Deinococcus radiodurans, one of which tolerates extremely high levels of oxidative stress and DNA-damaging reagents. Transcriptional levels of CRP were increased under hydrogen peroxide (H2O2) treatment during the stationary growth phase, indicating that CRPs function in response to oxidative stress. By constructing all CRP single knockout mutants, we found that the dr0997 mutant showed the lowest tolerance toward H2O2, ultraviolet radiation, ionizing radiation, and mitomycin C, while the phenotypes of the dr2362, dr0834, and dr1646 mutants showed slight or no significant differences from those of the wild-type strain. Taking advantage of the conservation of the CRP-binding site in many bacteria, we found that transcription of 18 genes, including genes encoding chromosome-partitioning protein (dr0998), Lon proteases (dr0349 and dr1974), NADH-quinone oxidoreductase (dr1506), thiosulfate sulfurtransferase (dr2531), the DNA repair protein UvsE (dr1819), PprA (dra0346), and RecN (dr1447), are directly regulated by DR0997. Quantitative real-time polymerase chain reaction (qRT-PCR) analyses showed that certain genes involved in anti-oxidative responses, DNA repair, and various cellular pathways are transcriptionally attenuated in the dr0997 mutant. Interestingly, DR0997 also regulate the transcriptional levels of all CRP genes in this bacterium. These data suggest that DR0997 contributes to the extreme stress resistance of D. radiodurans via its regulatory role in multiple cellular pathways, such as anti-oxidation and DNA repair pathways.The cyclic AMP receptor protein family of transcription factors regulates various metabolic pathways in bacteria, and also play roles in response to environmental changes. Here, we identify four homologs of the CRP family in  Cyclic AMP (cAMP) receptor proteins (CRPs) are global transcriptional regulators that are widely distributed in bacteria, 2. TheyEscherichia coli, CRP directly or indirectly controls the transcription of more than 400 genes, and it has been studied extensively[E. coli CRP have been performed to determine the structure of a CRP-cAMP complex, its target DNA, and the carboxyl-terminal domain of the RNA polymerase (RNAP) α-subunit[Corynebacterium glutamicum GlxR[Mycobacterium tuberculosis RV3676[Haemophilus influenza CRP[E. coli CRP. Biochemical analyses reveal that CRP interacts with the carboxyl-terminal domain of the RNAP α-subunit[In tensively, 13. It tensively. Crystalα-subunit–19. It iα-subunit–22. Bindα-subunit–25, whicα-subunit. Howevericum GlxR, Mycobacis RV3676, and Haeuenza CRP differ sα-subunit. This inDeinococcus radiodurans, which belongs to the phylum Deinococcus-Thermus, is characterized by its extreme resistance to ionizing radiation, ultraviolet (UV) irradiation, desiccation, hydrogen peroxide (H2O2), and other DNA-damaging agents[D. radiodurans is generally considered to be an ideal model organism for studying bacterial resistance mechanisms under various stress conditions. There exists many predicted open reading frames (ORFs) encoding transcriptional factors in the D. radiodurans genome [ng agents, 32. Becs genome , whereasD. radiodurans is predicted to contain four CRP family proteins, including DR0997, DR1646, DR2362, and DR0834. Interestingly, the transcriptional level of dr0997, also referred to as ddrI (DNA damage response gene I), increases 38-fold after ionizing radiation treatment[In this study, we constructed all the single mutants of CRPs and examined their roles in anti-oxidation, DNA repair, and other important cellular pathways. It was demonstrated that one of the CRP homologs, DR0997, acts as an important transcriptional activator that is involved in diverse cellular pathways, including cell growth, oxidative stress response, and DNA damage repair.D. radiodurans wild-type R1 , E. coli DH5α, and E. coli BL21 (DE3)pLysS strains were available in our laboratory. All D. radiodurans strains were grown at 30°C in tryptone-glucose-yeast extract (TGY) broth  with aeration, or on TGY medium solidified with 1.5% w/v agar. When necessary, antibiotics were added to D. radiodurans cultures as follows: 30 μg mL–1 kanamycin, 10 μg mL–1 streptomycin, and 3.4 μg mL–1 chloramphenicol. All E. coli strains were grown at 37°C in Luria–Bertani medium  and supplemented with 30 μg mL–1 chloramphenicol, 50 μg mL–1 kanamycin, or 50 μg mL–1 ampicillin where appropriate. All strains and plasmids used in this study are listed in The dr0997, dr2362, dr1646, and dr0834 null mutant, were constructed using the deletion replacement method as described previously[dr0997 null mutant for example. Briefly, ~500-bp DNA fragments immediately upstream and downstream of dr0997 were amplified from the genome of the wild-type R1 strain. After gel extraction, the two fragments were digested with BamHI and HindIII respectively, and ligated with a kanamycin resistance cassette fragment containing the groEL promoter, which was digested with the same enzymes. The kanamycin resistance cassette was obtained from pRADK, a shuttle plasmid derived from pRADZ3. The ligated product was then transformed into competent D. radiodurans cells in the exponential growth phase using CaCl2 as described previously[dr0997upF and dr0997downR . All strains were cultured in 5 ml of liquid TGY medium until the OD600 reached ~2.0, and then they were diluted 1:500 into new flasks containing 100 ml of TGY medium. The cultures were incubated at 30°C with shaking at 220 rpm, and samples were taken every 2 h to measure the OD600.Growth curves of the CRP null mutant strains were determined by measuring the optical density at 600 nm  were used to test the mutants’ stress resistances. The survival rates of the CRP mutants after exposure to H2O2 were determined with a previously described method [4. H2O2  was added to the cell suspension to a final concentration of 50 mM, and the cells were incubated at room temperature. Samples were obtained at 0, 5, 10, 20, and 40 min, and catalase (30 mg/ml) (Sigma-Aldrich) was added to inactive the H2O2. The survival fraction (%) was calculated using the following equation: survival fraction (%) = Nsample/Ncontrol × 100%, where Ncontrol is the number of wild-type bacteria and Nsample is the number of mutant bacteria. For the UV sensitivity test, cultures with an OD600 of ~1.0 were diluted with 10 mM MgSO4. After plating onto TGY agar plates, the plates were exposed to different dosages of UV radiation by calculating the exposure time as described previously[Hd method . Brieflyreviously. For the600 reached ~1.0. Half of the culture was treated with 30 mM H2O2 for 30 min, harvested, washed twice with phosphate buffer , and suspended in 2 ml of phosphate buffer.Cells were cultured at 30°C in 100 ml of TGY medium for at least 12 h until the OD2O2 remaining in the mixture is determined by the oxidative production of N-(4-antipyryl)-3-chloro-5-sulfonate-p-benzoquinonemonoimine in the presence of H2O2 when catalyzed by horseradish peroxidase in the chromogenic reagent mixture. After a 15-min incubation, the resultant product was quantified at 520 nm.Catalase activity was determined as described previously. The col.+ was measured at 734 nm. The total antioxidant capacity was presented by comparing it with the antioxidant activity of Trolox (Beyotime).The total antioxidant capacity was assayed as reported previously. Brieflydr0997 mutant strain were cultured until the OD600 reached 0.4, and then harvested. Total RNA was extracted using the Whole RNA Extraction kit . RNA quality and quantity were evaluated by measuring the A260/A280 ratio with a NanoDrop-1000 spectrophotometer  and Denaturing Agarose Gel Electrophoresis.Total RNA extraction and data processing were conducted as described previously. Briefly600 reached 0.5. Each culture was divided in two halves: one half of the culture was treated with H2O2 at a final concentration of 50 mM, while the other half was used as the non-treated control. Total RNA was extracted as mentioned previously. First-strand cDNA synthesis was conducted in 20-μl reactions containing 1 μg of purified RNA and 3 mg of random hexamers. The SYBR Green PCR kit  was used for PCR amplification according the manufacturer’s instructions, and all assays were performed using the Mx3005PTM Real-time Detection System .The qRT-PCR assay utilized RNA samples that obtained under different conditions. The wild-type and mutant strains were grown in TGY until the ODdr0997 fragment was obtained by digesting pRAD-dr0997 with NdeI and BamHI, and it was ligated with pET28a-HMT. pET28a-HMT is a pET-28a derivative that encodes maltose binding protein (MBP) containing specific tobacco etch virus (TEV) protease recognition sites. E. coli BL21 (DE3) pLysS cells carrying plasmid pET28a-HMTdr0997 were grown to an OD600 of 0.6 at 37°C and induced with 200 μM isopropyl β-D-1-thiogalactopyranoside at 37°C for at least 12 h. Cells were harvested by centrifugation, washed twice with phosphate buffer , and resuspended in binding buffer . Cells were disrupted on ice with an ultrasonic cell disruptor at an output of 350 W for 20 min. After centrifugation at 15,000 × g for 20 min at 4°C, the supernatant was loaded onto a HisTrap HP column . The purified DR0997-MBP fusion protein was digested with a proper concentration of TEV protease at 4°C overnight, and then loaded onto a maltose-affinity column. The eluted protein was applied to a HiTrap Q HP column to remove undigested protein and imidazole. The purity of the protein sample was determined using 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and only the fraction containing pure DR0997 protein was used for further experiments.Proteins were induced and purified as described previously, 43. Bri2, 5 mM MnCl2, 10 μg mL–1 bovine serum albumin, 200 μM cAMP, and 100 μM dithiothreitol[E. coli CRP-binding site was used to identify predicted CRP-regulated genes in D. radiodurans. The promoter of dr1998 was used as a positive control of DR0615. The dr0167 promoter and coding region of dr0167 (dr0167C) were used as negative controls. All primers are listed in EMSA was performed with DNA fragments (100 ng) mixed with 2 μM purified DR0997 or DR0615 protein in a total volume of 10 μl. The binding buffer contained 10 mM Tris-HCl (pH7.5), 10 mM KCl, 200 mM NaCl, 5 mM MgClothreitol. The readr0997 ORF is predicted to encode a 260-amino acid protein . However, based on our sequencing results, a base was missing at the 543-bp site, resulting in the shortening of the dr0997 ORF  analysis. The conserved helix-turn-helix and the nucleotide monophosphate-binding regions are underlined in E. coli CRP [D. radiodurans CRP that could sense oxygen or redox variations [The 0997 ORF . The corcoli CRP –49. Unliriations –52.D. radiodurans in response to H2O2 treatment were detected compared to the untreated sample, and statistical analyses were applied for data processing  .To identify the role of ocessing . The resr 20 min . The H2ORAD0997) , and thee stress  Table, wdr0997 resulted in a dramatic increase in sensitivity to UV, gamma radiation, and MMC, which was restored by complementation with the dr0997 gene  in catalase