PHO5 gene in Saccharomyces cerevisiae. Introducing a model for nucleosome kinetics that takes into account ATP-dependent remodeling activity, DNA sequence effects, and kinetics of TFs (Pho4p), we compute the probability of obtaining different “promoter states” having different nucleosome configurations. Comparing our results with experimental data, we argue that the presence of local remodeling activity (LRA) as opposed to basal remodeling activity (BRA) is crucial in determining transcriptionally active promoter states. By modulating the LRA and Pho4p binding rate, we obtain different mRNA distributions—Poisson, bimodal, and long-tail. Through this work we explain many features of the PHO5 promoter such as sequence-dependent TF accessibility and the role of correlated dynamics between nucleosomes and TFs in opening/coverage of the TATA box. We also obtain possible ranges for TF binding rates and the magnitude of LRA.Studying nucleosome dynamics in promoter regions is crucial for understanding gene regulation. Nucleosomes regulate gene expression by sterically occluding transcription factors (TFs) and other non–histone proteins accessing genomic DNA. How the binding competition between nucleosomes and TFs leads to transcriptionally compatible promoter states is an open question. Here, we present a computational study of the nucleosome dynamics and organization in the promoter region of  The eukaryotic chromatin in gene-rich regions resembles “beads on a string” made of nucleosomes—a DNA-protein complex with 147 base pairs (bp) of DNA wrapped around an octameric histone protein in nearly two turns134578One important effort in chromatin research in the past one or two decades has been to understand the precise organization of nucleosomes along the DNA and to examine what controls their positioning on the DNA3451161331541755Given that a large number of factors can influence nucleosome positioning, its organization is highly diverse and varies considerably from gene to gene and location to location82121GAL1/ 10 promoter, interesting interplay between nucleosomes, remodelers, and TFs are known to lead to strong nucleosome positioningHO and CLN2 promoters, the binding of activator SBF at their binding sites has been shown to correlate with nucleosome eviction and gene expression24PHO5, a gene that encodes acid phosphatase required for scavenging phosphate from the environment. Typically, nucleosomes at the PHO5 promoter are known to form a regular array with relatively high occupancy262930In yeast, a number of experiments have been carried out to investigate nucleosome positioning at various inducible promoters. For example, in PHO5 promoter of yeast Saccharomyces cerevisiae as a model systemEven though many experimental studies253031k bp and kt bp, respectively.To study the complex problem of nucleosome dynamics in the presence of non–histone proteins , we have made a stochastic model that has three layers: (i) nucleosome kinetics with sequence- and ATP-dependent effects (ii) transcription factor kinetics and (iii) coupling between transcription factor and nucleosome kinetics via enhanced local remodeling of nucleosomes. In the paper, DNA is modeled as a one-dimensional (1D) lattice of N bp; nucleosomes and TFs are modeled as sterically interacting (hard-core) particles of size k bp can bind with an intrinsic rate ron = r0. Since it is known that nearly any part of the genomic DNA can easily wrap around histones to form a nucleosome, we assume that the binding is sequence-independent. However, once bound, a nucleosome dissociates (disassembles) in a sequence-dependent manner. This dissociation occurs at a rate given byWe assume that nucleosomes can have three kinetic moves—binding, dissociation and sliding—as discussed earlier in the literatureVi is the sequence-dependent binding energy at ith position and r0 is the intrinsic removal rate per nucleosome. The potential energy function can be obtained using Vi = −kBTlnPi, where Pi is the equilibrium probability of finding a nucleosome at ith bp in the limit of very low nucleosome density4Pi’s by submitting a genomic sequence of our interest to the online server of Segal et al. (http://genie.weizmann.ac.il/software/nucleo_prediction.html) that computes the probabilities based on bioinformatic analysis of in vitro data. We set the nucleosome concentration low so that we obtain the potential “seen” by a single nucleosome. Given the binding energy function, the thermally driven adsorption–desorption process obeys the Boltzmann condition given bywhere Vi〉 is estimated to be ≈−42 kBT, which is nothing but the average binding energy of histone octamer onto DNA34Vi values implies that the the typical nucleosome removal rate Based on experimental data, 〈Va is the energy contribution from ATP-dependent “active” chromatin remodeling. Second, ATP-dependent machines can also slide the nucleosomes along the DNA. We assume each nucleosome is slid in a randomly chosen direction with a rate αs and stops when it sterically interacts with its nearest neighbour (histone or transcription factor). We also assume that ATPases can slide nucleosomes independent of the local DNA sequenceVa and αs, as basal remodeling activity (BRA). The underlying assumption here is that there is BRA all the time in the regions near TSS/promoters. This is the simplest kinetic model that would explain many of the experimentally seen phenomena. Our earlier theoretical work34where kt = 17 bp, and assume that it can only bind at specific binding sites in the promoter region, marked as UAS1 and UAS2 in ronp per binding site, whenever sterically permissible. The Pho4p at UAS1 and UAS2 dissociate with rates Apart from nucleosomes, we have also introduced the binding–dissociation dynamics of the transcriptional activator, Pho4p, into the model. We consider Pho4p as a particle of size Va was independent of the location. However, experiments suggest that nucleosomes at the promoters are evicted/remodeled more often when compared to other regions3929So far we have only considered a basal ATP-dependent remodeling of nucleosomes—that is, the contribution to the active nucleosome removal b” bp:We introduce h is the maximal local remodeling energy and b is the width of the potential energy. This potential is “activated” only when at least one transcription factor is bound at location j (j is the location of either at UAS1 or UAS2 (j spans the region (j − k − 150) to (j − k) because this leads to removal of nucleosomes immediately to the left of the location j. When both UAS1 and UAS2 are occupied by activators, the resulting potential for local activity has the form shown in Here cation j , where j or UAS2 . The locPHO5 accounting for both nucleosome and TF kinetics as described above, using kinetic Monte Carlo simulations with Gillespie algorithmet al.A) state and “inactive” (I) states. We consider a promoter state as active when at least one Pho4p is bound and the TATA binding site is empty . Any other promoter state is considered as inactive. Whenever the promoter is active, in the simulation, we produce mRNA at a constant rate ε (δ per mRNA. Let m be the number of mRNA molecules present at t = 1 hour after the addition of Pho4p proteins. From our simulations we computed the distribution of mRNA molecules P(m). We also computed the mean mRNA number 〈m〉, the variance σ2 = 〈m2〉 − 〈m〉2, and the Fano factor = σ2/〈m〉.We simulate a long region (5000 bp) around the promoter of the t rate ε . When thron, αs for nucleosome dynamics are determined from previous experiments, as done in the literature34ron = 12s−1. This value has been estimated from experiments using Xenopus egg extracts34Va, we set Veff, the net effective binding potential experienced by a nucleosome, as Veff = Va + 〈Vi〉; and use Veff as a free parameter, where, N = 5000 bp of PH05 gene when its full promoter region is taken into account. The active sliding parameter of the nucleosome is set to αs = 0.0024s−1 based on the estimates of earlier works34k = 147 bp. The size of the dimeric Pho4p is set as kt = 17 bp. We vary the protein binding rate, ronp, relative to the nucleosome adsorption rate such that it has the following range: ronp =  × k0s−1, where −4 and at UAS2 ≈ 7.5 × 10−3 as estimated by Lam et al.et al.b = 150 bp and h is kept as a variable. As previously describedPHO5 mRNA as 〈m〉 ≈ 12 per cell per transcriptionally active promoter state. Considering the half-life of an mRNA as thalf-life ≈ 5 minThe three kinetic parameters, namely, PHO5 promoter region taking into account only two factors—the effect of DNA sequence and the basal ATPase activity (34Veff = −7kBT curve and other curves). In the presence of sequence-dependent kinetics, the basal ATPase activity results in two effects: it controls the density of nucleosomes bound in the promoter region and it modulates the