the complexity of the lncRNA world promises the emergence of novel therapeutic opportunities.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.


Sorex araneus), the mouse, humans and a bat (Myotis blythii), were tested by cotransfection with a reporter gene into cells lacking MTF-1. Whereas shrews only live for 1.5–2.5 years, bats, although living on a very similar insect diet, have a lifespan of several decades. We find that bat MTF-1 is similarly metal-responsive as its human counterpart, while shrew MTF-1 is less responsive, similar to mouse MTF-1. We propose that in comparison to most other mammals, the short-lived shrews and rodents can afford a “lower-quality” system for heavy metal homeostasis and detoxification.Non-essential “heavy” metals such as cadmium tend to accumulate in an organism and thus are a particular threat for long-lived animals. Here we show that two unrelated, short-lived groups of mammals  each have independently acquired mutations in their metal-responsive transcription factor (MTF-1) in a domain relevant for robust transcriptional induction by zinc and cadmium. While key amino acids are mutated in rodents, in shrews an entire exon is skipped. Rodents and especially shrews are unique regarding the alterations of this region. To investigate the biological relevance of these alterations, MTF-1s from the common shrew ( Sorex araneus, has a lifespan of only 1.5 years and weighs a mere 9 g . The diet of most bats, which represent a distinct group of animals, also consists mainly of insects. But in marked contrast to shrews, bats are generally long-lived, three to four decades have been recorded for free-living bats of the Myotis genus . The common shrew, We were interested in comparing the activity of the metal-responsive transcription factor MTF-1) in these two types of mammals—shrews and bats. MTF-1, also referred to as metal regulatory transcription factor or metal response element binding transcription factor, is a key component of the heavy metal regulatory system (Lichtlen and Schaffner  in theseSorex araneus and the long-lived Myotis blythii (lesser mouse-eared bat). In transfection experiments with a MRE-containing reporter gene, we find that shrew MTF-1 is indeed poorly inducible by zinc and cadmium, due to loss of an entire exon domain involved in transcriptional activation and metal response, while bat MTF-1, similar to its human ortholog, responds well to these heavy metals.The main reason for the present investigation was our previous finding that, in marked contrast to the robust metal responsiveness of human MTF-1, mouse and capybara (a South American rodent) MTF-1s display a poor metal response when tested on MRE-containing reporter genes  and the two lysines replacing leucine and threonine in opossum (Monodelphis) might be worth a further investigation; both of them are relatively short-lived, rapidly multiplying species.Exploiting the (incomplete) genome sequence of the common shrew ain Fig. ). The MTain Fig. , and fugain Fig. , both ofain Fig. ). The exTo determine the metal responsiveness of shrew and bat MTF-1s, their cDNAs were cloned into an expression vector driven by the widely used HCMV enhancer–promoter region  and one from a long-lived European reptile, the slow worm Anguis fragilis. With regard to metal induction, the former responds poorly and the latter well  Fig. a, b. We Our experiments also addressed an apparent paradox: on the one hand we found weak, if any, metal inducibility of mouse and shrew MTF-1. However, mouse metallothionein-1 and 2 genes contain multiple MREs in their upstream enhancer–promoter region. Moreover, metallothionein transcript levels clearly increase upon heavy metal load ribose polymerase PARP-1  was shown to correlate with the lifespan of mammalian species. While this effect is mostly due to different expression levels, a higher specific activity of human PARP-1 compared to its rat counterpart seems to also contribute  can reach an age of 30 years  in subgroups of rodents, one shared by mouse, rat and Syrian hamster (suborder Myomorpha), the other by capybara, naked mole rat and guinea pig (suborder Hystricomorpha) , at variance with the solid response of the endogenous metallothionein gene to both mouse and shrew MTF-1, as shown in Fig. Sorex araneus (ENST00000373036) and Crocidura russula were kind gifts of Peter Vogel (University of Lausanne). From approximately 200 mg tissue, RNA was isolated using Trizol (Invitrogen) following the supplier’s recommendations. MTF-1 cDNA was obtained using the SMART RACE cDNA Amplification Kit (Clontech) and inserted into the same expression vector driven by the human cytomegalovirus enhancer–promoter as described for the MTF-1 clones containing human, mouse, capybara and fugu MTF-1 prepared previously in our lab  or cadmium (CdCl2) at the concentrations indicated in the figures, typically 100 μM and 50 µM for zinc and cadmium, respectively. After collection of the cells, RNA was isolated by phenol/DCM extraction; reporter and reference gene transcripts were quantified by the S1-nuclease protection assay as described (Weaver and Weissmann MTF-1 expression vectors and a reference gene (OVEC-ref) for normalization of transfection efficiency (Westin and Schaffner 32P-labeled MRE-s oligonucleotide and samples were separated under non-reducing gel conditions as described (Westin and Schaffner Exponentially growing Dko7 cells in a 10 cm dish were transfected with 5 µg of MTF-1 of each expression plasmid. Two days later, nuclear extracts were prepared as described in (Schreiber et al.