activity. However, catalase activity was barely induced in the dr0997 mutant, whereas the catalase activity in the dr0997-complemented strain was nearly equal to that of the wild-type strain, indicating that cells lacking the dr0997 gene lose the ability to cope with oxidative stress. The data were subjected to statistical analysis, and the differences were statistically significant , katA (dra0146), sodC (dr1546 and dra0202), sodA (dr1279), terB (dr2220), and NADH dehydrogenase (dr1506) were measured. As shown in 2O2 treatment, the transcriptional levels of katE (dr1998 and dra0259), katA (dra0146), and sodC (dra0202) were 2-fold lower in the mutant than in the wild-type strain. After treatment with 50 mM H2O2, the transcriptional levels in the mutant were still lower than those of the wild-type strain, which is in accordance with the aforementioned catalase activity.As a predicted global transcription regulator, DR0997 may control a series of genes that is directly or indirectly involved in oxidative resistance. Using reverse transcription-PCR method, the transcriptional levels of a series of anti-oxidative enzymes, including dr1506 and dr2220 genes were directly regulated by DR0997. DR1506 is an NADH-quinone oxidoreductase that is involved in redox maintenance. The DR2220 (TerB) knockout strain exhibited reduced H2O2 resistance compared with the wild-type strain , pprA (dra0346), and recN (dr1477) were shown to be directly regulated by DR0997  and glgC (dr1689) are involved in glycometabolism. Dr0349 and dr1974 encode proteins that belong to the Lon protease family, which can degrade and recycle damaged proteins, suggesting that DR0997 participates in protein degradation. CpdB (dr1736) encodes a cAMP phosphodiesterase that catalyzes the conversion of cAMP to AMP, while dra0006 encodes a CyaA-like cyclase, indicating that DR0997 might regulate cAMP metabolism. Dr0998 encodes an ATP-binding protein that is involved in chromosome partitioning, which implies that it functions in chromosome partitioning during cell division. Meanwhile, the dr2531 gene encodes a thiosulfate sulfurtransferase that was also regulated by DR0997. Because the oxidation of thiols would result in non-native disulfide bond formation in proteins[Of the identified genes,  proteins and causD. radiodurans, indicating that DR0997 can regulate its own expression to cope with changes in the environment.Furthermore, it is interesting that DR0997 could regulate not only its own expression, but also that of the other CRP homologs in D. radiodurans.The regulation of gene expression is critical for a cell’s response and survival in various environments. As global transcriptional factors, CRPs have been investigated in diverse bacteria, and they are considered to be important players in the responses to various environmental changes. In this study, an analysis of survival of CRP knockout strains, as well as the transcriptional responses of CRP-regulated genes, revealed that one of the CRPs (DR0997) plays vital roles in E. col several decades ago[E. coli CRP, D. radiodurans CRPs contain a cyclic nucleotide-binding domain in their amino-terminus, as well as a helix-turn-helix domain that bindings DNA and recruits RNAP to promoters to activate transcription. The crystal structure of an E. coli CRP-DNA complex showed that the CRP dimer binds to the TGTGA and TCACA blocks of the 22-bp consensus CRP binding site. The consensus CRP-binding sites in other CRP homologs, such as C. glutamicum GlxR, M. tuberculosis Rv3676, and Thermus thermophilus HB8 TTHA1437, are highly similar. Using WebLogo, we obtained the sequence logo of DR0997-binding sites (TGTGA-N6-TCACA) based on the aforementioned EMSA data (E. coli CRP (AAATGTGA-N6-TCACATTT) and other CRP family proteins[E. coli CRP, cAMP acts as an essential signaling molecule, and it helps to form the CRP-DNA complex. FNR uses the redox states of bound metals as signals to regulate the expression of other genes[M. tuberculosis genome encodes 15 adenylate cyclases, showing the importance of cAMP[D. radiodurans does not contain a classical adenylate cyclase that catalyzes the synthesis of cAMP. Nevertheless, an alternation of the cAMP concentration after radiation has been reported[dra0006, which contains domains that are similar to those of adenylate cyclases, and dr1736 (cpdB), which catalyzes the conversion of cAMP to AMP, implying a connection between DR0997-mediated gene regulation and cAMP metabolism.The first CRP regulation model was in cades ago. Just liMSA data , which i proteins, 69. Theher genes The M. te of cAMP. However reported, indicatE. coli CRP has been demonstrated to play its anti-oxidation role by regulating the expression of rpoS, which activates the general stress response[T. thermophilus HB8 CRP/FNR family protein SdrP is regarded as an oxidative stress-responsive transcriptional activator[sodA and a thioredoxin reductase-encoding gene, which are known to participate in redox control and oxidative resistance. However, there are no rpoS homologs in D. radiodurans, and the CRP family protein DR0997 does not bind the promoters of sodA (dr1279) and katE (data not shown). Nevertheless, it was demonstrated that DR0997 binds the promoters of dr1506, which encodes NADH-quinone oxidoreductase subunit A, and terB (dr2220). DR1506 is involved in redox control, while DR2220 was reported to respond to oxidative stress[ response. Using a response. The T. activator, and it ve stress. These drecN, pprA, and uvsE. Many DNA damage response and repair genes were indirectly regulated by DR0997. It will be interesting to further clarify the function of DR0997 in DNA damage responses and DNA repair.For decades, CRPs have been regarded to be involved in metabolism and anti-oxidation pathways. However, our investigations revealed that DR0997 also directly regulates the expression of DNA repair-related genes, including Beyond the impact of DR0997 on cells via its regulatory role in oxidation and DNA repair responses, its ability to regulate other cellular pathways could also affect cell survival. The regulation of expression of Lon protease and thiosulfate sulfurtransferase would directly affect cell viability in response to damage, while regulating glycometabolism would affect energy production. The regulation of the cAMP pathway also affects many cell activities. Further studies of DR0997 will contribute to a detailed understanding of its functions on the regulatory network during stress responses.S1 Fig(TIF)Click here for additional data file.S2 Fig(A) All of the mutants were identified using their upF and downR Click here for additional data file.S3 Figdr2362 mutant were exposed to 50 mM H2O2 for different periods of time. Data represent the averages and standard deviations of three independent experiments.Wild-type and (TIF)Click here for additional data file.S4 FigLane 1, 100 ng of the indicated DNA fragment; lanes 2 to 6, 100 ng of the indicated DNA fragment with increasing concentrations of DR0997 .(TIF)Click here for additional data file.S5 Fig(TIF)Click here for additional data file.S6 FigLane 1, protein ladder; lane 2, DR0997 protein.(TIF)Click here for additional data file.S7 FigLane 1, protein ladder; lane 2, DR0615 protein.(TIF)Click here for additional data file.S8 FigLane 1, 100 ng of the indicated DNA fragment; lanes 2 to 6, 100 ng of the indicated DNA fragment with increasing concentrations of DR0997 .(TIF)Click here for additional data file.S1 File(DOC)Click here for additional data file.S2 File(DOC)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.


The field of long noncoding RNA (lncRNA) research has been rapidly advancing in recent years. Technological advancements and deep-sequencing of the transcriptome have facilitated the identification of numerous new lncRNAs, many with unusual properties, however, the function of most of these molecules is still largely unknown. Some evidence suggests that several of these lncRNAs may regulate their own transcription in cis, and that of nearby genes, by recruiting remodeling factors to local chromatin. Notably, lncRNAs are known to exist at many imprinted gene clusters. Genomic imprinting is a complex and highly regulated process resulting in the monoallelic silencing of certain genes, based on the parent-of-origin of the allele. It is thought that lncRNAs may regulate many imprinted loci, however, the mechanism by which they exert such influence is poorly understood. This review will discuss what is known about the lncRNAs of major imprinted loci, and the roles they play in the regulation of imprinting. Genomic imprinting is an epigenetic phenomenon, whereby differential expression of alleles occurs with respect to the parent-of-origin. It is thought to have evolved in placental mammals as a biological/ecological mechanism for dosage compensation, whereby a conflict of interest exists over maternal investment of resources during gestation, and, thus, dosage mediation (particularly of growth-related genes) ,2,3. Thede novo DNA methyltransferases DNMT3A and DNMT3L, and later propagated by the maintenance DNA methyltransferase DNMT1 . Th. ThIGF2  regions . The precription ,13.de novo DNA methyltransferase DNMT3L . A . A SNURFe humans , but hase humans . This anSNORD115 and SNORD116 clusters) derived from this imprinted locus, but we would like to note some interesting lncRNAs recently identified in this region. Increased attention has been paid to the region containing the SNORD116 cluster, as a deletion encompassing these snoRNAs has been found to be the minimal deletion sufficient to cause PWS [Snord116 host gene (116HG) RNA as a chromatin binding lncRNA cloud that interacts with genes important for metabolic regulation [Snord116 deletion mouse model of PWS, and overlap between significantly altered transcripts and 116HG interacting genes in the cortex indicates that 116HG has a role in the dysregulated metabolic phenotype observed in the mouse [In keeping with the theme of this review, we will not discuss in detail the short RNAs , with the intervening sequence undigested, leaving highly stable 1–3 kb lncRNA molecules with a snoRNA at each end, and no 5’ cap or polyA tail. Additional characterizations of the five sno-lncRNAs arising from the SNORD116 cluster (in the 15q11-13 locus) revealed that they were rich in binding sites for members of the FOX family of splicing factors, and knockdown of these sno-lncRNAs produced specific splicing changes, so they may function as protein sinks to regulate gene expression [A third recent report revealed a novel class of lncRNAs, called sno-lncRNAs, which arise from the unusual presence of two snoRNAs within the same intron . These Rpression . More pepression .in cis regulation that takes place at imprinted loci, such as directing chromatin modifiers to specific locations, as they remain tethered to the site of transcription for a significant amount of time, and provide unmatched temporal and spatial specificity [The first long noncoding RNAs discovered were those involved in genomic imprinting and X-inactivation, but our knowledge of both lncRNA biology and the mechanisms of imprinting