sequence-dependent positioning. For instance, the NFR at the UAS1 site (grey stripes in Veff) increasesVeff < −8kBT) the nucleosome density is very high, and the promoter is covered by nucleosomes.First, we have simulated the nucleosome positioning in the activity . In the kBT > Veff > −8kBT  or too low (Veff < −8kBT) (beyond the range discussed above), the spatial organization of the promoter nucleosomes (curve from simulation) is very different from the experimental data (squares and circles)—e.g., Veff = 0kBT (blue curve) and Veff = −10kBT (black curve). The CRB1 data points (circles) represent promoter configurations for constitutively inactive PHO5 gene in pho4Δpho80Δtata mutant cells. The data points marked as CRB2 (squares) represent promoter for pho2Δ mutant cells grown under the repressing condition of a high phosphate growth medium. Except for the third and fourth states, all the probabilities of states obtained for Veff = −7kBT and Veff = −4kBT are comparable with CRB1 and CRB2 data points respectively  gene. It has been reported, experimentally, that the average number of nucleosomes in the promoter region, in repressed states, is between ≈2.4 and 2.65 > −8kBT . This am > −8kBT . We furton et al.. We compon et al.. When thectively . Note thVeff = 0 . Evidenth = 0kBT) and computed the probabilities of different promoter states (ronp ~ 0.1 × k0s−1) makes our curves comparable to the experimental data (CRB1 and CRB2) by redistributing the frequencies of the first, third, and fourth promoter states. One can understand this by noting the following: as the TFs start competing with nucleosomes, the −2 nucleosome region—the region corresponding to the UAS1 and UAS2—gets depleted of nucleosomes and the probability of the third promoter state increases. This is a direct consequence of competition between TFs and nucleosomes. Our study suggests how stochastic switching from promoter state 1 to state 3 is modulated by competitive binding of Pho4p. As seen here, in this range of low Pho4p binding rates, the promoter is shown to be inactive. The above results also suggest another interesting point: the non–uniform nature of probabilities seen here (in the repressed state) is due to an interplay between basal remodeling activity, DNA sequence-effcts and effects due to “barrier”-like non–histone proteins binding. In the absence of sequence effects and TF binding, one would obtain same probability for different 2-nucleosome promoter states  and single-nucleosome promoter states . To quantify the inactive promoter we investigated mRNA production and its statistical distribution. To do this, we extended our simulations and introduced mRNA production into the model—whenever the promoter is in one of the active or ON states, mRNA will be produced with a given rate (see Models and Methods for details). Reflecting the inactive nature of the promoter, the mRNA distribution is found to be skewed towards a major peak at zero  and inactive (CRB1 and CRB2) promoter states in h”. We define h as the maximal remodeling energy from bound Pho4p , as discussed in the model section  obtained for constitutively active PHO4 pho80Δ cells, as reported in ref. ronp ~ 0.3–0.6 × k0s−1  of promoter nucleosomes ~0.1 s, which corresponds to nucleosome removal rate roff ~ 10 s−1  state of the promoter? So far, in our simulations, we have accounted for basal ATPase activity and Pho4p binding. However, from the high coverage of TATA discussed above and from 49 section . Our sim × k0s−1 . Interes~ 10 s−1 . In othem ≈ 10 representing cells in the ON state, and another peak near mRNA number m = 0 indicating a population of cells in the OFF state , we find that the Fano factor F → 1 (blue region). This implies that the underlying process of gene expression has a Poisson distribution; this is indeed seen in h > 26kBT) and the protein binding is strong (ronp > k0s−1), the probability of the promoter being in the ON state is also very high (PON > 90%). The corresponding gene expression is constitutive and we get high mRNA production. For other values of h and ronp, different types of non-Poissonian distributions can be obtained. The region given by the parametric values h ≥ 16 kBT and ronp ~ 0.2–0.5 × k0s−1 (green region) displays bimodal distribution , the expression distribution is a monotonically decreasing function with a long-tail. In this case, the probability of having non-zero mRNA expression is high and decreases slowly for higher expressions. For protein binding rate ronp < 0.1 × k0s−1 (red region), we obtained an OFF-like longtail distribution. In this phase-space, the promoter is OFF-like and has an expression with a very high probability of having no mRNA (OFF) and low or null probability of having non-zero mRNA (ON) , derived for a two-state model of gene transcription when the promoter activation rate, say kon, is modulated  Finally, our work fills a major gap in the literature to provide a combined understanding of nucleosome dynamics in the promoter region and in the coding region. The models that explain nucleosome positioning in the coding region cannot explain the nucleosome organization in the promoters16Despite the fact that there are studies trying to understand gene regulation in yeast promoters47The literature on nucleosome kinetics is dominated by nucleosome sliding444755From the current study we learn two things: (i) transcription factor binding alone may not be sufficient to switch ON the gene, and (ii) location-dependent remodeling is absolutely necessary. Pho4p binding can only occlude the −2 nucleosome. Therefore, one has to go beyond a passive model of activator binding, and a good candidate for this would be the recruitment modelet al. have suggested that there is no steric exclusion of nucleosomes by TFs, which means that nucleosome formation is independent of TF bindingin vitro reconstitution experiments of PHO5 promoter chromatin suggest that there is competition between Pho4p and nucleosomeThe precise role of steric exclusion by TFs in nucleosome remodeling is still needed to be fully understood. Brown We present one of the first computational studies that explicitly incorporates nucleosome and TF dynamics accounting for DNA sequence-dependent and ATPase effects. Through this study we show how nucleosome dynamics coupled with TF binding kinetics can give us gene expression profiles that capture most of the essential features of a typical eukaryotic gene expression. By tuning the parameters like LRA and TF binding rate, we can get the Poisson, bimodal, long-tail, OFF-like long-tail, and other intermediate distributions. For sensible parameters , we get a bimodal distribution for mRNA expression. Bimodal gene expressions are typically associated with inducible promoters2451et al.PHO5 promoter. In a recent study by Small et al.PHO5 promoter states. They show that there is a redistribution of the state probabilities upon altering cellular conditions from a nutrient rich (repressed) to phosphate starvation (induced)—the former has states with many nucleosomes while the latter is predominantly depleted of nucleosomes. Broadly, even our simulations agree with this picture where the inactive state is dominated by bound nucleosomes and the active state is relatively nucleosome free. However, one of the major differences between the Small et al. data and Brown et al. data is that, in the Small et al. experiments, UAS1 is covered with nucleosomes more frequently, even in the active state. We find that we can obtain this feature by appropriately modifying the LRA . Under these conditions, one can also vary Pho4p expression (overexpress) and measure nucleosome occupancy in the PHO5 promoter  One can test our prediction for nucleosome occupancy shown in promoter . Anotherpromoter . This maPHO5 gene in Saccharomyces cerevisiae. In the model, we include nucleosome and Pho4p dynamics taking into account the effects of DNA sequence and ATP-dependent remodeling activity. We first validate our model by making sure that our results are comparable with known experimental data of promoter states having different nucleosome configurations. Then we go on to predict a number of features such as the necessity for LRA with nucleosome removal. We show that only the binding–dissociation interaction of Pho4p with DNA does not give rise to active promoter states; we require Pho4p-mediated “local remodeling activity” on promoter nucleosomes by ATP-dependent remodeling complexes. We also show that the mRNA expression profile for high LRA is a Poisson distribution, and for low LRA the expression is a non-Poisson distribution.In summary, we have studied a computational model for promoter dynamics of the How to cite this article: Kharerin, H. et al. Role of transcription factor-mediated nucleosome disassembly in PHO5 gene expression. Sci. Rep.6, 20319; doi: 10.1038/srep20319 (2016).