Mycobacterium tuberculosis cAMP receptor protein (CRPMt) at endogenous expression levels using a specific α-CRPMt antibody. Under these native conditions an equal distribution between intragenic and intergenic locations was observed. CRPMt binding overlapped a palindromic consensus sequence. Analysis by RNA sequencing revealed widespread changes in transcriptional profile in a mutant strain lacking CRPMt during exponential growth, and in response to nutrient starvation. Differential expression of genes with a CRPMt-binding site represented only a minor portion of this transcriptional reprogramming with ∼19% of those representing transcriptional regulators potentially controlled by CRPMt. The subset of genes that are differentially expressed in the deletion mutant under both culture conditions conformed to a pattern resembling canonical CRP regulation in Escherichia coli, with binding close to the transcriptional start site associated with repression and upstream binding with activation. CRPMt can function as a classical transcription factor in M. tuberculosis, though this occurs at only a subset of CRPMt-binding sites.Chromatin immunoprecipitation identified 191 binding sites of  Mycobacterium tuberculosis as one of the most deadly human pathogens depends on its ability to adapt to diverse intracellular and extracellular environments encountered during infection, persistence and transmission [reviewed in  of M. tuberculosis with a particular emphasis on the localization of CRPMt-binding sites relative to transcription start sites (TSSs).The success of iewed in ]. This iiewed in . Defininrategies . A combirategies , a recenrategies ,8. High-profiles . In the Escherichia coli, with a role in regulation of around 200 transcriptional units. CRP binds to a palindromic sequence (TGTGAN6TCACA) in the promoters of target genes and, depending on the distance between the binding site and the transcriptional start site, enhances or restricts recruitment of ribonucleic acid (RNA) polymerase . In rganizer . A key fM. tuberculosis CRPMt, encoded by Rv3676, recognizes a similar binding motif and has been shown to regulate transcription of several promoters—for example, the serC promoter  and M. tuberculosis Δcrp, an H37Rv mutant in which Rv3676 has been deleted (E. coli cultures were grown in Luria-Bertani broth and agar (15 g/l). Where needed, ampicillin and kanamycin were used at final concentrations of 100 and 50 μg/ml, respectively. M. tuberculosis cultures were grown in Dubos broth containing 0.05% (v/v) Tween, Middlebrook 7H9 broth or Middlebrook 7H11 agar and supplemented with 0.5% (v/v) glycerol and 4% albumin. To monitor the response to nutrient starvation , the cells pelleted and washed twice in phosphate buffered saline (PBS) supplemented with 0.025% tyloxapol. Cells were then resuspended in 100 ml PBS plus tyloxapol and incubated without shaking at 37 °C.The strains used were  deleted . E. coliarvation , M. tubeE. coli as described previously  was administered subcutaneously in Freund's complete adjuvant, followed by two booster injections in Freund's incomplete adjuvant after 14 and 30 days. Sera were prepared and then stored at −20°C until required. Immunoglobulin G (IgG) was purified from the serum using T-Gel purification kit (Pierce) as per manufacturer's instructions. Purified IgG was used for ChIP-seq analysis. Rv3597c antibody was raised by Cambridge Research Biochemicals, using a peptide antigen.ChIP was performed as previously described  with someviously  and usedM. tuberculosis H37Rv (wild type) and M. tuberculosis Δcrp cells were grown in roller bottles to mid-exponential phase (OD600 0.6) and formaldehyde was added to a final concentration of 1%. After 10 min of incubation, glycine was added to a final concentration of 0.5 M to quench the reaction and incubated for a further 5 min. Cross-linked cells were harvested by centrifugation and washed twice with ice-cold Tris-buffered saline . Cell pellets were resuspended in 4-ml immunoprecipitation (IP) buffer . Cells were lysed and the DNA sheared to an average size of ∼300 base pairs (bp) using a Bioruptor (Diagenode) with 40 cycles of 30-s on/off at high setting. Insoluble matter was removed by centrifugation for 10 min at 4°C, and the supernatant was split into two 1.8-ml aliquots. The remaining ∼400 μl was kept to check the size of the DNA fragments and for sequencing to remove any sequencing bias (input).Mt antibody raised against purified CRPMt (Rv3676) protein  and 50-μl Protein A/G UltraLink Resin, pre-incubated with 1-mg/ml bovine serum albumin in TBS, on a rotary shaker at 4°C overnight. Samples were washed once with IP buffer, twice with IP buffer containing 500-mM NaCl, once with wash buffer  and once with TE (pH 7.5). Immunoprecipitated complexes were eluted from the resin in 100-μl elution buffer  at 65°C for 30 min. Immunoprecipitated samples and the input DNA were de-cross-linked in 0.5x elution buffer containing 0.8-mg/ml Pronase at 42°C for 2 h followed by 65°C for 6 h. DNA was purified using a polymerase chain reaction (PCR) purification kit (QIAGEN). Prior to library preparation and sequencing, the DNA fragment sizes were checked by agarose gel electrophoresis and gene-specific quantitative PCR was carried out.Each 1.8-ml aliquot was incubated with 20-μl Protein A/G UltraLink Resin (Pierce) on a rotary shaker for 45 min at room temperature to eliminate complexes bound to the resin non-specifically. The supernatant was then removed and incubated with either no antibody (mock-IP), specific α-CRPΔcrp M. tuberculosis H37Rv in Middlebrook 7H9 media, as previously described . Library construction and sequencing were performed using the ChIP-seq Sample Prep kit, Reagent Preparation kit and Cluster Station kit (Illumina). Samples were sequenced on an Illumina Genome Analyzer IIx (GAIIx) instrument and loaded at a concentration of 10 pM. For RNA sequencing (RNA-seq), strand-specific cDNA libraries were constructed using the Illumina Small RNA Sample preparation kit with the v1.5 small RNA (sRNA) 3′ Adaptor and sequenced on a GAIIx (Illumina).M. tuberculosis H37Rv (EMBL accession number AL123456) as single-end data using the Burrows-Wheeler aligner (BWA)  . The gener (BWA)  and visuer (BWA)  and the er (BWA) . For evaer (BWA)  were anaMt. To validate the RNA-seq data, specific primers were used to quantify the messenger RNA targets showing up- or downregulation, and control targets showing no differential expression. RNA was extracted as described above, and 30 ng total RNA from wild-type and Δcrp cells was used with the Express One-Step SYBR GreenER kit (Invitrogen) according to the manufacturer's guidelines. All primer sequences and qRT-PCR results are in Supplementary Tables S1 and S2.Quantitative real-time PCR (qRT-PCR) was used to determine whether the ChIP experiment had worked prior to library construction and to validate both the ChIP-seq and RNA-seq data. To measure the enrichment of TF-binding targets in the immunoprecipitated DNA samples, 1 µl of IP or mock-IP DNA was used with Quantitect SYBR Green (QIAGEN) together with specific primers to the upstream region of Rv3616c, known to be bound by CRPSamples for cAMP measurement during the starvation response were taken at time points 0 h, 24 h, 48 h and 96 h. At each time point, 2 ml of culture was spun down, resuspended in 0.1-M HCl and boiled for 10 min. The samples were lysed in the presence of glass beads  using a Ribolyser (Hybaid) at a speed setting of 6.0 for 40 s. The supernatant was collected by centrifugation and stored at −20°C until the assay was performed. Levels of cAMP in the cells were measured using the Direct cAMP Enzyme Immunoassay kit (Sigma), following the acetylated version according to the manufacturer's guidelines, and normalized to the total amount of protein in the samples (measured using a Nanodrop ND-1000).Mt binding to the M. tuberculosis chromosome by ChIP combined with high-throughput sequencing (ChIP-seq) and to integrate these data with transcriptional profiling by RNA-seq. ChIP-seq was done using a specific antibody against CRPMt, thus enabling us to study the binding under native conditions without the need to tag and overexpress the protein. Under these native conditions, CRPMt is expressed at high levels in the cell; based on published quantitative mass spectrometry and electron microscopy (Mt molecules to be approximately stoichiometric to the number of ribosomes per cell (∼3500).The aim of this work was to investigate CRPcroscopy ,37 and wM. tuberculosis chromosome was calculated and the traces visualized in the Artemis genome browser. Peaks were called using the CisGenome software (Mt-IP compared to the mock-IP (performed in the absence of antibody) and input (sheared genomic DNA). To validate the results, the data were also analysed using the BayesPeak package