are still in its infancy. In theory, lncRNAs are an ideal candidate for the seemingly impossible and highly complex cificity ,64.XIST, SNURF-SNRPN, and DLK1, as well as non-clustered imprinted genes [H19 locus [One of the greatest mysteries of imprinting is how the ICRs retain their methylation status in the face of global demethylation events after fertilization. To date, no universal factor has been identified, but ZFP57 has been shown to be important for maintenance of ICR methylation at ed genes , while M19 locus . In addi19 locus . Another19 locus .nuclear dsRNA pathway may be at work; that is, whether these RNAs are edited by the ADAR family of enzymes [In many cases, it is difficult to determine whether RNA duplexes occur between the characteristic sense-antisense transcription that occurs in imprinted clusters. Although the RNAi pathway is often ruled out immediately, due to its predominantly cytoplasmic realm of function , to our  enzymes .SNURF-SNRPN) [H19) [MEG3) [As mentioned above, some of these imprinted clusters not only house small RNAs, but their lncRNAs are also the precursors for those small RNAs, including snoRNAs (F-SNRPN) , miRNAs N) [H19) ,58, or b) [MEG3) , which atrans-acting enzymes. Imprinted genes are often found in large clusters, and their expression is regulated by the transcriptional activity of an lncRNA in cis. Some imprinted genes also bear tissue-specific and/or temporally-regulated imprinted gene expression, particularly in extraembryonic  and neuronal  tissues. Despite this breadth of knowledge on imprinting, there are still many aspects that are poorly understood.In summary, genomic imprinting involves allele-specific epigenetic regulation of gene expression, based on parent-of-origin. Imprints are established during gametogenesis in a sex-specific manner, and are characterized primarily by DNA methylation on special DMRs called ICRs. After fertilization, these marks resist the genome-wide waves of methylation erasure, and are later complemented and strengthened by further epigenetic changes via chromatin remodeling factors and other


AhR has become an interesting and actual area of research mainly boosted by a significant number of recent studies analyzing its contribution to the proper functioning of the immune, hepatic, cardiovascular, vascular and reproductive systems. At the cellular level, AhR establishes functional interactions with signaling pathways governing cell proliferation and cell cycle, cell morphology, cell adhesion and cell migration. Two exciting new aspects in AhR biology deal with its implication in the control of cell differentiation and its more than likely involvement in cell pluripotency and stemness. In fact, it is possible that AhR could help modulate the balance between differentiation and pluripotency in normal and transformed tumor cells. At the molecular level, AhR regulates an increasingly large array of physiologically relevant genes either by traditional transcription-dependent mechanisms or by unforeseen processes involving genomic insulators, chromatin dynamics and the transcription of mobile genetic elements. AhR is also closely related to epigenetics, not only from the point of view of target gene expression but also with respect to its own regulation by promoter methylation. It is reasonable to consider that deregulation of these many functions could have a causative role, or at least contribute to, human disease. Consequently, several laboratories have proposed that AhR could be a valuable tool as diagnostic marker and/or therapeutic target in human pathologies. An additional point of interest is the possibility of regulating AhR activity by endogenous non-toxic low weight molecules agonist or antagonist molecules that could be present or included in the diet. In this review, we will address these molecular and functional features of AhR biology within physiological and pathological contexts.Traditionally considered as a critical intermediate in the toxic and carcinogenic response to dioxin (2,3,7,8-tetrachlorodibenzo- AhRR) has been identified as a target gene of AhR, providing a novel mechanism of feedback inhibition of AhR function in that a transcription factor directly induces the expression of its repressor through binding to its cognate regulatory sequence located in the promoter of the target gene. This regulatory circuit involves the activation of AhR by xenobiotics in order to induce AhRR expression, which, in turn, will compete with AhR for ARNT heterodimerization. As AhRR-ARNT heterodimers are transcriptionally inactive, AhR-dependent gene expression results abolished by reduced AhR-ARNT recruitment to the XRE sites located in the promoter of responsive genes  has been defined as an environmental-sensor PAS (Period [Per]-Aryl hydrocarbon receptor nuclear translocator [Arnt]-Single minded [Sim]) protein structurally included within the class I of basic helix-loop-helix transcriptional regulators  showed developmental defects in the hepatic, hematopoietic, cardiovascular and immune systems  does not have detoxifying functions but it is instead required for eye, leg and wing development  and human Alu subfamilies account for close to 13% of their respective genomes and, more importantly, they are highly abundant in intronic and upstream promoter regions of target genes  induced the expression of LINE-1 (long interspersed nuclear element-1) retrotransposons in human cell lines from cervical carcinoma (HeLa) and microvascular endothelium (HMEC), and in mouse cells from smooth muscle (mVSMC) and embryonic kidney (mK4). Interestingly, when the prototypical AhR ligand TCDD  was used, LINE-1 induction was only observed in HeLa cells, suggesting that LINE-1 retrotransposons may be modulated by cell type-specific mechanisms of AhR activation  and 2-amino-3,8-dimethyl-imidazo quinoxaline (MeIQx) were shown to induce LINE-1 mobilization by a mechanism that requires AhR, MAPK (mitogen-activated protein kinase) and C/EBPβ (CCAAT enhancer binding protein-β), suggesting a link between carcinogens, LINE-1 elements and AhR  induced LINE-1 retrotransposition in human hepatocellular carcinoma HuH-7 cells by a mechanism requiring ARNT and MAPK but not AhR , widely represented in the mouse genome, and that was particularly enriched in upstream promoter regions  and for the E-box binding proteins Snail and Slug/Snai2. Importantly, the AhR/ARNT heterodimer and Slug/Snai2 were co-recruited under basal and ligand-induced conditions to B1-X35S elements located in the promoter of developmental and differentiation-related genes Lpp (cell-adhesion related lipoma preferred partner), Tbc1d1 (Obesity risk gene), and Dad1 (Defender against cell death 1)  required the transcription of B1-X35S by a switch mechanism involving RNA polymerase III and RNA polymerase II. That is, the repression of target genes does not take place by a direct effect of AhR on the transcription of their mRNAs (by RNA polymerase II) but rather by a mechanism triggered by the non-coding RNA transcripts produced from the B1-X35S retrotransposon , that is responsible for its insulator activity, was suggestive of the existence of a local change in chromatin structure flanking the retrotransposon  allowed the identification of NANOG-Alu and OCT4-Alu derived transcripts. The analysis of the microRNA machinery of NTERA-2 cells using RISC (RNA-induced silencing complex) inhibitors, dominant-negative forms of Microprocessor components DROSHA and DGCR8 and AGO-2 driven RNA immunoprecipitation (RIP), led us to propose a model in which NANOG-Alu derived transcripts were processed through the miRNA pathway to generate small non-coding RNAs complementary to the 3′UTR region (unstranslated region) of NANOG and OCT4. Such complementarity would eventually reduce the mRNA levels of both genes in cis and trans, respectively. Consistently, NTERA-2 cells engineered to stably express a small-hairpin RNA for AHR (sh-AHR) had a basal undifferentiated status, did not respond to RA-induced differentiation and had a marked reduction in non-coding RNA transcripts from NANOG and OCT4 Alus  produced a marked increase in AHR protein levels that was followed by its nuclear translocation and transcriptional activation  REH and chronic myeloid leukemia K562 cell lines had high levels of AhR promoter methylation and did not express detectable levels of receptor under basal conditions , but also by the histone deacetylase inhibitor Trichostatin A, indicating that differences in histone acetylation may also alter AhR expression  were analyzed in different disorders such as human vascular senescence and atopic dermatitis. AhR regulates cellular senescence of human endothelial cells mediated by oxidative stress. Specifically, AhR activation by indoxyl sulfate (IS) in human umbilical vein endothelial cells (HUVECs) accelerates senescence by suppressing the NAD+-dependent SIRT1 with a concomitant decrease in NAD+ levels and in intracellular nicotinamide phospho-ribosyl-transferase (iNampt) activity   in estrogen receptor (ERα)-negative breast cancer cells. miR-335 over-expression inhibited invasion and metastasis of MDA-MB-231 LM2 cells in vivo  as epigenetic and post-transcriptional regulators add another level of control to AhR functions in normal physiology and pathogenesis. miRNAs are small, 19-25 nucleotides long non-protein-coding RNAs that, once matured, bind to 3′-untranslated regions (3′UTR) of target messenger RNA molecules (mRNAs). miRNA binding to its cognate mRNA results in posttranscriptional suppression of gene expression through cleavage of the target mRNA molecule. miRNAs are involved in normal eukaryotic cellular functions like proliferation, differentiation, apoptosis and development and, as a result, their deregulation is associated with various diseases   for long-term self-renewal . Consequently, alterations in AhR-dependent signaling may cause different developmental and homeostatic disorders in the organism. In this review, we will focus in alterations affecting the cardiac, hematopoietic, epithelial and hepatic systems.OCT4 and ALDH1 levels and exacerbated RA-induced granulocytic differentiation of HL-60 cells as determined by the upregulation of CD38 and Cd11b markers , angiotensin-2, β-myosin heavy chain and atrial natriuretic factor  induces the expression of AhR in rat liver pre-neoplastic foci and in human hepatocarcinoma HepG2 cells , in which a ligand exhibits tissue-specific AhR agonistic or antagonistic activity , and dogs. Potent effects of the candidate AhR ligand bilirubin  mediate an agonist/antagonist synergistic effect of AhR activity in different cell types , the Junta de Extremadura (GR10008 and GR150088) and the Red Temática de Investigación Cooperativa en Cáncer (RTICC), Carlos III Institute, Ministerio de Economía y Competitividad (RD12/0036/0032). All the Spanish funding is co-sponsored by the European Union FEDER program.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. The Associate Editor JL declares that, despite having collaborated with the author PF, the review process was handled objectively and no conflict of interest exists.