S. coelicolor genome that have no obvious resemblance to previously identified DasR-responsive elements. Thus, the regulon of DasR extends well beyond what was previously predicted and includes a large number of genes with functions far removed from N-acetylglucosamine metabolism, such as genes for small RNAs and DNA transposases. Conversely, the DasR regulon during vegetative growth largely correlates to the presence of canonical DasR-responsive elements. The changes in DasR binding in vivo following N-acetylglucosamine induction were studied in detail and a possible molecular mechanism by which the influence of DasR is extended is discussed. Discussion of DasR binding was further informed by a parallel transcriptome analysis of the respective cultures. Evidence is provided that DasR binds directly to the promoters of all genes encoding pathway-specific regulators of antibiotic production in S. coelicolor, thereby providing an exquisitely simple link between nutritional control and secondary metabolism.Streptomycetes produce a wealth of natural products, including over half of all known antibiotics. It was previously demonstrated that N-acetylglucosamine and secondary metabolism are closely entwined in streptomycetes. Here we show that DNA recognition by the N-acetylglucosamine-responsive regulator DasR is growth-phase dependent, and that DasR can bind to sites in the  Streptomyces coelicolor genome by 45 ECF (extracytoplasmic function) sigma factors genes and some 70 two-component regulatory systems [S. coelicolor genome sequence—the first from an antibiotic-producing actinomycete [The ability of bacteria to adapt to a wide range of nutritional environments requires intricate regulatory systems to facilitate the key routes of primary metabolism. The flux of carbon and nitrogen is typically controlled by a number of global, pleiotropic regulators, each targetting a number of specific, non-pleiotropic regulators in their response regulon. The global regulators ensure that the flux of essential metabolites is closely monitored and tuned ,2. Strep systems . Typicalnomycete —was a lanomycete ,9,10,11,nomycete . Many ofnomycete ,14. Manynomycete ,15,16.Escherichia coli and Bacillus subtilis, CCR is mediated via the phosphoenolpyruvate-dependent phosphotransferase system  uptake [S. coelicolor [in vitro by glucosamine-6-phosphate [in silico and in vitro evidence revealed that direct repression of the actII-ORF4 and redZ, the pathway-specific regulators of actinorhodin and prodiginine production, respectively [To allow activation and screening of cryptic antibiotic biosynthetic clusters, detailed insight is required into the linkages between environmental  signals and secondary metabolite production. Carbon source utilization is a major determining factor in the metabolic control of antibiotic production ,18. The iewed in ,20,21). iewed in ,23. Addiiewed in . In stre) uptake ,26 as we) uptake . This swelicolor ,28. GlcNelicolor ,29,30, ahosphate , a metabdre, for DasR responsive element), namely the palindromic 16-bp consensus sequence A(G/C)TGGTCTAGACCA(G/C)T. These target sequences are primarily associated with genes related to primary metabolism, sugar transport and extracellular polysaccharide hydrolysis, as well as antibiotic production [in vitro and changes in the level of associated transcript(s) in a DasR knockout.Previous studies showed the involvement of DasR in the control of genes involved in GlcNAc/chitin catabolism ,31, antioduction . Moreovein vivo and correlate them with changes in gene expression. This has highlighted major and highly dynamic changes in its binding pattern both in a growth phase-dependent manner (binding during vegetative growth or sporulation) and after the exogenous addition of the ligand GlcNAc.In this work we use a genome-wide analysis to determine directly the sites of DasR binding dasR null mutants and the extracellular accumulation of N-acetylglucosamine resulted in similar phenotypes, with enhanced antibiotic production and accelerated development under poor growth conditions, and blocked development and antibiotic production on rich media [dasR null mutants is most likely provided by GlcN-6P, which acts as a ligand for DasR [in vivo is therefore a prerequisite to better understand the biological role of DasR. We therefore conducted ChIP-on-chip analysis on wild-type S. coelicolor M145 carrying the integrative vector pGAM29, which expresses C-terminally 3xFLAG-tagged DasR, using the dasR null mutant GAM29 as the control. Two different types of genome-wide DNA-binding studies were performed; first we analysed DasR binding in cultures grown on minimal media (MM) agar plates with mannitol as the sole carbon source for the comparative analysis of DasR binding during vegetative and developmental growth, and subsequently we performed induction experiments, adding GlcNAc to liquid-grown cultures to monitor the changes on DasR binding in both rich and poor media.We previously showed that the phenotype of ch media . Linkagefor DasR . HowevernagE1, nagE2, nagKA, scr3092, dasR-dasA intergenic region, scr5239, tRNAGln-Glu, ptsH, SCO6032-6033 intergenic region and SCO7056) were shared between the 24 and 54 h samples. It should be noted at this point that we also performed eight additional ChIP-on-chip experiments on liquid-grown NMMP and R5 cultures, to analyse the effect of GlcNAc on in vivo binding (see below). Taken together, this provided very good validation for the in vivo binding of DasR, with up to eight independent verifications of the target genes  and 54 h (sporulation), with similar growth rates and timing of development for both strains. Using a combined approach of statistical (two or more significantly enriched probes) and manual inspection , DasR binding events were identified at 21 and 51 promoter-proximal regions bound in the 24 h and 54 h old samples, respectively , SCO6300 (for a secreted β-N-acetyl-glucosaminidase), SCO6345, the SCO6005-6007 operon (the SCO6004-SCO6005 intergenic region has only one probe significantly enriched), dasA and dasR itself  with ge and chb ,30,31 alnag, ptsH, chi) that were bound by DasR at 24 h were either not bound after 54 h of growth, or  showed reduced binding. From the perspective of developmental control, only after 54 h did we see binding of DasR to any of the genes involved in development and secondary metabolism, as well as many other targets outside the core regulon. Binding was observed to the intragenic regions of three biosynthetic genes (SCO6273-6275) of the cpk gene cluster for the cryptic type I polyketide synthase (Cpk) and of cdaPSI and cdaPSII (SCO3230 and SCO3231) encoding the peptide synthetases I and II for the synthesis of calcium-dependent antibiotic (CDA). Additionally, in liquid-grown cultures binding of DasR was detected in the promoter proximal region of actII-ORF4 and redD, redZ, encoding the pathway-specific activator genes for Act and Red biosynthetic clusters, respectively  at the post-transcriptional level [dre elements—and in several independent ChIP-on-chip experiments—within the five-membered tRNAGln/Glu gene cluster, to a gene for tRNAGly upstream of SCO3679, to tRNAMet and to the tRNAGlu/Asp/Phe gene cluster upstream of SCO4092. Additionally, after 54 h of growth on MM agar plates, DasR binding was observed upstream of SCO2504 (glyS) encoding glycyl-tRNA synthetase . The negative control (a fragment of the bla gene for a beta-lactamase) was not bound by DasR.DasR binding was observed for the promoter regions of many genes for transfer-RNA (tRNA) and noncoding RNA (ncRNA). As for ncRNAs, binding was observed to  scr3092  and scr5 scr3092   and famine (poor media) conditions, with block or stimulation of development and antibiotic production, respectively. To address this important question, we performed ChIP-on-chip experiments for DasR following the extracellular addition of GlcNAc. For this purpose, mycelia of S. coelicolor M145 harbouring pGAM29 and the dasR mutant were each grown in liquid NMMP mannitol or R5 cultures, and cells were induced by the addition of 50 mM GlcNAc. Samples were collected prior to (T0) and 30 (T1), 60 (T2) or 120 min (T3) after GlcNAc addition, and DasR-enriched chromatin was analysed as described in the Experimental Procedures section. Prior to GlcNAc induction (T0) high ChIP-enrichment ratios demonstrated DasR binding at 72 and 66 sites in NMMP- and R5-grown mycelia, respectively ; (ii) the transcription unit that includes nagA which is necessary to provide the DasR effector GlcN-6P; and (iii) actII-ORF4 and of redD and redZ, encoding the pathway-specific activator genes for Act and Red biosynthetic clusters, respectively, as well as from the coding sequences of cdaPSI and cdaPSII (SCO3230-3231) for CDA peptide synthetases I and II, and for cpkBC . FurthenagE2 product is the first step, while control of pathway-specific regulators is the last step. We previously showed that DasR and AtrA have antagonistic actions towards nagE2 and actII-ORF4. AtrA is required for actinorhodin production via the direct activation of actII-ORF4 [nagE2 [atrA, and that this binding is reduced by the addition of GlcNAc to NMMP-grown mycelia and relieved when GlcNAc was added to R5 cultures. Thus, AtrA and DasR antagonise each other in terms of their influence on this important signaling cascade.In the above mentioned DasR-mediated GlcNAc signaling cascade from nutrient availability to antibiotic production, internalization via the tII-ORF4 , and als4 [nagE2 , while DpyrR-bldD, the upstream regions of the tRNA genes/operons tRNAGlu/Asp/Phe, tRNAGln/Glu and tRNAMet, and secE for the preprotein translocase subunit SecE . Some 100 genes showed more than three-fold and 26 genes showed more than five-fold change in transcription relative to the parental strain  and small peptides with unknown functions; transposases and DNA recombination. The most notable changes are discussed below; for details see The ChIP-on-chip experiments provided insight into the global DasR binding. To obtain complementary insight into the global changes at the transcriptional level following the deletion of elopment . RNA wasl strain . ClassesdasR mutant and mostly at all time points, including nagE2, nagF, ptsH (encoding PTS components); nagB, nagK (encoding GlcNAc metabolic enzymes); glmS1 and glmS2 ; and dasA-dasBC   for γ-butyrolactone synthase implicated in the control of the antibiotic clusters act, red and cpk in S. coelicolor [cpkO (SCO6280), for the transcriptional activator of the cpk cluster, was up-regulated at 24 h, followed by enhanced transcription of the cpk cluster in the following time point (30 h) (nsdA (SCO5582), a negative regulator of antibiotic production in S. coelicolor [nsdB (SCO7252) [ste gene cluster of Streptomyces sp. 139 required for Ebosin biosynthesis, were also up-regulated in the dasR mutant  and prodiginines (SCO5877-SCO5898) was enhanced in the R mutant . Howeverelicolor , and cpkt (30 h) . Other aelicolor  and its SCO7252) . SeveralR mutant . Ebosin  was left unchanged. The pentacistronic bldK operon encodes an oligopeptide transporter required for early stages of development [bldN encodes a developmentally regulated σ factor, σBldN, required for aerial mycelium formation, which in turn is required for the transcription of the response regulator gene bldM [crp is involved in the control of spore germination [wblC (SCO5190) and its flanking genes SCO5189 and SCO5191.The DasR regulon includes a large number of putative transcriptional regulators, with around 40 regulatory genes at least two-fold up- or down-regulated in the mutant at one or more time points, which forms a likely explanation for the large number of differentially expressed genes in the mutant. Several early developmental genes were up-regulated in the and wblA , while eelopment ,49, bldNene bldM , crp is mination ,52, and mination . Some ofchpABCDEFGH, rdlAB and ramS, encoding chaplins, rodlins and SapB, respectively, were very strongly down-regulated during early time points. The chaplins and rodlins are hydrophobic ‘coat’ proteins that assemble into a rodlet layer that provides surface hydrophobicity to aerial hyphae and spores, thus allowing them to break through the moist soil surface [Strikingly,  surface ,55; the  surface . SeveralgntR regulatory gene SCO3986, encoding the likely regulator of this cluster, was identified as a DasR target in the ChIP-on-chip experiments on MM agar plates with mannitol at 54h  secreted proteins and a GntR regulator , was alsl at 54h . Having nag and pts genes for GlcNAc metabolism and transport, respectively, and the chi regulon involved in the catabolism of polymers of GlcNAc. Since Fructose-6P, acetate and ammonia are end products of GlcNAc catabolism, the DasR core regulon is immediately connected to major metabolic pathways such as glycolysis, nucleic acids, nitrogen, and fatty acid metabolism, the tricarboxylic acid (TCA) cycle and cell-wall biosynthesis. There is an excellent correlation between the presence of a dre motif and DasR binding in vitro and in vivo, and indeed we have never observed a predicted dre with a high statistical score that is not bound by DasR in vitro. There were however a few examples of known core target genes that did not display ChIP-enrichment peaks across multiple samples (e.g. binding to crr-ptsI was only seen once); false negatives are not uncommon in ChIP-based studies and may reflect cases where the DasR is physically hidden within the cross-linked chromatin (e.g. encompassed by looped DNA and/or other transcription factors). A notable example in this context is the failure to detect a ChIP-enrichment peak for CRP bound to the E. coli lac operon promoter [Streptomycetes usually live in an environmental context of poor nitrogen and rich carbon availability . In thispromoter .i.e. preceded by dre elements) identified here include genes for ncRNAs and tRNAs. Additionally, we here provide evidence that in S. coelicolor all of the known antibiotic biosynthetic gene clusters are directly controlled by DasR, namely act, red, cpk and cda for actinorhodin, undecylprodigiosin, a cryptic polyketide and calcium-dependent antibiotic, respectively. Most of these are associated with dre elements. However, many binding events, referred to as ‘Class II’ targets, were identified in vivo that are not associated with a canonical dre element. Some of these were identified as highly-enriched probes in as many as eight biologically independent ChIP-on-chip experiments, providing a strong case that these are indeed bona fide binding sites. Genome-wide analysis of the regulons of several other globally acting bacterial transcription factors show similar duality, with binding to both their cognate (consensus) target sequences and to non-canonical sites in vivo, even though the latter binding sites show no similarity to the consensus sequence and they are not bound in vitro. Other examples include LexA [E. coli, Spo0A in Bacillus subtilis [Caulobacter crescentus [Streptomyces. For B. subtilis Spo0A, some 15% of the total number of binding sites was not bound in vitro [Streptomyces griseus and PhoP of S. coelicolor represent examples of master regulators in streptomycetes with a significant proportion of putative target genes without recognisable consensus binding sites [E. coli, which binds to a noncanonical site in the melAB promoter in concert with MelR associated with adjacent target sites [Novel functional categories of Class I targets  were bound by DasR at 24 h, but after 54 h, using the same detection methods, no evident binding was found for chiC, chiH, SCO6300, SCO6345 and SCO7225. Conversely, DasR binding to genes involved development and antibiotic production (and other processes) was only found after 54 h. The pleiotropic role of DasR in the control of antibiotic production at least in S. coelicolor was further underlined by the binding of DasR to the promoter-proximal regions of actII-ORF4, redD and redZ, encoding the activator genes for Act and Red biosynthetic clusters, respectively [The ectively .Bacillus also revolves around GlcNAc metabolism, but in contrast to dre elements, the NagR-responsive elements are found scarcely (up to three sites per genome [Streptococcus and Listeria (not shown). Our work shows that streptomycetes have adopted the GlcNAc regulatory system to build a much more intricate system that controls a wide range of genes. More than 1,200 genes were significantly differentially expressed in the dasR null mutant of S. coelicolor (i.e. 15% of the genome) and transcription of over 400 genes altered more than two-fold, which indicates the large changes in gene expression resulting from the absence of the metabolic master regulator DasR. In a recent environmental study, microarray data on the dasR null mutant in soil-grown cultures in the presence of chitin revealed some 700 genes that were differentially expressed [Streptomyces, exemplified by the more than 1000 significantly differentially expressed genes in mutants lacking the morphogene ssgA, which controls processes relating to cell-wall remodeling such as germination, tip growth, branching and cell division [The core regulon of the DasR homologue NagR in r genome ). Comparxpressed . In genedivision ,72, or tdivision .dasR was not anticipated. The specific environmental conditions are an important parameter for the response of the regulon, and additional control systems operate, such as substrate induction for induction of sugar utilization operons, or growth phase-dependent control of antibiotic production. The additional requirement of substrate induction is exemplified by the fact that despite DasR binding to almost all of the genes of the chi regulon, transcription of the genes was not notably changed in dasR mutant cells due to the lack of chitin as inducer. Indeed, strong induction of the chitinolytic system by chitin was observed in soil-grown S. coelicolor cultures, as well as enhanced antibiotic production [The DasR regulon incorporates around 40 transcriptional regulatory genes, suggesting an extensive secondary response. Global transcriptional regulators provide an additional layer of control on top of the control mediated by specific activators and/or repressors . Therefooduction ,74. The actII-ORF4 as an example, this gene is controlled by more than 10 transcriptional regulators. Besides DasR these include among others ActII-ORF4, AfsR, AtrA, SCO6008 and the stringent response mediated through ppGpp during growth cessation ([S. coelicolor M145 (which lacks the plasmid-borne methylenomycin biosynthetic cluster) may have evolved to ensure global control of natural product formation that is modulated by changes in nutritional conditions. The relatively small changes in gene expression of the antibiotic-related genes seen under the chosen experimental conditions are a consequence of the choice of media, which only allow minor induction of antibiotic production, but were chosen here to allow proper comparison between wild type and mutant, which requires similar (and synchronized) growth and development; however, major changes in antibiotic production are seen under conditions of feast (rich media) and famine (MM with agar as the sole carbon source) [Antibiotic production is typically extensively controlled, which ensures correct growth phase-dependent expression. Taking ssation  .in vitro [n degradation, GlcNAc transport and intracellular metabolism, glycolysis, nitrogen and lipid metabolism, as well as peptidoglycan synthesis [in vivo. The relief of DasR binding to its core regulon following the addition of GlcNAc to cultures provides in vivo evidence that DasR binding indeed undergoes major changes following binding of GlcNAc-derived metabolites. In terms of antibiotic production, the same relief of binding was seen for the pathway-specific activator genes redD and redZ for prodiginine production, and actII-ORF4 and atrA for actinorhodin production, as well as biosynthetic genes for the cryptic polyketide Cpk and the calcium-dependent antibiotic CDA. This provides experimental in vivo proof that the activation of antibiotic production by GlcNAc is directly mediated through DasR in S. coelicolor and also shows that this goes further than the control of actII-ORF4 and redZ (which were previously identified as direct targets in vitro [We have previously shown that glucosamine-6P (GlcN-6P) acts as an effector of DasR in vitro . This isynthesis ,80, and in vitro ).bldKc, bldM, bldN, wblA, wblC and crp. Of these, bldKc, bldN and wblC were identified as direct targets by ChIP-on-chip analysis, forming Class II targets as none of them is preceded by a dre element. Of these, binding to bldN was only seen for one experiment, and care has therefore to be taken as to whether this reflects true binding. Interestingly, however, the bldN gene product σbldN is required for transcription of the chp, rdl and ramS genes [bldN transcription in dasR null mutants apparently connects to the observed increase in chp, rdl and ramS expression during sporulation.Our data indicate that DasR exerts developmental control by targeting the expression of a number of key regulatory genes for the control of early developmental processes, including mS genes , and thenagE2 and actII-ORF4. Their gene products form core members in the signalling cascade from GlcNAc accumulation outside the cell to the activation of actinorhodin production, with NagE2 importing the inducer and ActII-ORF4 as terminal activator. In contrast, there was surprisingly little overlap between the highly pleiotropic regulatory networks of BldD and DasR. Regulators of later sporulation events do not feature among the direct DasR targets, while, conversely, the primary BldD regulon contains a plethora of such developmental regulatory genes [bldD may be a Class II target for DasR, with binding upstream of bldD verified independently in samples obtained after 54 h of growth on MM agar plates as well as from liquid-grown R5 cultures. Transcription of bldD was not changed in the dasR deletion mutant on MM mannitol, but under these conditions both the bldD and the dasR mutants sporulate. Whether and if so when DasR controls bldD transcription awaits further analysis.In terms of cross-talk with other developmental regulators, AtrA and DasR apparently counteract each other, with activating and repressing activity, respectively, on ry genes , suggestin vivo in response to the extracellular signalling nutrient GlcNAc , which has hitherto not been identified. Alternatively, these sites may require conformational changes in the DNA, or even a combination of the two. DasR mediates a higher order level of control, and relief of, or induction of, binding to its target sites does not necessarily result in an immediate change