in R/Bioconductor, and peaks called in only one of the two methods were discarded. As seen in Figure M. tuberculosis chromosome in the mock-IP  indicating negligible non-specific binding to the resin or the antibody. In the CRPMt-IP, however, 191 peaks were found, denoting CRPMt-binding sites (here abbreviated to CBSs) on the chromosome . No ChIP-seq signals were detected for the crp-deletion strain.We were able to map 98% of the sequences uniquely to the H37Rv genome  and achieved a near-complete representation of the entire genome (98% of the genome was mapped). The remaining 2% of the genome includes PE/PPE genes, which contain highly repetitive sequences that are poorly resolved by short-read sequencing. To visualize the genome coverage, the number of reads mapping to each position on the software  to idente Figure . The aveDe novo motif discovery by MEME-ChIP , we integrated the ChIP data set with a ibraries . The spaibraries  and B. GMt may act in concert with other regulators. The promoter region of Rv1057, for example, has binding sites for MprA, EspR and TrcR in addition to CRPMt (fadD26 (Rv2930) with an EspR-binding site (espA (Rv3616c) with binding sites for EspR, MprA and CRP . Filtering based on an adjusted P-value of <0.05 identified 453 genes with >2-fold increased abundance in the knockout and 412 with >2-fold decrease. CBS genes comprised only a minor fraction of the differential expression profile, with statistically significant upregulation of 37 genes and downregulation of 15 genes. Forty eight per cent of the CBS-regulated genes corresponded to genes annotated as key metabolic enzymes or genes with predicted roles on transcription regulation that could amplify the CRPMt regulatory signal (Supplementary Table S5). Fifty percent of the differential expression profile (212 of the upregulated genes and 211 of the downregulated genes) was shared with the response to nutrient starvation (Supplementary Table S4), and is likely to reflect secondary effects associated with the marked growth defect of the crp mutant.Previous microarray and targeted qRT-PCR analyses have demonstrated differential expression of CBS genes following deletion of the crp gene . Using aMt was acting together with other transcription factors, differential expression of CBS genes may be enhanced under alternative growth conditions. ChIP-seq analysis revealed a general decrease in CRPMt binding to DNA after incubation for 24 h in PBS , but that this paradigm operates at only a subset of CRPMt-binding sites. Two models can be proposed to reconcile the limited direct effect of crp deletion on expression of CBS genes with the extensive impact of crp deletion on the total transcriptome and growth phenotype. It can be envisaged that the primary transcription changes are amplified through their effect on secondary networks and co-regulation with additional transcription factors. This model is illustrated by recent mapping of the multiple regulatory layers that contribute to the overall function of the FNR transcription factor in E. coli  that can readily switch to a conformation that promotes DNA interaction, even when cAMP is not bound. cAMP binding to CRP may increase the fraction of protein in the DNA-binding state, via conformational selection, which is reflected in ‘enhanced’ DNA binding. By lowering cAMP levels during starvation, the fraction of CRP in the DNA-binding conformation is lowered, thereby reducing occupancy at CRP-binding sites. This significant DNA binding by CRPMt in the absence of cAMP suggests an evolutionary adaptation given the high levels of cAMP seen in mycobacteria. Therefore CRPMt could have evolved to act as a DNA-coating protein or a recruiting protein for other transcription factors or RNA polymerase. CRPMt levels are quite high in the cell, so any decrease in cAMP levels could also be offset by the high levels of CRPMt present at any given time. However, cAMP binding to CRP resulting in the sustained occupancy of a CRP site may influence the interaction of CRPMt with other transcriptional regulators and chromosome organizing proteins.There is a need for further analysis of the effects of cAMP on CRPgulation , cAMP bigulation  and has gulation . However of cAMP , biochem of cAMP . Therefo of cAMP , it is lChIPseq and RNAseq data have been submitted to the EBI ArrayExpress under accession number E-MTAB-2390.Supplementary Data are available at NAR Online.