Bertero et al. used human embryonic stem cells (hESCs) to show that the Activin–SMAD2/3 signaling pathway cooperates with the core pluripotency factor NANOG to recruit the DPY30-COMPASS histone modifiers onto key developmental genes. Functional studies demonstrate the importance of these interactions for correct histone 3 Lys4 trimethylation and also for self-renewal and differentiation. In mice, Dpy30 is also necessary to maintain pluripotency in the pregastrulation embryo. Stem cells can self-renew and differentiate into multiple cell types. These characteristics are maintained by the combination of specific signaling pathways and transcription factors that cooperate to establish a unique epigenetic state. Despite the broad interest of these mechanisms, the precise molecular controls by which extracellular signals organize epigenetic marks to confer multipotency remain to be uncovered. Here, we use human embryonic stem cells (hESCs) to show that the Activin–SMAD2/3 signaling pathway cooperates with the core pluripotency factor NANOG to recruit the DPY30-COMPASS histone modifiers onto key developmental genes. Functional studies demonstrate the importance of these interactions for correct histone 3 Lys4 trimethylation and also self-renewal and differentiation. Finally, genetic studies in mice show that Dpy30 is also necessary to maintain pluripotency in the pregastrulation embryo, thereby confirming the existence of similar regulations in vivo during early embryonic development. Our results reveal the mechanisms by which extracellular factors coordinate chromatin status and cell fate decisions in hESCs. Stem cells are defined by their ability to propagate indefinitely while retaining the capacity to differentiate into multiple cell types. Such remarkable characteristics are dictated by specific cellular signaling pathways, transcriptional networks, and epigenetic regulators. These elements ultimately direct stem cell fate decisions and thus are essential to generate cells required for not only embryonic development but also adult tissue homeostasis and repair . Human pThe signaling pathways controlling hPSC cell fate are relatively well known, and the TGFβ superfamily has a central function in these mechanisms ,c. In paIndeed, hPSCs differ from somatic cells due to a specific epigenetic state characterized by a highly dynamic and accessible chromatin structure, which is lost upon induction of differentiation. This epigenetic landscape includes numerous genes that are simultaneously marked by trimethylation of histone 3 Lys4 (H3K4me3) and H3K27me3, two histone marks that are usually found on active and inactive genes, respectively, and thus are associated with opposite transcriptional activities . ConsequDespite the wealth of studies describing the dynamic changes of chromatin histone marks in pluripotent cells and their derivatives, there are several important unanswered questions. First, whether and how extracellular signaling cues control the H3K4me3 and H3K27me3 epigenetic landscape remain to be uncovered. Second, H3K4me3 and H3K27me3 marks could have a direct functional importance in the transcriptional regulation of genes directing cell fate decisions or could be just a consequence of such regulations . FinallyConsidering the central role of Activin/Nodal signaling in pluripotency, we decided to explore its possible function in the regulation of H3K4me3 and H3K27me3 in hESCs. For that, we performed detailed gene expression profile experiments combined with H3K4me3 and H3K27me3 ChIP-seq (chromatin immunoprecipitation [ChIP] followed by deep sequencing) on hESCs grown in the absence of Activin/Nodal signaling. These experiments showed that inhibition of Activin/Nodal results in a rapid loss of H3K4me3 (but not H3K27me3) on a specific subset of genomic regions associated to master regulators of pluripotency and mesendoderm specification that are subsequently silenced, thus leading to neuroectoderm differentiation. Activin/Nodal signaling achieves this regulation through its effector, SMAD2/3, which cooperates with NANOG to recruit DPY30, a subunit of the COMPASS methyltransferase complexes, on key developmental regulators. Importantly, the relevance of these mechanisms was demonstrated by loss-of-function experiments showing that DPY30 is essential for pluripotency and mesendoderm specification of hESCs. Finally, we demonstrated the importance of our findings in the context of embryonic development by showing that the absence of Dpy30 in the mouse embryo impairs gastrulation of the pluripotent epiblast, thus blocking mesendoderm specification while promoting neuroectoderm differentiation. Taken together, our results identify the fundamental dynamics by which signaling pathways orchestrate transcriptional responses via epigenetic changes to establish the capacity of stem cells to differentiate into specific lineages.P ≤ 1 × 10−3), while only 14 were up-regulated , an antagonist of the ALK4 and ALK7 type I Activin/Nodal receptors A, on theegulated C. Thus, NANOG, POU5F1/OCT4, LEFTY1, and NODAL , demonstrating that the observed reduction of H3K4me3 levels was not due to nucleosome repositioning. Overall, these results suggest that inhibition of Activin/Nodal results in specific H3K4me3 demethylation.Importantly, inhibition of Activin/Nodal signaling for 2 h specifically impaired H3K4me3 but not H3K4me2 or H3K4me1 levels on both promoter and enhancer regions . Indeed, H3K4me2 and H3K4me1 levels were either unchanged or increased on most of the regions that we analyzed, with the only exception being P < 1 × 10−4 as measured by genomic association test [GAT]). Among others, canonical SMAD2/3 target genes such as NANOG, POU5F1/OCT4, NODAL, and LEFTY1 showed this association . This observation is in agreement with previous reports that showed how SMAD2/3 regulates the expression of its target genes mostly by binding to distal enhancers rather than proximal promoters  identified three main clusters, two of them containing genes whose expression was decreased after inhibition of Activin/Nodal  and POU5F1/OCT4, DPPA4, and EPCAM (cluster 2). Importantly, these two clusters included not only several pluripotency factors but also regulators of mesendoderm differentiation  that are expressed at background levels in hESCs , in agreement with the known “primed” pluripotency status of hESCs  as well as regulators of mesendoderm differentiation . These genes belonged to either cluster 1 or cluster 2 described above and were transcriptionally repressed upon inhibition of Activin/Nodal signaling . These included several genes crucially required for pluripotency  or slowly (cluster 2) transcriptionally down-regulated genes, while up-regulated ones (cluster 3) were not affected . Interestingly, none of the genes analyzed showed any significant change in the levels of H3K27me3 . These observations confirmed that loss of H3K4me3 associated with SMAD2/3 disappearance precedes transcriptional impairment on a subset of genomics targets. This suggests a functional importance of H3K4me3 histone mark regulation downstream from Activin/Nodal signaling to maintain the expression of a core network of genes controlling pluripotency and differentiation potency.Having defined the importance of Activin/Nodal signaling in maintaining H3K4me3 on key regulatory genes, we sought to understand the underlying molecular mechanism. The association between decreased H3K4me3 and SMAD2/3-binding sites suggested that Activin/Nodal signaling effectors could be directly involved in the deposition of H3K4me3. Thus, we decided to test whether SMAD2/3 could interact with the COMPASS complexes, which are responsible for the deposition of this histone modification. The COMPASS H3K4 methyltransferases belong to the MLL/SETD1 family and form six functional complexes classified into three subgroups, all of which include common WRAD  cofactors . Among tIn order to evaluate the functional relevance of such interactions in the context of Activin/Nodal-dependent H3K4me3 marks and gene expression, we decided to knock down the expression of the common COMPASS cofactor DPY30. Indeed, DPY30 is required for efficient COMPASS-dependent H3K4me3 but not H3K4 mono- and dimethylation, in contrast with the other members of the WRAD module, which are also important for H3K4me2 and H3K4me1 deposition . MoreoveP < 1 × 10−500, as calculated using rank–rank hypergeometric overlap [RRHO] analysis). Indeed, all of the genes that we validated by qPCR to be increased or decreased during SB treatment  followed the same trend in DPY30 knockdown cells, including well-known SMAD2/3 targets such as LEFTY1, NODAL, NANOG, POU5F1/OCT4, CDX2, and WNT8A , thereby suggesting that the requirement for high levels of DPY30 in hESCs is gene-specific. Transient transfection for 48 h of a DPY30 shRNA also resulted in a similar gene-specific reduction of H3K4me3 on SMAD2/3 target genes . Interestingly, DPY30 knockdown did not affect H3K4me3 on a diversity of loci, including housekeeping genes such as et genes H, indicaConsidering the effect of DPY30 knockdown in undifferentiated hESCs, we decided to assess the capacity of differentiation of DPY30 knockdown hESCs by performing in vivo teratoma assays . Strikingly, teratomas derived from DPY30 knockdown hESCs were small and failed to completely invade the testicular capsule of immuno-deficient mice, in contrast to those grown from SCR shRNA control hESCs   , they alctively) C, furtheTo further validate this hypothesis, we performed sequential ChIP experiments that demonstrated that DPY30 cobinds with both SMAD2/3 and NANOG onto chromatin regions associated to key regulators of hESC pluripotency and differentiation D. We theWe then investigated whether S/N/D binding correlated with histone methyltransferase recruitment. First, we confirmed that COMPASS complexes are recruited in an Activin/Nodal-dependent manner onto chromatin regions bound by S/N/D, as measured by ChIP for WDR5 before and after inhibition of Activin/Nodal signaling for 2 h G. FurtheP-value, 1.00 × 10−6; FDR, 0.1%; fold change, >1.5 ). Nevertheless, we observed a number of regions specifically affected by either protein, which was particularly large for DPY30 , in agreement with its general role in promoting the activity of COMPASS complexes . While both conditions induced a large number of significant differences, deregulation of H3K4me3 was still specific to selected loci: Out of 31,923 peaks in DPY30 knockdown hESCs, 6482 (20.3%) were decreased, and 1394 (4.4%) were increased, while 4028 (12.3%) and 1510 (4.6%) of the 32,642 peaks in NANOG knockdown hESCs were decreased and increased, respectively . Interestingly, regions with decreased H3K4me3 after DPY30 or NANOG knockdown were associated with genes involved in the development of tissues from all germ layers , suggesting a role for DPY30 and NANOG in the regulation of developmental processes. Importantly, we observed a large overlap in the H3K4me3 peaks impaired after DPY30 or NANOG knockdown, implying that these two factors control a large set of common genes . These regions were associated to genes that include the key pluripotency markers NANOG, POU5F1/OCT4, DPPA4, and PRDM14 and the mesendoderm regulators LEFTY1, WNT3, CER1, FGF8 , and NODAL  of H3K4me3 peaks decreasing after 2 h of SB significantly overlapped with those decreasing after both NANOG and DPY30 knockdown  could be controlled independently of SMAD2/3. However, the expression of several of these genes was significantly impaired after 48 h of inhibition of Activin/Nodal signaling with SB D, arguinIn addition to H3K4me3, we also performed ChIP-seq for H3K27me3 after DPY30 knockdown or NANOG knockdown . In sharp contrast to the results from treatment with SB for 2 h, we identified many regions of differential H3K27me3 . Moreover, the regions in which H3K27me3 changed after DPY30 or NANOG knockdown did not show any significant difference in the H3K27me3 levels after 2 h of SB . These data implied that this repressive histone mark required more time to change when compared with H3K4me3. This suggests that Activin/Nodal signaling might not be directly involved in the regulation of H3K27me3 in hESCs, while NANOG and DPY30 could have a more important role in this process. In summary, these results confirmed and validated a model in which SMAD2/3, NANOG, and DPY30 collaborate to maintain H3K4me3 on a subset of genes, which can be divided into two classes defined by the speed at which they lose H3K4me3, thereby suggesting an epigenetic hierarchy between different regulators of cell fate decisions.We next decided to investigate whether the functional interactions described above could imply collaborative mechanisms between SMAD2/3, NANOG, and DPY30 at a genome-wide level. For that, we