in gene expression. For that, the environmental conditions are an important determining factor—such as the presence of chitin for induction of chi gene expression—and further experimental data need to be obtained from genome-wide studies under different growth conditions. For example, in approaches to activate poorly expressed (or even dormant) antibiotic biosynthetic genes, relief of DasR-binding may well be an important step, but better understanding of the triggers that must be in place to effectively activate their biosynthetic gene clusters should aid their discovery and exploitation.In conclusion, our work provides detailed insights into how DasR links nutritional control to antibiotic production and development and shows how its affinity for different binding sites changes t GlcNAc . The canE. coli JM109 [S. coelicolor M145 was obtained from the John Innes Centre strain collection and is the parent for the dasR null mutant GAM29, which had its entire coding region replaced by the apramycin resistance cassette as described previously [Streptomyces techniques are described in the Streptomyces manual [E. coli were grown in Luria–Bertani broth (LB) at 37°C. SFM (mannitol soya flour) agar plates were used to prepare spore suspensions. Strains were cultivated on MM agar plates with mannitol (1% w/v) (covered with cellophane discs) or in liquid R5 or NMMP supplemented with mannitol (1% w/v). For induction experiments, liquid cultures were grown at 30°C until mid-logarithmic phase (OD600 approximately 0.4 for NMMP cultures and 0.8 for R5 cultures), followed by the addition of GlcNAc (50 mM). For the ChIP-on-chip studies on MM mannitol solid medium mycelium was harvested after 24 h and 54 h.li JM109  and ET12li JM109  were useeviously . All meds manual . R5 . The DNA fragment contained the dasR gene along with 306 bp promoter region, with the stop codon of the gene replaced by the sequence 5’-ATGGACTACAAGGACCACGACGGCGACTACAAGGACCACGACATCGACTACAAGGACGACGACGACAAGTAGACCAGAGCCCGCTCACCCGGCCCCAGATTGCGGTTGAAGTCC-3’ (stop codon underlined). In this way, the DasR protein was extended with the amino acid sequence MDYKDHDGDYKDHDIDYKDDDDK. The fragment was cloned into the integrative vector pSET152 [For the ChIP-on-chip studies  pSET152 . This re6-tagged DasR was overexpressed in E. coli BL21 (DE3) as described previously [N-terminally Hiseviously . Proteineviously  and peakeviously .S. coelicolor M145 containing the integrative vector pGAM29 expressing 3xFLAG-tagged DasR and the dasR null mutant GAM29 were grown in duplicate . Cultures were treated with formaldehyde  in order to crosslink proteins to DNA. Subsequently glycine  was added to quench the formaldehyde and cultures were incubated for further 5 minutes at 30°C. Mycelium was harvested by centrifugation, washed twice with 1xPBS and stored at -70°C. Chromatin isolation, labeling and hybridization were performed as described previously [Streptomyces arrays manufactured by Agilent Technologies [eviously ,86. Biolnologies .Total RNA was purified using the Kirby-mix protocol . DNaseI S. coelicolor M145 and dasR mutant (GAM29) were cultivated on MM mannitol agar plates overlaid with cellophane discs. Samples were taken at 24 h, 30 h, 36 h, 42 h and 54 h. Two biological replicates were analysed for all samples and for each strain. cDNA incorporating Cy3 dCTP was synthesized as described in http://www.surrey.ac.uk/fhms/microarrays/Downloads/Protocols/Strep_hyb_protocol_1005.pdf, from 10 μg of total RNA extracted from each time point. Labeled cDNA was co-hybridized with genomic DNA, used as a common reference, labeled with Cy5-dCTP as described in http://www.surrey.ac.uk/fhms/microarrays/Downloads/Protocols/Strep_hyb_protocol_1005.pdf. The fluorescently labeled samples were hybridized onto whole genome custom Streptomyces microarrays 4 x 44K as described in ref.35. For the ChIP-chip experiment, chromatin samples were either immuno-precipitated using a monoclonal anti-FLAG tag M2 antibody (Sigma cat. Number F1804)(sample) or incubated in absence of antibody (mock IP control). When mycelium from solid medium was used in the ChIP-chip experiment, the mycelium was harvested quickly with a spatula and immersed in 1% formaldeyde solution for 20 min at 30°C before adding glycine to 0.5 M final concentration to quench the formaldeyde. In the case of liquid cultures, the cells were treated with formaldeyde by adding the reagent directly to the culture at a final concentration of 1% and continuing the incubation for 20 min before adding the glycine as quencher. After the formaldeyde treatment the protocol followed was as described in [For the time-course gene expression studies ribed in .dasR mutant. ChIP-on-chip data, allowing identification of genes/regions directly bound by DasR were analysed essentially as described previously [2 (WT-DasR antibody/mock-antibody) and log2 (Mutant-DasR antibody/mock-antibody) ratios for each time-point (using the R package ‘multtest’). A probe was scored as significant if the associated t-statistic was greater than 2 which, given the number of replicates in this study, corresponds to a false discovery rate of less than 10%; this was considered to be a suitable initial threshold given our application of additional subsequent thresholds and manual inspection. Additional thresholds applied to highlight putative DasR binding events for manual inspection were: (1) a log2 (WT-DasR antibody/mock-antibody)—log2 (Mutant-DasR antibody/mock-antibody) enrichment ratio of >0.3, where 0.3 is the point at which the distribution of these WT vs Mutant ratios depart from the typical Gaussian curve; (2) the presence of two significantly enriched DasR-ChIP probes within a 3.5 kb window. These DasR ChIP-enrichment regions containing putative DasR binding sites were then manually inspected to identify associated genes. Here, clear peaks within the 3.5 kb region were located by visual inspection of profile data using a genome browser.Expression array data were normalized using the Limma package . Brieflyeviously ; the arrS1 Fig(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 Fig(PDF)Click here for additional data file.S4 Fig(PDF)Click here for additional data file.S5 Fig(PDF)Click here for additional data file.S1 TableExpression, trend in microarray analysis.(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.


The seemingly simple, constitutive Hedgehog response of patched/Ptch1 is driven by a complex regulatory architecture, with batteries of context-specific enhancers engaged in promoter-specific interactions to tune signaling individually in each tissue, without disturbing patterning elsewhere. This structure—one of the oldest cis-regulatory features discovered in animal genomes—explains how patched/Ptch1 can drive dramatic adaptations in animal morphology while maintaining its essential core function. It may also suggest a general model for the evolutionary flexibility of conserved regulators and pathways.The Hedgehog signaling pathway is part of the ancient developmental-evolutionary animal toolkit. Frequently co-opted to pattern new structures, the pathway is conserved among eumetazoans yet flexible and pleiotropic in its effects. The Hedgehog receptor, Patched, is transcriptionally activated by Hedgehog, providing essential negative feedback in all tissues. Our locus-wide dissections of the DOI:http://dx.doi.org/10.7554/eLife.13550.001 Hh signignaling . Althougorganism —the basiorganism , so far patched gene, a universal transcriptional target of Hh/Gli signaling in Drosophila, and its mouse ortholog Ptch1, which has an equally universal response to signaling. The Hh receptor Patched (Ptc) normally inhibits the function of Smoothened, blocking signal transduction and favoring the production of the repressor isoform of the Ci/Gli transcription factor (GliR). This inhibition is relieved upon binding of Hh ligand to Ptc, allowing the activator isoform (GliA) to accumulate. Ptc is expressed broadly, keeping the Hh pathway silent in the absence of ligand, but it is also directly transcriptionally activated by Gli in all Hh-regulated tissues, where it moderates signal levels  can repress enhancer activity in embryos, even in Hh-responding cells  that optimal Gli motifs can either activate or repress transcription in the same cells, depending on their cis-context, or (2) that the proxptc element is not a universally Hh/Gli-responsive enhancer. To distinguish between these possibilities, we examined the prox ptcenhancer in the embryonic ectoderm, and found that it fails to respond to Hh/Gli in this context  and the first intron into regions 1A through 1I, and then naming each sequence fragment after the 5’- and 3’-most regions included in that fragment   and poorly conserved and the 3’ neighboring gene is located less than 700 bp downstream of ptc.For this study, we created a nomenclature of sequence fragments by dividing the 5’ intergenic sequences into regions A through Z (with A containing the fragment : for exament BA) . We focupatched expression may be controlled by this region—but it is not able, on its own, to drive Hh-responsive patterns in any tissue  driving GFP. When paired with the hspmin promoter, three ptc enhancers drive ptc-like responses in the embryonic ectoderm , the Drosophila Gli, using an in vitro binding dataset  which co in vivo , demonst in vivo —are requ in vivo .wingless and stripe genes causes a decrease in enhancer activation in vivo , with its cluster of three well-conserved optimal sites, is a weaker wing enhancer than several other regions of the ptc locus with fewer and/or lower-affinity Gli motifs  were tested for Gli responsiveness in Gli1-expressing NIH/3T3 cells as luciferase reporters, we found that their levels of activation were not well correlated with either their expression levels in wing discs or the number or quality of Gli sites . Howeverpatched wing enhancer proxptc/DB, with its cluster of optimal Gli motifs, drives relatively weak expression in vivo (though it is strongly responsive to Hh/Gli in vitro). This result led us to hypothesize that the high-affinity Gli sites in the DB module may require additional flanking sequence in order to exert a strong influence on gene expression in vivo. We first addressed this by testing a nested set of fragments from the -5.5 kb region upstream of ptc, all containing the 3 proximal optimal GBS (top). All of the fragments  drove ptc-like stripes in the wing, but while the anterior-posterior positions of these stripes were the same, the levels and dorsal-ventral extents of gene expression increased greatly with the addition of 5’ flanking sequence  contribution to the Hh/Gli response of ptc locus, we found evidence of higher-level control of gene expression via specialized enhancer-promoter interactions. Some ptc enhancers drove qualitative and quantitatively different in vivo expression patterns when placed in cis to a homologous vs. a heterologous promoter  factors Pleiohomeotic (Pho), Polyhomeotic (Ph), Polycomb (Pc), and Posterior sex combs (Psc), as well as the Trithorax group (TrxG) factor Trl/GAF  and low H3K27me3 (ptc promoter using the CIS-BP database (ptc core promoter region lacks a TATA box but contains a Drosophila Initiator element (Inr) and multiple GAF-binding GAGA motifs, as well as a pause button (PB) and a Downstream Promoter Element (DPE) 3’ of the transcription start site  (left). However, when