performed ChIP-seq for DPY30 on control (SCR shRNA) hESCs and DPY30 knockdown hESCs. These analyses revealed that DPY30 could be found on 26,387 genomic regions in hESCs, while only 6893 peaks were identified in DPY30 knockdown hESCs . Moreover, DPY30 ChIP-seq coverage was strongly impaired in DPY30 knockdown hESCs compared with control hESCs . Finally, DPY30 binding correlated with H3K4me3 deposition, as expected and previously shown for mESCs . Moreover, Dpy30 knockout embryos dissected at E6.5 showed prematurely reduced expression of epiblast markers (Nanog and Pou5f1/Oct4) and failed to properly induce mesendoderm genes , while neuroectoderm markers were either unaffected (Sox2 and Sox1) or even increased (Dlx5 and Hesx1) Wtsi>] . Mice carrying heterozygous mutations were healthy, and we did not observe any obvious phenotype. However, viable homozygous mutant mice could never be recovered, and further analysis revealed that the absence of Dpy30 was embryonic-lethal between embryonic day 7.5 (E7.5) and E9.5 A. In addd Hesx1) C. Therefd Hesx1) D.Here we uncovered the mechanisms by which extracellular signals are converted into epigenetic and transcriptional regulations necessary for hESC pluripotency and cell fate decisions. The effectors of the Activin/Nodal signaling pathway SMAD2/3 and NANOG recruit DPY30-containing COMPASS complexes onto specific genomic regions where they maintain H3K4me3. This event is essential to maintain the expression of a core set of factors necessary for pluripotency. Accordingly, inhibition of Activin/Nodal signaling results in a rapid decrease in both H3K4me3 and expression of several master regulators of hESC pluripotency, including NANOG itself. NANOG is involved in a feed-forward regulatory network that maintains its own expression and that of other core pluripotency factors, such as OCT4, all of which are part of a broader SMAD2/3-dependent transcriptional network characterizing hPSCs . As suchOur data show that MLL2/KMT2B could be the COMPASS complex regulating a broad part of the transcriptional network downstream from Activin/Nodal signaling. Interestingly, MLL2/KMT2B was reported to regulate H3K4me3 on bivalent promoters in mESCs . NonetheInterestingly, the prompt and selective decrease of H3K4me3 on pluripotency genes upon signaling inhibition suggests the existence of an active process to erase positive histone marks on these specific genomic regions. Likely candidates for such a role are the H3K4-specific histone demethylases of the JARID1 family. Indeed, the profile of histone methylation after inhibition of Activin/Nodal signaling is consistent with the sequential H3K4me3 and H3K4me2 demethylation that is known to be mediated by this class of demethylases . InteresOur results also show that Activin/Nodal signaling controls H3K4me3 presence on “bivalent genes” known to be master regulators of mesendoderm specification. Inhibition of Activin/Nodal, NANOG knockdown, and DPY30 knockdown are all associated with a decrease of H3K4me3 on these loci, which correlates with an impaired capacity of hESCs to differentiate toward mesendoderm. These results suggest that positive marks are maintained by active mechanisms on key bivalent genes and that the deposition of these marks could be indispensable for proper induction of differentiation. This supports the hypothesis that bivalent marks are necessary to poise developmental genes for efficient and synchronous cell fate decisions in response to extracellular signaling .Importantly, our study suggests that the presence of H3K4me3 on Activin/Nodal target genes not only is a consequence of the level of transcription but might also be a causal event that directly influences gene expression. Indeed, knockdown of the epigenetic remodeler DPY30 is sufficient to mimic the transcriptional effect of Activin/Nodal inhibition. Several epigenetic readers are known to link H3K4me3 with various factors that can promote gene expression, including TFIID, SAGA, NuRF/BPTF, and Sin3B/HDAC complexes . Thus, iThe role of H3K4me3 in pluripotency and differentiation has been a matter of open controversy . StudiesThe interconnection between Activin/Nodal signaling and SMAD2/3-dependent deposition of H3K4me3 on master regulators of cell fate could be relevant for many cell types. Indeed, TGFβ is a widespread pathway , and H3KFeeder-free H9 hESCs (WiCell) were grown in chemically defined culture medium with 10 ng/mL Activin A and 12 ng/mL FGF2 as described in P < 0.05 (*), P < 0.01 (**), and P < 0.001 (***).Standard methods for qPCR, immunostaining, Western blot, and flow cytometry were previously described , and detImmunoprecipitation and ChIP and were performed as described previously in Microarrays were performed on biological triplicates using Illumina HT12-v4.0 bead chips, and the raw data were processed using Genome Studio (Illumina) and normalized using the R/Bioconductor package lumi. The raw and processed microarray data are available on ArrayExpress (E-MTAB-2749). Microarray bioinformatic analyses are described in the Supplemental Material.ChIP samples were sequenced using Illumina HiSeq 2000, and the raw data are publicly available on ArrayExpress . ChIP-seq bioinformatic analyses are described in the Supplemental Material.Dpy30 knockout mice were generated by the Sanger Institute Mouse Genetics Project. Dysmorphology assessments were performed on embryos dissected from the decidua of pregnant females after removal of extraembryonic tissues, and qPCR analyses were done on RNA extracted from individual embryos.All animal procedures were performed in agreement with the United Kingdom Home Office regulations, United Kingdom Animals (Scientific Procedures) Act of 1996. Detailed descriptions and additional references for all the experimental and bioinformatics procedures presented here are provided in the Supplemental Material.


Arabidopsis, is the regulator of many sulfur deficiency responsive genes. Among the five other proteins of the family, three regulate ethylene (ET) responses and two have unassigned functions. Contrary to the well-defined ET signaling, the pathway leading from sensing sulfate status to the activation of its acquisition via SLIM1 is completely unknown. SLIM1 binds to the 20 nt-long specific UPE-box sequence; however, it also recognizes the shorter TEIL sequence, unique for the whole EIL family. SLIM1 takes part in the upregulation and downregulation of various sulfur metabolism genes, but also it controls the degradation of glucosinolates under sulfur deficient conditions. Besides facilitating the increased flux through the sulfate assimilation pathway, SLIM1 induces microRNA395, specifically targeting ATP sulfurylases and a low-affinity sulfate transporter, SULTR2;1, thus affecting sulfate translocation to the shoot. Here, we briefly review the identification, structural characteristics, and molecular function of SLIM1 from the perspective of the whole EIL protein family.Sulfur limitation 1 (SLIM1), a member of the EIN3-like (EIL) family of transcription factors in  Arabidopsis thaliana, so far the only described transcription factor strictly assigned to this pathway, was found in an elegant genetic approach exploiting the fluorescent sulfur deficiency responsive reporter  together with fungi and bacteria are capable of sulfate assimilation and its reduction, therefore playing a pivotal role in the biogeochemical sulfur cycle. The availability of sulfur in the soil fluctuates, therefore plants constantly have to adapt to the changing environment by reprogramming their metabolism. Modulation of gene expression at the level of transcription is a major control point in multiple biological processes, thus the main interest of many researchers was to identify transcriptional regulators specific for sulfur deficiency signaling. Sulfur limitation 1 (SLIM1) from reporter . It has ETHYLENE-INSENSITIVE-LIKE 3 (EIL3) coding for a putative transcription factor of unknown function  and EIL1–EIL5; slim1 mutants , is required for ET binding . ET binding leads to the functional inactivation of receptors and the disability of CTR1 to phosphorylate a positive component of the pathway – the membrane located EIN2. The non-phosphorylated C-terminal end of EIN2 is cleaved off by an unknown mechanism and is transferred to the nucleus . Upon perception of ET, the C-terminal end of EIN2 stabilizes EIN3/EIL1 and induces degradation of EBF1 and EBF2. Additionally, the levels of mRNAs encoding EBF1 and EBF2 are negatively regulated by the exoribonuclease EIN5 in the presence of ET. The transcription factor EIN3 dimerizes and then activates the expression of target genes, including the transcription factor gene ethylene-response-factor1 (ERF1). ERF1, in turn, starts a transcriptional cascade of 100s of ET-regulated genes. The mechanism of ET signaling in plants is probably universal as all the elements identified in Arabidopsis are conserved in evolutionary distant plant species .Moreover, EIN3 protein seems also to be quantitatively controlled by other signals. It has been found that glucose can promote EIN3 degradation by an unknown mechanism , whereasFigure 1). Through this interaction, proteasomal degradation of FIT is reduced and leads to a higher level of expression of the iron acquisition genes. Another factor directly interacting with EIN3 is the Mediator complex subunit MED25 . MYB72, which is involved in induced systemic resistance, has been shown to interact physically with SLIM1 in the yeast two-hybrid assay , showing differences in the binding preference between EIL family members . The metabolite analysis further suggests that slim1 mutants may be experiencing the lowered supply of sulfate to the reduction pathway as evidenced by the significant decrease of glutathione content and overaccumulation of O-acetylserine in their shoots. Such metabolite profiles are characteristic for sulfur-deficient plants and caused by an insufficient sulfur amount for the cysteine synthesis pathway. Degradation of glucosinolates is another important aspect of sulfur limitation response. Glucosinolates are characteristic compounds for Brassicales participating in the defense against herbivores and pathogens . The cell-type specific expression pattern between miR395 and its target transcripts enables the fine-tuning of the sulfur assimilation rate , which are a class of naturally occurring, small non-coding RNA molecules. They are partially complementary to one or more mRNA molecules, and their main function is to affect the stability of these molecules in a variety of manners, including translational repression, mRNA cleavage, and deadenylation . Functio sulfate . The expion rate . Especiao shoots . Additioo shoots . Recentlo shoots , again p cascade . HoweverInterestingly, EIN3 also participates in the control of miRNA by integrating different developmental and environmental cues and directly binding to the promoters of miR164 activating leaf senescence processes .Figure 1). It is of interest as to whether the same level of complexity can be expected in sulfur deficiency signaling. Still, the exact signaling cascade leading from sensing to activating the expression of the SLIM1-dependent gene set, resulting in sulfur metabolism reprogramming, needs to be clarified and future studies are required to reveal the molecular components, with a special emphasis on the role of O-acetylserine . Such studies should also concentrate on the investigation of post-transcriptional modifications of SLIM1 influencing its functionality under different sulfur regimes, as well as its direct interaction with specific DNA sequences. We must be cautions, however, in drawing general conclusions and remember that SLIM1 is present mostly in the vascular tissues thus its action might be predominantly connected with the translocation of sulfate between plant parts rather than governing the whole plant sulfur metabolism.Due to the sessile life cycle, plants have developed different strategies to adapt to adverse environmental stresses. Plant growth and development is largely impaired by nutrient deficiencies; therefore to maintain good productivity in plant breeding, it is essential to understand fully those mechanisms. In this review, we focused on SLIM1 as the only described transcriptional regulator dedicated to plant response to sulfur deficiency. It belongs to the same protein family of transcription factors as EIN3. Contrary to SLIM1, regulation of EIN3 stability, interaction with other proteins as well as the whole signaling pathway leading to transcriptional response is already well described (Anna Wawrzyńska drafted the manuscript and prepared the figure; Agnieszka Sirko revised it critically for important intellectual content.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.


Bacillus subtilis from the late log phase to the stationary phase without an inducer. Eleven candidate promoters were selected based on B. subtilis microarray data and the quantitative PCR analysis. Among the selected promoters, Pylb exhibited the highest activity with the reporter bgaB during the stationary phase. Compared with P43 (a commonly used constitutive promoter), promoter Pylb could express two reporter genes (egfp and mApple), and the expression levels of EGFP and RFP were 7.8- and 11.3-fold higher than that of P43, respectively. This finding was verified by overexpression of the genes encoding pullulanase and organophosphorus hydrolase, the activities of which were 7.4- and 2.3-fold higher, respectively, when driven by Pylb compared with P43. Therefore, our results suggest that the Pylb promoter could be used to overexpress target genes without an inducer; this method could facilitate the identification and evaluation of attractive promoters in the genome.A promoter that enabled high-level expression of the target gene during the stationary phase in the absence of an inducer would facilitate the efficient production of heterogeneous proteins at a low cost. In this study, a genome-scale microarray-based approach was employed to identify promoters that induced high-level expression of the target genes in  Bacillus subtilis is an attractive host for the production of heterologous secretory proteins. B. subtilis is a nonpathogenic bacterium that can efficiently secrete a target protein into the culture medium. Much information concerning large-scale fermentation and production technology using this bacterium is available12B. subtilis have been determined45As a Gram-positive bacterium, B. subtilis: constitutive promoters, inducer-specific promoters, and auto-inducible promoters1789spac and Pxyl) are the most widely used type, the requirement for inducer compounds—such as IPTG and xylose—could increase the cost of their industrial application11121314151Promoters are important genomic regulatory elements that directly affect gene expression levels. In bacteria, RNA polymerases and associated sigma factors recognize promoters and are recruited by the binding of regulatory proteins to specific sites within promoters. To date, three types of promoter have been used for the high-level expression of heterologous proteins in B. subtilis based on the report by Evert-Jan et al.cdd under the control of the P43 promoter (a commonly used constitutive promoter)319ylb was the most potential promoter. Circularized RNA reverse-transcription PCR indicated that the transcription initiation site of the Pylb promoter was an adenine, and the key elements (−10 box and −35 box) of the promoter were determined by site-directed mutagenesis. These results indicated Pylb to be an attractive and highly active promoter.Here, we described a genome-wide microarray-based approach to identifying auto-inducible promoters in B. subtilis genome-wide microarray data were downloaded from NCBI (Accession No. GSE19831) and analyzed using the SAM software  represented the ‘lag’, log, early stationary, and late stationary growth phases, respectively. As shown in cdd gene which controlled by P43 (indicated by an arrowhead). The P43 promoter was selected as the positive control as it was commonly used strong promoter for B. subtilis212223To identify the target promoter, software . The miccdd gene under the control of the P43 promoter was used as a positive control. The results showed that the selected 21 genes could be divided into three categories according to their expression level  were selected based on the results of SAM and qRT-PCR. Among the eleven promoters, the gene transcriptional levels driven by Ppbp, Prap, Psig, Pylb, Pyqe and PyddF were higher than that of P43, the gene transcriptional levels driven by Pmsm, Pyvl and Pyob were near to that of P43, and the gene transcriptional levels driven by Pydc and PyddA were lower than that of P43. To assess the activity of the eleven candidate promoters, 500–600 base pairs (bp)B. subtilis WB600 and inserted into the region upstream of the reporter gene bgaB. The promoter P43 was used as a positive control. The β-galactosidase activities of these eleven recombinants after culture for 18 h were shown in ylb was highest, 8.2-fold higher than that of P43. However, the β-galactosidase activity driven by PyddF was only 2.4-fold higher than that driven by P43. This may be because PyddF is a log-phase-specific promoter; Indeed, it resulted in the highest β-galactosidase activity in a log phase, 7.2-fold higher than that driven by P43  and red (mApple) fluorescent proteins as reporters. To prevent any influence of plasmid copy number and cell concentration on promoter activity, three fusion plasmids , each of which contained two promoters and two reporter genes, were constructed and transformed into B. subtilis WB600. EGFP and mApple have similar fluorescence intensities; These proteins exhibit relative brightness levels of 100 and 109, respectivelyylb-R-P43-G-pUBC19, the Pylb and P43 promoters drove the mApple and egfp reporter genes, respectively. Green and red fluorescence were measured in the recombinant strain harboring the Pylb-R-P43-G-pUBC19 plasmid. If the activity of the Pylb promoter were greater than that of P43, the red fluorescence intensity would be greater than that of green fluorescence. The recombinant strain containing the plasmid E-R-pUBC19 was used as a negative control; no fluorescence was detected in this strain. As shown in ylb-G-P43-R-pUBC19 and Pylb-R-P43-G-pUBC19 exhibited green and red fluorescence after culture for 18 h. These results indicated that the activity of the Pylb promoter was greater than that of P43.To compare the activity level of the Pylb-G-P43-R-pUBC19 or Pylb-R-P43-G-pUBC19 plasmid was monitored spectrophotometrically every hour. The results of the strain containing the recombinant plasmid Pylb-G-P43-R-pUBC19 indicated that the green fluorescence intensity driven by Pylb was higher than the red fluorescence intensity driven by P43  and those with deletions  exhibited similar β-galactosidase activities. However, the B. subtilis strain containing the F5 fusion plasmid exhibited negligible β-galactosidase activity. These results revealed that the F4 strain contained the main core element, but F5 was deficient in the −35 element. Therefore, the putative recognition element (−35 TTGGAT −30) and a −10 box (−12 TACAAT −7) were presented in the Pylb promoter.In the istronic . The CR- adenine . To expemeasured , and SDSmeasured . The B. ylb was F4 and revealed its putative recognition element (−35 box and −10 box). To experimentally analyze the cis-acting elements of Pylb in detail, site-directed mutagenesis was performed using the EGFP fusion reporter system with the F4-egfp-pUC19 plasmid . Thus the promoter region from bp −48 to −1 of F4 was analyzed using site-directed scanning mutagenesis to pinpoint the regions that mediate the transcriptional activity of Pylb. The resulting constructs were named pM4843 to pM0301; the numbers indicated the mutated region. For example, pM4843 indicated mutagenesis from bp −48 to −43 relative to the transcription start site of Pylb. These plasmids were transformed into B. subtilis WB600 to generate the reporter strains M4843 to M0301, which exhibited green fluorescence  and II (bp −18 to −3) were particularly important for the transcriptional activity of Pylb.The above results demonstrated that the shortest functional sequence of Prescence . Mutatioylb promoter’s potential for application, Pylb-ZDs/P43-ZDs  and two foreign genes, pul/ophc2, were cloned into the shuttle vector pUBC19. The Pul activities of the Pylb-ZDs-Pul-pUBC19 and P43-ZDs-Pul-pUBC19 recombinants after 18 h of culture were shown in ylb promoter induced significantly higher levels of expression of Pul and OPHC2 than did P43. The Pylb-driven Pul activity was 7.4-fold higher than that driven by P43, and the Pylb-driven OPHC2 activity was 2.3-fold higher than that driven by P43. The results of SDS-PAGE analysis of Pul and OPHC2 in the culture supernatant after concentrated about 15 folds were shown in B. subtilis WB600 strains (data not shown). In addition, cultivations were also performed in SB medium, as shown the ylb promoter was stronger than that of P43 when driving Pul and OPHC2. Thus, the Pylb promoter may have potential for the overexpression of proteins in B. subtilis.To evaluate the PB. subtilis is usually mediated by inducer-specific promoters. In comparison, auto-inducible promoters have a distinct advantage in terms of not requiring an inducer compound, which simplifies the industrial production of the target protein and reduces the cost. Thus, identification of potential auto-inducible promoters would be beneficial.One of the key factors for achieving high-level expression of heterologous genes is the use of a strong and controllable promoter. Despite the fact that several strong promoters have been reported71028293031Nowadays, much information about transcriptome data of many species is available on line, which will help to identify the useful promoters6ylb promoter induced sufficiently high levels of expression in B. subtilis of active β-galactosidase, EGFP, RFP, pullulanase, and organophosphorus hydrolase from the late log phase to the stationary phase. In addition, the production of those foreign proteins was higher than that induced by the P43 promoter, which has been used widely in B. subtilis. Moreover, the high expression level of heterologous proteins did not significantly affect the growth of B. subtilis. Thus, the Pylb promoter may have potential for the overexpression of useful proteins in B. subtilis.Our results indicated that the PB. subtilis genome sequence, the Pylb promoter controls the ylbP gene, which has a length of 483 bp and encodes an N-acetyltransferase. The gene could be expressed in the stationary stage, which has also been confirmed by the transcriptome study in the different conditions, as shown in SubwikiylbP is capable of catalyzing the transfer of acetyl groups between acetyl coenzyme A and amines. Thus, it may be involved in post-translational modification in B. subtilis during the stationary phase. However, the function of and the reason that the ylbP gene was highly expressed from the late log phase to the stationary phase remain unclear.According to the This study is an example of the discovery of an interesting promoter using a genome-scale microarray-based approach. Genomic and gene expression profile data contain a large quantity of biological information, which could be exploited in various fields. Compared with the traditional promoter trap methodB. subtilis WB600 and Bacillus amyloliquefaciens were stored in our laboratory. Escherichia coli Trans1-T1 was used as the host strain for the cloning experiment . The plasmids used in this study are listed in B. subtilis microarray data (NCBI Accession No. GSE19831) were reported by Evert-Jan et al.B. subtilis exhibits four distinct growth phases: the lag phase, the log growth phase, and the early and late stationary growth phases. The microarray data were grouped into four growth phases, and the Significant Analysis of Microarray software  was employed to identify genes specifically expressed from the late log growth phase to the stationary phase.The B. subtilis WB600 was cultured at 37 °C overnight in LB medium. Then, a 1:100 volume of a fresh overnight B. subtilis culture was inoculated into 50 mL LB medium in a 100 mL flask and cultured at 37 °C with shaking at 200 rpm. Growth was monitored by measurement of the optical density at 550 nm (OD550)  according to the manufacturer’s instructions. To remove DNA contamination, 1 μg of diluted RNA was digested with DNase I . Specific primers for the target DNA regions ( (OD550) . B. subt regions  and suitB. subtilis WB600 using the respective primer pairs, and the commonly used strong constitutive promoter P43 was amplified as the control promoter. The amplified promoters were digested with Pst I and Xho I and inserted into the plasmid P-free-bgaB-pUBC19, which had been treated with the same restriction endonucleases, and then transformed