paired with the native ptc promoter fragment BA—which alone is broadly and weakly active (like the ptc gene itself) but unresponsive to Hh/Gli (top)—these enhancers drive stronger and more complete, ptc-like expression patterns in embryos (right). Neither the core ptc promoter, fragment A, nor the upstream element, fragment B, were sufficient to enhance the Hh/Gli response of module PN  , left. Ho Hh/Gli , top—the embryos , right. odule PN , suggestptc enhancer function in larval imaginal discs—where, in contrast to the embryo, we found strong PcG binding and the Polycomb-produced repressive mark H3K27me3 at and upstream of the promoter , as well as a complex multi-enhancer organization in which the universal response to Hedgehog signaling is not constituted in a master Gli response element, but distributed across many sub-tissue-specific Gli-regulated modules  and a far more remote 5’ boundary .ptc enhancers were amplified by PCR (Roche Expand High Fidelity PCR System) from BAC DNA (CH322-170A-12 or CH322-188E13). PCR primers are provided in D. melanogaster Hsp70 gene, were subsequently cloned into the pHPdesteGFP transgenesis vector via LR Cloning (Life Technologies). Enhancers tested with the endogenous ptc promoter were cloned by traditional methods into the pStinger transgenesis vector . AlexaFluor488, AlexaFluor555, and AlexaFluo568 conjugates with secondary antibodies from Invitrogen were used at 1:2000 dilutions. DAPI was included in the Prolong Gold antifade mountant (Life technologies). Confocal images were captured on an Olympus FluoView 500 Laser Scanning Confocal Microscope mounted on an Olympus IX-71 inverted microscope, and on a Nikon A1 confocal microscope. Samples to be directly compared were fixed, prepared, and imaged under identical confocal microscopy conditions and settings. Quantitative GFP expression data from imaginal discs were collected from confocal images: all GFP-expressing fly stocks were crossed to the same reference line, dppD-Ciptc, which drives DsRed expression in wing discs, as an internal normalization reference  containing 10% bovine calf serum  and penicillin/streptomycin/glutamine . Luciferase assays were performed by plating 2.5 x 104 cells/well in 24 well plates. The next day, cells were co-transfected using Lipofectamine2000 with the DNA constructs indicated in each experiment in addition to ptc∆136-GL3  and pSV-Beta-galactosidase (Promega) constructs to report Hh pathway activation and normalize transfections, respectively. Gli1 cDNA was added where relevant to activate the Hh pathway. Cells were changed to low-serum media  48 hr after transfection and cultured at 37°C in 5%CO2 for an additional 48 hr. Cells were harvested and luciferase and beta-galactosidase activities were measured using Luciferase Assay System  and BetaFluor β-gal assay kit . Multiple assays were performed and each treatment group was assayed in triplicate. Two-tailed t tests for two samples with unequal variances were used to compare samples.NIH/3T3 cells were cultured at 37°C, 5% COIcarus   . Each exElectromobility shift competition assays were performed as previously described . EMSA olhh) transgenic flies . All ChIP-seq experiments were performed with 2 biological replicates.The protocol for carrying out ChIP in larval tissue has been described previously , with mihttp://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=yrifkssspfuzbsp&acc=GSE76892.ChIP-seq data can be accessed at this private link for reviewers: Ptch1 corresponds to ) have been deposited under accession number GSE71199. Gli3 ChIP-seq data correspond to GSE52939. Individual enhancer regions were defined by the conservation block encompassing the detected Gli binding region, amplified by PCR and cloned into a Gateway compatible version of the reporter construct previously described (E10.5 embryos were obtained from timed matings of Swiss Webster (Taconic) mice and micro-dissected in cold PBS to isolate the head, trunk  and limb buds. Each tissue was pooled separately and fixed for 30 min in 1% formaldehyde/PBS at room temp. Chromatin immunoprecipitation and embryoid body differentiation were performed as previously described . Antibodescribed . Mouse e In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.cis-regulatory architecture allows localized Hedgehog tuning by patched/Ptch1" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by Janet Rossant as the Senior Editor and Reviewing Editor.Thank you for submitting your article "An ancient yet flexible The reviewers are all very enthusiastic about the paper and its insights into gene regulatory architecture and want to see it published without delay. One reviewer suggested some additional transgenic experiments which could add to the paper. However, the consensus was that these were not necessary for publication, unless you happen to have the data in hand. Thus we ask that you respond to the minor comments below. We have included the comment on additional experiments for your information and comment only.Summary:Patched and Ptch1 gene regulation in flies and mice, respectively. Patched is a core component of the important hedgehog signaling pathway. It inhibits the action of Smoothened, which is required for the synthesis of activator forms of the transcriptional effectors Ci and Gli. This repression is relieved by Hedgehog-Patched interactions, leading to the induction of a variety of target genes, including Patched itself.Lorberbaum et al. present a detailed analysis of Past studies led to the view that Patched is regulated by simple upstream activator elements consisting of a linked Gli/Ci binding sites. Here, the authors elegantly demonstrate that this is not the actual mechanism of Patched regulation. Instead, the locus contains a series of upstream and downstream enhancers that mediate restricted induction in specific tissues at different times and in response to a variety of different signals, including Hedgehog.This is an excellent paper. The scope of the analysis, which includes both flies and mice, is unusually broad. The findings are striking and convincing, and the analysis is thoughtful and penetrating. The paper was a pleasure to read. This type of work lays the foundation for future studies into the basis for synergy between enhancers, the relationship between binding site affinity and tissue specificity and expression level, and regulatory evolution at the locus level.Suggested new experiments (not necessary for publication):Ptch1, it would be important to assay if equivalent or similar constructs containing the Ptch1 proximal and core promoter are also able to drive low/broad expression of the reporter in mouse transgenic embryos (by lacZ staining or qPCR). The analysis of the Ptch1 locus shown in 1) A key aspect of this article is the identification and characterization of the ptc proximal promoter (region BA) that drives tonic low levels of expression in a broad manner. This is particularly relevant because such Hh-independent expression is required to prevent ectopic pathway activation in the absence of the ligand. As this is also the case for 2) The authors should compare what is the impact of the endogenous promoter on the expression driven by the DB element . This data could be added to Minor points (need to be addressed):1) In relation with 2) It is not always clear how the different mutated constructs were made. A table with all the mutations introduced  should be provided. For example, in In addition, the sequences of the main regions tested should be provided as supplementary data and the GBS color-annotated according to predicted affinity.3) If possible, colors should be consistent, in particular for enhancers that are described in several figures. For example, ZY is green in 4) What is exactly the Ptc-prox construct shown in patched” it should read LK instead of VT. The legend for 5) Given that the nomenclature is complicated, the text should be carefully proofed for mistakes. For example, in the second to last sentence of the subsection “Bypassing enhancer-promoter specificity reveals diverse sub-patterns among segmentation stripe enhancers of 6) Introduction, first paragraph. It took me a few reads through these sentences to understand the contrast of patterned vs. constitutive targets. Maybe there is a way to clarify this further? Perhaps simply state that patterned targets respond in a subset of cells with an active Hh pathway, whereas universal targets respond in all Hh active cells.A priori, there are two extreme possibilities (which is explained more clearly later in the paper). The universal pattern is either encoded by a bunch of separate enhancers, one or more for each tissue, or the universal pattern is encoded by a single regulatory element that is expressed in multiple contexts. The results of the paper demonstrate the first scenario, but it might be nice to set up these two possibilities at the outset to give some context.7) Introduction, second paragraph. This set up was a little hard for me to follow on the first read, as it contains a lot of specific results. I would suggest a new paragraph here  enhancer sufficient to drive the universal expression pattern. Then, you hypothesize no, based on previous results where placing high affinity Gli sites into embryonic ectoderm enhancers isn't sufficient to turn them on. You then test this hypothesis by examining expression of ptc(prox) in the embryonic ectoderm.8) Results, first paragraph. I suggest "In this study, we define a nomenclature[…] “rather than "we determined the nomenclature", which could be confused for uncovering an existing nomenclature.9) 10) Drosophila enhancers, which is confusing. The heatmap representation conveys more information, so I suggest changing this figure to that convention in both panels.11)  Suggested new experiments (not necessary for publication):1) A key aspect of this article is the identification and characterization of the ptc proximal promoter (region BA) that drives tonic low levels of expression in a broad manner. This is particularly relevant because such Hh-independent expression is required to prevent ectopic pathway activation in the absence of the ligand. As this is also the case for Ptch1, it would be important to assay if equivalent or similar constructs containing the Ptch1 proximal and core promoter are also able to drive low/broad expression of the reporter in mouse transgenic embryos (by lacZ staining or qPCR). The analysis of the Ptch1 locus shown in 2) The authors should compare what is the impact of the endogenous promoter on the expression driven by the DB element . This data could be added to ptc promoter, but “the consensus was that these were not necessary for publication, unless you happen to have the data in hand. We have included the comment on additional experiments for your information and comment only.” Although we strongly agree that these experiments are worth performing and fully intend to do so, it’s our opinion that this analysis would be better suited to a separate follow-up report in which we can more fully characterize this promoter’s function, its interactions with a panel of enhancer elements, its chromatin states in vivo, and its regulation by PcG/TrxG.One reviewer suggested some additional transgenic experiments concerning the special properties of the Minor points (need to be addressed):1) In relation with This has been