into E. coli strain Trans1-T1 to construct the corresponding fusion expression plasmid Promoter-bgaB-pUBC19. The fusion plasmid Promoter-bgaB-pUBC19 was constructed by means of the experimental procedure shown in All primers used in this study are listed in egfp (GenBank Accession No. JQ627826.1) and mApple (GenBank Accession No. HM771700.1) genes, and one termination sequence was added between the two genes. Moreover, the direction of the two genes was reversed. Multiple cloning sites were then added to the ends of egfp and mApple . The synthetic egfp-mApple gene was located on the pUC57-Simple plasmid. The fusion egfp-mApple-pUC57 Simple plasmid was digested with Hind III and BamH I and inserted into the plasmid pUBC19, which had been treated with the same restriction endonucleases, and then transformed into E. coli Trans1-T1 to construct the corresponding fusion plasmid (G-R-pUBC19). Next, the promoters P43 and Pylb were inserted upstream of the egfp and mApple, respectively, and then transformed into E. coli Trans1-T1 to construct the corresponding fusion plasmids (Pylb-R-P43-G-pUBC19). Moreover, the promoters P43 and Pylb were inserted prior to mApple and egfp, respectively, and then transformed into E. coli Trans1-T1 to construct the corresponding fusion plasmids  promoter −381– + 43 (F1), −233– + 43 (F2), −154– + 43 (F3), −78– + 43 (F4), and −21– + 43 (F5)—were amplified using the cognate primer pairs F1-up, F2-up, F3-up, F4-up, F5-up and Pylb-down  was amplified from the plasmid Pylb-G-P43-R-pUBC19 using the primers F4-egfp-up and F4-egfp-down. The amplified product F4-egfp was digested with Xba I and Hind III and inserted into the plasmid pUC19, which had been treated with the same restriction endonucleases, to construct the fusion plasmid F4-egfp-pUC19. Based on the plasmid F4-egfp-pUC19, 10 scanning site-directed mutational plasmids of the F4 fragment were constructed using a previously described two-step PCR methodXba I and Hind III and inserted into the plasmid Pylb-G-P43-R-pUBC19, which had been treated with the same restriction endonucleases, to construct mutational plasmids (pM4843 to pM0301).To analyze the regulatory region of the Pophc2) genes were selected to evaluate the Pylb promoter’s potential for application in B. subtilis. The signal peptide sequence (ZDs) of α-amylase from B. amyloliquefaciens was inserted into the region upstream of the target genes. Four recombinant plasmids were constructed; the fusion plasmids Pylb-ZDs-Pul-pUBC19 and P43-ZDs-Pul-pUBC19 contained the F4 and P43 promoters, respectively, together with the pullulanase gene from Bacillus naganoensis. The other two fusion plasmids, Pylb-ZDs-OPHC2-pUBC19 and P43-ZDs-OPHC2-pUBC19, contained the organophosphorus hydrolase gene (ophc2) from Pseudomonas pseudoalcaligenes. The details of fusion plasmid construction are provided in the The pullulanase and organophosphorus hydrolase  was used to identify the transcription start site of the PThe methods for determination of pullulanase, organophosphorus hydrolase, and β-galactosidase activities are described in the B. subtilis WB600 cells containing the eGFP or mApple reporter gene were cultured for ~18 h, washed three times, and resuspended in 0.9% NaCl. A 5 μL volume of cells at an appropriate dilution in 0.9% NaCl was placed on a microscope slide and covered with a coverslip. Fluorescence microscopy was performed using a 100 × oil immersion lens  to visualize cell fluorescence. To measure the fluorescence intensity, the constructed B. subtilis WB600 strains harboring the Pylb-R-P43-G-pUBC19 and Pylb-G-P43-R-pUBC19 plasmids were cultured in black, 96-well plates with a clear, flat bottom  at 37 °C with shaking at 750 rpm in incubator 1000 . The fluorescence intensity was measured at 1-h intervals using a SpectraMax M2 instrument . The excitation and emission wavelengths were 484 and 507 nm and 586 and 592 nm, for enhanced green fluorescent protein (EGFP)How to cite this article: Yu, X. et al. Identification of a highly efficient stationary phase promoter in Bacillus subtilis. Sci. Rep.5, 18405; doi: 10.1038/srep18405 (2015).


Nuclear pore proteins interact dynamically with chromatin to regulate gene activities. A key question is how nucleoporin interactions mechanistically alter a gene's intranuclear position and transcriptional output. We reported recently on a direct interaction between the nuclear pore-associated TREX-2 complex and promoter-bound Mediator. This highlights how nuclear-pore associated adaptors gain regulatory access to the core transcription machinery. In this Extra View, we discuss an additional implication that arises from our work and the recent literature: how promoter elements may regulate mRNA metabolism beyond transcription initiation. S.cerevisiae TREX-2 complex was found to regulate a surprisingly diverse number of chromatin-associated processes including transcriptionNuclear pore complexes (NPCs) are gatekeepers at the nuclear envelope mediating traffic between the nucleus and cytoplasm. Beyond transport, NPCs dynamically interact with chromatin to regulate its architecture and activity. Genome-wide studies in yeast, flies and humans have shown that nuclear pore proteins (nucleoporins) interact with numerous active genes, but also with heterochromatin boundaries and repressed genes.Mediator, on the other hand, is the central scaffold for transcription initiation in eukaryotes. Transcription initiation at protein-coding genes requires RNA Pol II, general transcription factors, and Mediator. Assembly of these factors on promoter DNA results in a core initiation complex, which recruits the TFIIH complex to unwind DNA and to phosphorylate the RNA Polymerase (Pol) II C-terminal domain (CTD) at Ser5. Mediator is recruited by transcription activators, stabilizes the initiation complex and stimulates TFIIH kinase activity.GAL1 and HXK1) genes in yeast. Moreover, the interaction between the 2 complexes is required for the dynamic targeting of activated genes to NPCs. In essence, Mediator is a relay for communication between TREX-2 and Pol II. Here, we discuss the emerging role of promoters in regulating different aspects of mRNA metabolism and how promoter-bound Mediator and TREX-2 could impact on these transactions.We recently reported that TREX-2 (1) interacts directly with Mediator via a specific interface of its PCI domain, (2) regulates Mediator association with the Cdk8 kinase, a central activity switch on Mediator and (3) influences RNA Pol II phosphorylation at Ser5, the defining mark of transcription initiation .15 This The biogenesis of eukaryotic mRNAs involves not only pre-mRNA synthesis by RNA Pol II, but also cotranscriptional RNA processing comprising 5′ capping, intron splicing, 3′ RNA cleavage and polyadenylation (3′ processing). The mature mRNA is packaged with RNA-binding proteins into messenger ribonucleoprotein particles (mRNPs), which is essential for its export to the cytoplasm and protein synthesis.A prime example for sequential coupling reactions is the RNA Pol II C-terminal domain (CTD), which undergoes dynamic phosphorylation as the Pol II progresses through initiation, elongation, and termination.Xenopus already in 1990, which showed that the 5′ cap and the interacting cap binding complex (CBC20/CBC80 in metazoa) are required for mRNA export.A common model is that acquisition of export competence begins with the recruitment of the conserved TREX (Transcription-coupled Export) complex.Our study revealed an unexpected link between TREX-2 and Mediator and the phosphorylation status of RNA Pol II. Specifically, we found that a loss of TREX-2 function leads to an increase of Ser5 phosphorylation of the Pol II CTD, which indicated that TREX-2 may have a regulatory impact when bound to Mediator. In support of this notion, we found that TREX-2 binds directly to the Med31/Med7 submodule, which is positioned in proximity to the Pol II CTD binding site on Mediator. Whether and how the impact of TREX-2 on the Pol II “phospho-code” plays a role in choreographing subsequent mRNA processing events will require further analysis.SWI5 and CLB2 mRNAs at the onset of metaphase in S.cerevisiae.S.cerevisiae responds to glucose starvation by translating a subset of transcriptionally activated mRNAs while decreasing translation of others. The information specifying the differential localization and protein production of these 2 classes of mRNAs was found to depend on specific promoters targeted by heat-shock factor 1 (Hsf1). Last but not least, promoter-proximal pausing was shown to be involved in regulating alternative cleavage and polyadenylation of mRNAs in Drosophila neurons.Since TREX-2 is necessary for mRNA export, its interaction with promoter-bound Mediator raises the interesting question whether the mRNA export function of TREX-2 is linked to Mediator's role in initiating transcription. This would imply that cells connect the earliest and latest steps of nuclear gene expression. Notably, a growing number of studies has reported promoter-dependent effects on downstream mRNA metabolism, which altogether raise the question whether transcription initiation is indeed a promoter's only task. In 2011, 2 studies showed that transcription factors and DNA promoters can directly influence the stability of the transcripts that they produce, independent of the transcript sequence. One paper described destabilization of The emerging theme of all these studies is an impact of promoters on gene expression that clearly goes beyond transcription initiation. Promoters appear to reach out to influence later steps of mRNA processing and mRNP formation. A central assumption underlying promoter-coupled mRNA processing events is that promoter elements are somehow brought into physical proximity with the nascent mRNA to allow several types of molecular cross-talk. However, the molecular details of these transactions are largely unclear.Given the influence of promoter-bound Mediator on Pol II Ser5 phosphorylation and the role of the CTD in scaffolding various mRNA processing and packaging events, it is conceivable that Mediator impacts on the mRNA life cycle through setting the proper RNA Pol II “phospho code.” But is there any evidence for a direct role of Mediator in dictating mRNA fate? Surprising insights came from a study, which reported direct physical interactions between the Mediator subunit MED23 and the hnRNP L protein, a regulator of alternative splicing in metazoans.A surprising finding of our study was that TREX-2 employs identical Sac3 PCI domain residues to promote both transcription and mRNA export. TREX-2 directly interacts with the Mediator Med31/Med7 submodule via conserved polar residues on the Sac3 PCI domain surface. Notably, TREX-2 uses the same polar residues to contact Mediator and to regulate mRNA export. This could indicate that TREX-2 is involved in a sequential coupling reaction, in which binding to Med31/Med7 is displaced by an interaction with a yet unknown mRNP factor. TREX-2 could therefore either promote the loading of factors onto the nascent mRNA or become itself loaded onto RNA. These possibilities remain to be explored, but are conceivable given that mutually exclusive protein-protein and protein-RNA interactions are a common feature of many co-transcriptional events. Such handover reactions are thought to impose directionality and a sequential order to mRNP formation.in vitro.in vitro.TREX-2 also binds to the general mRNA exporter Mex67/Mtr2 in vitro biochemical reconstitution and various in vivo functional analyses. These findings are key to understanding how NPC-associated adaptors modulate transcription, mRNA export and nuclear gene localization. This has revealed a molecular mechanism of TREX-2 function and opens new avenues for exploring how promoters can regulate multiple aspects of mRNA biology beyond transcription initiation.We have recently described a direct link between the TREX-2 complex and Mediator using structural biology,