done. See revised 2) It is not always clear how the different mutated constructs were made. A table with all the mutations introduced  should be provided. For example, in In addition, the sequences of the main regions tested should be provided as supplementary data and the GBS color-annotated according to predicted affinity.This has been done. We created a new Supplementary file  that con3) If possible, colors should be consistent, in particular for enhancers that are described in several figures. For example, ZY is green in This has been done. See revisions to all relevant figures and figure supplements  to match4) What is exactly the Ptc-prox construct shown in We added a sentence in the legend for 5) Given that the nomenclature is complicated, the text should be carefully proofed for mistakes. For example, in the second to last sentence of the subsection “Bypassing enhancer-promoter specificity reveals diverse sub-patterns among segmentation stripe enhancers of patched” it should read LK instead of VT. The legend for This typo has been corrected, and the manuscript has been carefully proofread. Forexample, we identified a mis-labeling of an oligonucleotide in the original Figure 2—figure supplement 2, which has been corrected (those sequences are now in part B of 6) Introduction, first paragraph. It took me a few reads through these sentences to understand the contrast of patterned vs. constitutive targets. Maybe there is a way to clarify this further? Perhaps simply state that patterned targets respond in a subset of cells with an active Hh pathway, whereas universal targets respond in all Hh active cells.A priori, there are two extreme possibilities (which is explained more clearly later in the paper). The universal pattern is either encoded by a bunch of separate enhancers, one or more for each tissue, or the universal pattern is encoded by a single regulatory element that is expressed in multiple contexts. The results of the paper demonstrate the first scenario, but it might be nice to set up these two possibilities at the outset to give some context.We have revised the text in this section to try to clarify this point .7) Introduction, second paragraph. This set up was a little hard for me to follow on the first read, as it contains a lot of specific results. I would suggest a new paragraph here  enhancer sufficient to drive the universal expression pattern. Then, you hypothesize no, based on previous results where placing high affinity Gli sites into embryonic ectoderm enhancers isn't sufficient to turn them on. You then test this hypothesis by examining expression of ptc(prox) in the embryonic ectoderm.We have revised the text in this section , includingreorganizing the argument along the lines suggested by the reviewer.8) Results, first paragraph. I suggest "In this study, we define a nomenclature[…] “rather than "we determined the nomenclature", which could be confused for uncovering an existing nomenclature.We have revised the text in this section . We chose to use“we created a nomenclature[…]”, which we think accomplishes the reviewer’s goal.9) We added the line “GFP signals are isolated  in the lower half of K'-N' to showthe lack of stripe enhancer activity in the posterior (En-positive) compartment.” to the legend of 10) We have spent a lot of time working on the best way to visuallyindicate binding motif affinity. In earlier drafts of our figures, enhancer diagrams had colored backgrounds, exactly as the reviewer suggests. However, because the indicated motifs range from dark to light, most background tones mask the medium-gray sites, which are the ones most relevant to this report . Also, this effect depends on the background color used, with the result that the same site looks different in different enhancers. The advantage of the white background is that, by the visual logic of the greyscale heatmap, white represents the lowest, or background, affinity. Thus Gli motifs fade into the white background as their predicted affinity decreases.11) We have significantly revised


The integrin β6 (ITGB6) gene, which encodes the limiting subunit of the integrin αvβ6 heterodimer, plays an important role in wound healing and carcinogenesis. The mechanism underlying ITGB6 regulation, including the identification of DNA elements and cognate transcription factors responsible for basic transcription of human ITGB6 gene, remains unknown. This report describes the cloning and characterization of the human ITGB6 promoter. Using 5′-RACE (rapid amplification of cDNA ends) analysis, the transcriptional initiation site was identified. Promoter deletion analysis identified and functionally validated a TATA box located in the region −24 to −18 base pairs upstream of the ITGB6 promoter. The regulatory elements for transcription of the ITGB6 gene were predominantly located −289 to −150 from the ITGB6 promoter and contained putative binding sites for transcription factors such as STAT3 and C/EBPα. Using chromatin immunoprecipitation assays, this study has demonstrated, for the first time, that transcription factors STAT3 and C/EBPα are involved in the positive regulation of ITGB6 transcription in oral squamous cell carcinoma cells. These findings have important implications for unraveling the mechanism of abnormal ITGB6 activation in tissue remodeling and tumorigenesis. About 10 μl of the diluted DNA fraction was kept aside as template for PCR. The remaining DNA fraction was precleared using 25 μl of protein G magnetic beads for 2 hr at 4°C. Immunoprecipitation was performed by adding 2 μg of the antibody . After appropriate washing, the antibody-transcription factor-DNA complex was eluted from the beads, formaldehyde cross-links were reversed, and proteins were digested with proteinase K at 67°C for 2 hr. DNA was purified and used for PCR using the following primers for TBP  and C/EBPα and STAT3  experiments.In order to analyze the escribed . About 1Proteins were isolated with RIPA buffer  containing a cocktail of protease inhibitors (Sigma-Aldrich), separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane. The membrane was blocked by non-fat milk followed by incubation with anti-TBP (GeneTex), anti-β-actin , anti-GAPDH , anti-STAT3, or anti-C/EBPα antibody at 4°C overnight. After incubating with a peroxidase-conjugated goat anti-rabbit or anti-mouse IgG, protein bands were visualized by Western Bright ECL .-ΔΔCt method) resulted in a significant reduction in the promoter activity when compared with the wild type construct (pGL2-B6[−150/+21]) in both 293T and TCA8113 cells .in vivo and is required for human ITGB6 promoter transcriptional activity.TATA-binding protein (TBP) is the main DNA-binding protein that recognizes and binds a TATA box in TATA-containing gene promoters,. To detTo further investigate the role of TBP in the regulation of human ITGB6 mRNA expression, we knocked down TBP in SAS cells that express high levels of ITGB6 mRNA. RT-qPCR analysis demonstrated that the relative mRNA of TBP and ITGB6 was reduced by 73.3% and 27.3%, respectively .CCCGAT-3′, Antisense 5′-bio-ATCGGGAGTGAAATATTG-3′) did not form a complex with nuclear extract protein , namely, pGL2-B6-M-STAT3(1), pGL2-B6-M-STAT3(2), and pGL2-B6-M-STAT3(3) . After t protein . These rin vitro analysis were functional in the native chromatin, ChIP was performed on chromatin extracted from TCA8113 cells by using anti-STAT3 antibody. PCR targeting the −289 to −150 bp region showed that STAT3 immunoprecipitates were enriched for the ITGB6 promoter DNA compared to the non-specific IgG  did not form a complex with nuclear extract protein  was generated . After t protein . These rin vivo and C/EBPα is required for human ITGB6 promoter transcriptional activity.C/EBPα has been previously reported to be associated with integrin CD11c promoter activation , and theTo further determine the role of C/EBPα on the regulation of ITGB6 mRNA expression, the effect of C/EBPα knockdown on ITGB6 mRNA level in SAS cells was investigated. The RT-qPCR result showed that the mRNA expression of C/EBPα was reduced by 74.4% and ITGB6 mRNA expression was reduced by 32.9% . These rITGB6 is a critical subunit of the αvβ6 integrin heterodimer and is involved in regulating the expression of the heterodimer during cancer and wound healing. To understand the transcriptional regulation of the ITGB6 gene in cancer cells, we identified and characterized the human ITGB6 promoter and its activity. Our results revealed, for the first time, the presence of functional TATA box located from −24 to −18 bp upstream of the ITGB6 promoter. Furthermore, this study has identified that the transcription factors STAT3 and C/EBPα are both involved in the positive regulation of ITGB6 basic transcription in OSCC cells.Identification of the TSS has been previously described as the first step in defining the promoter and studying its regulatory mechanisms . Using 5To begin to functionally characterize the sequence upstream of the TSS, a series of 5′-deletions of the ITGB6 promoter were generated. We found that the region between −150 and −3 is minimally required for basal promoter activity. Bioinformatic analyses identified a potential TATA box located −24 to −18 upstream of the TSS. As the core promoter element, the TATA box is the binding site of either general transcription factors or histones, and is involved in transcription by RNA polymerase . Our resA region further upstream of the TSS, −289 and −150 bp, was identified as the minimal sequence that holds the most influence over promoter activity. A number of putative binding sites for transcription factors were identified in this region, including STAT3, C/EBPα, AP-1 and c-Myb, however, empirical data from the mutation analysis of the binding sites suggests that only STAT3 and C/EBPα function as the positive regulators of ITGB6 basal transcriptional activity Figs. . These rThe transcription factor STAT3 is not only persistently activated in many types of cancer, including oral cancer ,28, but The transcription factor C/EBPα belongs to the CCAAT enhancer-binding protein (C/EBPs) family of basic leucine zipper transcription factors that recognize the CCAAT motif of their target gene promoter ,30. As tOur findings do not preclude other transcription factor-binding sites that are located beyond the −289/−150 region characterized that may also be involved in ITGB6 regulation. Indeed, putative binding sequences for transcription factors such as Ets-1 and Smad were identified within the region between −146 and +1, which have been shown to robustly induce ITGB6 promoter activity ,13. HoweIn conclusion, we have characterized the promoter region of the human ITGB6 gene. Our results demonstrated, for the first time, that ITGB6 promoter contains a functional TATA box and that the transcriptional factors STAT3 and C/EBPα are involved in the positive regulation of ITGB6 transcription. These findings offer new insights toward understanding the temporal and disease-specific regulation of the human ITGB6 gene in OSCC cells.S1 FigLuciferase activity expressed by TCA8113 and 293T cells after transfection with the potential AP1-binding site-directed mutant pGL2-B6-M-AP-1(A), the potential c-Myb-binding site-directed mutant pGL2-B6-M-cMyb(B), and wild-type construct pGL2-B6(−289/−150) 48 hr post transfection.(DOC)Click here for additional data file.S1 File(RAR)Click here for additional data file.