NFAT and AP‐1 which created thousands of new DNase I‐hypersensitive sites (DHSs), enabling ETS‐1 and RUNX1 recruitment to previously inaccessible sites. Significantly, these DHSs remained stable long after activation ceased, were preserved following replication, and were maintained in memory‐phenotype cells. We show that primed DHSs maintain regions of active chromatin in the vicinity of inducible genes and enhancers that regulate immune responses. We suggest that this priming mechanism may contribute to immunological memory in T cells by facilitating the induction of nearby inducible regulatory elements in previously activated T cells.Immunological memory is a defining feature of vertebrate physiology, allowing rapid responses to repeat infections. However, the molecular mechanisms required for its establishment and maintenance remain poorly understood. Here, we demonstrated that the first steps in the acquisition of T‐cell memory occurred during the initial activation phase of naïve T cells by an antigenic stimulus. This event initiated extensive chromatin remodeling that reprogrammed immune response genes toward a stably maintained primed state, prior to terminal differentiation. Activation induced the transcription factors  M) arise as a consequence of previous immune reactions and are primed to efficiently upregulate key response genes when re‐challenged, while antigen‐inexperienced naive T cells (TN) take much longer to respond to the same stimuli  involves an interaction of the T‐cell receptor (TCR) with a specific antigen bound to the major histocompatibility complex on the APC. This occurs in parallel with additional interactions with co‐stimulatory molecules, such as CD28  and in the regulation of TCR‐inducible genes such as IL2, IL3, and CSF2   and chromatin immunoprecipitation assays (ChIP‐Seq) of TFs and histone modifications in TN, TM, and TB before and after stimulation. We identified over 2,000 pDHSs which were absent in TN but present in both TM and TB and were shared by CD4+ T helper cells and CD8+ cytotoxic T cells. In actively dividing TB cells, many of these pDHSs were stably bound by ETS‐1 and RUNX1, which we propose function to maintain chromatin priming. Upon re‐stimulation, they were also bound by AP‐1 which we propose is required to both establish priming and promote subsequent inducible responses. Significantly, the pDHSs were embedded within islands of active chromatin marked by H3K4me2 and H3K27ac which often encompassed inducible enhancers that could be activated in TB, but not in TN. In contrast to some genes which are silenced in naïve T cells by the Polycomb‐regulated H3K27me3 modification, the pDHSs we identified were not marked by H3K27me3 in naïve T cells.In the current study, our aim was to globally identify and characterize the regulatory elements and factors that maintain alternate chromatin states and underpin the recall response characteristic of both TN cells until they become primed toward an active chromatin state that is permissive for rapid enhancer induction by NFAT and AP‐1, and accessible to additional differentiation‐inducing TFs. Our model provides a simple chromatin‐based mechanism for maintaining the priming of TM‐specific regulatory elements using preexisting TFs which might allow T cells to retain immunological memory without the need for additional specific factors to silence genes in TN.We therefore propose a model whereby many inducible immune response genes exist within inaccessible chromatin in TM and TB. Because we wanted to investigate global mechanisms of T‐cell regulation, we chose to examine naturally arising memory‐phenotype cells (TM) instead of TCR‐specific T cells that may be restricted to a single T‐cell lineage and differentiation state. We first purified different populations of CD4+ T helper cells and CD8+ cytotoxic T cells from mice and then analyzed their responses to stimulation of TCR signaling pathways. For this purpose, we used the C42 transgenic mouse model which contains the intact human IL3/CSF2 locus encoding IL‐3 and GM‐CSF  to activate surface receptors, followed by a period of rapid proliferation for 2–3 days in the presence of IL‐2  ligand1 gene (Ccl1) were induced much more slowly over a period of 16 h  TM were predominantly memory‐phenotype cells expressing CD44 but lacking CD62L and CD25, thereby resembling effector memory T cells, and (iii) TB predominantly expressed the activation markers CD25 and CD44, consistent with activated effector T cells  to activate TCR signaling pathways that induce AP‐1 and NFAT activity. By directly activating pathways downstream of the TCR, we ensured that we investigated mechanisms directly associated with epigenetic and transcriptional responses and not just differences in the expression or function of molecules mediating signaling from the cell surface. Analyses of mRNA expression for human tics Fig A. This dcis‐regulatory elements in T cells from C42 mice, we performed global DNase‐Seq which identifies accessible regions of chromatin bound by transcription factors  contribute to the long‐term maintenance of memory in non‐dividing circulating TM. These analyses also confirmed that preparation of TB by stimulation with ConA gave an identical pattern of DHSs in the human IL3/CSF2 locus to that seen previously for TB prepared by specific stimulation of the TCR complex and CD28 , we detected additional inducible DHSs (iDHSs) which included the well‐defined −37‐kb and −4.5‐kb IL3 enhancers and the IL3 promoter, and novel iDHSs 25 and 28 kb downstream of CSF2. Significantly, these iDHSs existed in close proximity to pDHSs and zones of active chromatin modifications. Each of these iDHSs, plus the iDHS at the −3‐kb CSF2 enhancer, was highly inducible in TB but not in TN, consistent with a mechanism whereby the stable pDHSs function to maintain chromatin spanning inducible enhancers in a primed state that allows their rapid response to TCR signaling.To further define the features of the stably maintained pDHSs, we performed ChIP‐Seq assays for histone H3K4me2 and H3K27ac in CD4Tn, B Fig D. In stiIL3 locus lack enhancer activity, whereas the −37‐kb iDHS is a powerful inducible enhancer  CD4 TM relative to CD4 TN  TB and TM shared many specific DHSs that were absent in TN, highlighting the similarity between the recently activated proliferating TB and the quiescent primary TM; (ii) we reproducibly observed global enrichment of the same population of DHSs in TB compared to TN for both CD4 and CD8 T cells when all the data, including independent replicates of the CD4 TN and TB samples, were plotted using the same ranking as used for the original CD4 TN and TB data shown in Fig + T helper cells and CD8+ cytotoxic T cells utilize a common set of DHSs to maintain epigenetic priming in previously activated T cells. It is likely that these sites become established soon after cells first become activated, prior to terminal T‐cell differentiation to specific subtypes such as Th1 and Th2. The persistence of pDHSs in CD4 TM raised the interesting possibility that they may serve the additional purpose of enabling the rapid reactivation of inducible genes in memory T cells.We began by evaluating the different properties of DHSs in CD4 and CD8 T4 TN Fig A and the4 TN Fig B, and daM relative to CD4 TN. After excluding minor peaks, we selected a reproducible subset of 2,882 of the CD4 TM pDHSs that were also present in the CD4 TB DHS dataset  and the replicate CD4 TB (85%) datasets shown in Fig + cells  and in CD8 TN and TB by microarray analysis in both untreated cells and cells after 2 h of stimulation with PMA/I. The comparison of the fold induction for each gene in CD4 TN and TB showed 1,895 TM‐specific genes being at least twofold induced in TM and less than twofold induced in TN , indicating a strong correlation between proximity to preexisting pDHSs and inducible gene expression in memory T cells. Of the remaining inducible genes, 91% did nevertheless contain a constitutive DHS within 150 kb.To globally assess the role of pDHSs in regulating inducible gene expression, we mapped the distances from the TSSs of the 1,895 T83 genes  that hadB which had been stimulated for 2 h with PMA/I (TB+). In parallel, we performed ChIP‐Seq for H3K4me2, H3K27ac, and BRD4. The two sets of DHS data were plotted as density maps ranked according to the fold change of DNase‐Seq tag counts for the combined TB+ and TB datasets  throughout the genome in CD4 TB   Fig E, furtheM‐specific inducible gene . These data reinforce the view that pDHSs function at close range to support inducible enhancer function. In addition, we found that the most highly induced genes also displayed the shortest distances between the pDHSs and iDHSs   also lacked independent enhancer function  locus which contained multiple TM/TB‐specific DHSs. At the −3.2‐kb and +21‐kb pDHSs, ChIP peaks were detected for RUNX1 and ETS‐1 in TB but not TN. Importantly, motifs for both these factors were present within the hypersensitive region. At the −2.4‐kb DHS, we identified a RUNX motif, but no ETS motif, and this was reflected by binding of RUNX1 but not ETS‐1. Furthermore, at least 6 DHSs within this locus encompass AP‐1 motifs, and this was reflected by specific JUNB binding in TB+ and not in TB  but not inducible genes or constitutive genes believed to be independent of pDHSs (Tnf and Cd2)  . These det al, in silico DNase I footprinting on the DNase‐Seq data from TB. This algorithm statistically scores an imbalanced pattern of positive strand reads starting immediately 5′ of the footprint and negative strand reads starting immediately 3′ of the footprint, spanning the binding site which is detected as a region with reduced reads. The distribution of motifs detected by HOMER within these footprints  contained occupied AP‐1 motifs compared to the number of occupied RUNX and ETS motifs  and NFAT (56%). Many of these were composite NFAT/AP‐1 motifs of the type first described for the Il2 promoter and CSF2 enhancer  pDHSs to iDHSs, and (ii) iDHSs to the TSSs of these inducible genes. Significantly, the presence or absence pDHSs has little impact on steady‐state transcription of nearby genes, in contrast to classical enhancers. Others have also recently observed that evidence of TF binding is a much more reliable guide to enhancer function than the mere presence of open chromatin or active histone modifications  and Easy Sep (Stem Cell Technologies) purification kits  for an additional 2–3 days before harvesting. Cells were stimulated with 20 ng/ml phorbol myristate acetate (PMA) and 2 μM calcium ionophore (I) for up to 4 h.Tkits see . TB wereet al, DNase I digestion assays were carried out as previously described . RNA was either (i) reverse‐transcribed to cDNA using M‐MLV Reverse Transcriptase (Invitrogen) for analysis by quantitative PCR or (ii) prepared for microarray analysis .N and TB were prepared for sequencing on the SOLiD™ platform according to the manufacturer's instructions (Applied Biosystems). All other libraries were prepared using the Tru‐Seq library preparation kit according to the manufacturer's instructions (Illumina).DNA libraries for sequencing were prepared from ~10 ng DNA from DNase I or ChIP samples. Libraries for DNase‐Seq of untreated CD4 and CD8 TDetailed data analysis is provided in the ChIP‐Seq, DNase‐Seq, and expression microarray datasets have been deposited on the Gene Expression Omnibus (GEO) as a super series under GEO accession number GSE67465. Individual datasets were deposited as follows: ChIP‐Seq under accession number GSE67443, DNase‐Seq under accession number GSE67451, and expression microarrays under accession number GSE67464.et al, et al, et al, et al, et al, H3K27me3 ChIP‐Seq datasets in naïve CD4 T cells [(Wei SLB, EB, RCJ, LNG, and PNC performed the laboratory studies, PC, JP, NL, and SO performed bioinformatics analyses, and SLB, PC, CB, and PNC designed the experiments and wrote the manuscript.The authors declare that they have no conflict of interest.AppendixClick here for additional data file.Expanded View Figures PDFClick here for additional data file.Dataset EV1Click here for additional data file.Dataset EV2Click here for additional data file.Dataset EV3Click here for additional data file.Dataset EV4Click here for additional data file.Dataset EV5Click here for additional data file.Review Process FileClick here for additional data file.