PEPCK1 mRNA was associated with DNA hypermethylation and more enriched repression histone mark H3K27me3, while the up-regulated PEPCK2 and FBP1 mRNA was associated with DNA hypomethylation and more enriched activation histone mark H3K4me3. Furthermore, the expression of two miRNAs predicted to target PC and 6 miRNAs predicted to target PEPCK1 was dramatically suppressed in the liver of piglets born to betaine-supplemented sows. Our results provide the first evidence that maternal betaine supplementation affects hepatic gluconeogenic genes expression in newborn piglets through enhanced hepatic methionine metabolism and epigenetic regulations, which involve DNA and histone methylations, and possibly miRNAs-mediated post-transcriptional mechanism.In this study, gestational sows were fed control or betaine-supplemented diets (3 g/kg) throughout the pregnancy, and the newborn piglets were used to elucidate whether maternal dietary betaine affected offspring hepatic gluconeogenic genes through epigenetic mechanisms. Neonatal piglets born to betaine-supplemented sows had significantly higher serum and hepatic betaine contents, together with significantly greater expression of methionine metabolic enzymes in the liver. Interestingly, significantly higher serum concentrations of lactic acid and glucogenic amino acids, including serine, glutamate, methionine and histidine, were detected in the piglets born to betaine-supplemented sows, which were coincident with higher hepatic glycogen content and PEPCK1 enzyme activity, as well as greater protein expression of gluconeogenic enzymes, pyruvate carboxylase (PC), cytoplasmic phosphoenolpyruvate carboxykinase (PEPCK1), mitochondrional phosphoenolpyruvate carboxykinase (PEPCK2) and fructose-1, 6-bisphosphatase (FBP1). Moreover, maternal betaine significantly changed the methylation status of both CpGs and histones on the promoter of gluconeogenic genes. The lower  Gluconeogenesis is an important metabolic pathway for endogenous glucose generation from substrates such as lactic acid and glucogenic amino acids The rate of gluconeogenesis is controlled by key enzymes including pyruvate carboxylase (PC), phosphoenolpyruvate carboxykinase (PEPCK1) , fructose-1, 6-bisphosphatase (FBP1), and glucose 6-phosphatase (G6PC) Betaine functions as a methyl donor to convert homocysteine to methionine in a reaction catalyzed by betaine homocysteine methyltransferase (BHMT) Betaine is derived from either choline oxidation or dietary intake, and is critical for embryonic and fetal development Therefore, here we use pigs as model to investigate whether feeding gestational sows with betaine-supplemented diet may affect hepatic gluconeogenesis in newborn piglets. To explore the possible epigenetic mechanisms underlying such effects, we detected hepatic expression of genes involved in gluconeogenesis and methionine metabolism, as well as the status of DNA methylation and histone modifications on the promoter of gluconeogenic genes, together with the expression of microRNAs which are predicted to target gluconeogenic genes in the liver of neonatal piglets.Landrace×Yorkshire crossbred sows in the second parity were artificially inseminated, at the observation of estrus, with a mixture of Duroc semen samples obtained from two littermate boars. One week after the artificial insemination, sows were randomly divided into control and betaine groups (8 per group). Sows in control group received basal diet while those in betaine group were fed betaine-supplemented (3 g/kg) diet throughout the pregnancy. Betaine was in the form of betaine hydrochlorides with 98% purity, purchased from SKYSTONE FEED CO., LTD . The diet composition is shown in The experimental protocol was approved by the Animal Ethics Committee of Nanjing Agricultural University, with the project number 2012CB124703. The slaughter and sampling procedures complied with the “Guidelines on Ethical Treatment of Experimental Animals” (2006) No. 398 set by the Ministry of Science and Technology, China.Frozen serum and liver samples of the newborn piglets were shipped to the China National Feed Quality Control Center, Chinese Academy of Agricultural Sciences, Beijing, China and were prepared for the determination of betaine as described previously Serum concentrations of biochemical metabolites, including glucose and lactic acid, were detected with the enzymatic colorimetric methods using commercial kits for glucose  and lactic acid . Serum concentrations of insulin and glucagon were measured with respective commercial RIA kits  with assay sensitivities of 0.29 pmol/L and 16.1 ng/L, respectively. The intra- and inter-assay variations were 10 and 15%, respectively, for both assays.Serum samples for measuring the free amino acids concentrations were prepared according to a previous publication The hepatic glycogen content was determined as previously described Total RNA was isolated from liver samples using TRIzol Reagent  according to the manufacturer's instruction and reverse transcribed with the PrimeScript 1st Strand cDNA Synthesis kit . Two microliters of diluted cDNA (1∶25) were used in each real-time PCR assay with Mx3000P (Stratagene). It is a routine practice to confirm the absence of genomic DNA contamination in samples used for Real Time PCR analysis. Firstly, RNA samples were treated with DNase before reverse transcription to cDNA; secondly, DNase-treated RNA samples were directly used as template for QPCR to ensure that there was no specific amplification; thirdly, primers were designed to span an intron so any genomic DNA contamination can be easily reported by an extra PCR product in melting curve analysis. Peptidylprolyl isomerase A (PPIA) was chosen as a reference gene, because it is expressed in similar abundance to the genes of interest and its expression was not affected by the experimental factor. All primers were synthesized by Generay Biotech and listed in Liver samples were homogenized in RIPA buffer . Protein concentrations were determined with a Pierce BCA Protein Assay kit . Western-blot analysis for target proteins was carried out according to the protocols provided by the primary antibody suppliers. The sources of the primary antibodies used in Western blotting are listed in Hepatic PEPCK1 enzyme activity was detected as previously described with minor modifications FBP1 promoter absent of CpG sites. MeDIP results were calculated relative to the negative control and presented as the fold change relative to the average value of control group. The specific and negative control primers were designed with Primer 5 software and shown in High-quality genomic DNA isolated from liver was sonicated to produce small fragments ranging from 300 to 1000 bp. Two micrograms of fragmented DNA were heat denatured to produce single-stranded DNA. A mouse monoclonal antibody against 5-methyl cytidine  was used to immunoprecipitate the methylated DNA fragments. The immune complexes were captured with protein G agarose beads . The beads bound to immune complexes were washed to eliminate nonspecific binding and resuspended in 250 µL digestion buffer containing proteinase K. Finally, the MeDIP DNA was purified. A small aliquot of MeDIP DNA was used to amplify the proximal promoter sequences of the target genes by real-time PCR. A pair of negative control primers is used to amplify a fragment of ChIP analysis was performed according to our previous publication U6 small nuclear RNA (U6 snRNA) was used as a reference gene to normalize the expression of miRNAs.Two micrograms of total RNA treated with RNase-free DNase I (Promega) was polyadenylated by poly (A) polymerase using Poly (A) Tailing Kit  according to the manufacturer's instruction. Polyadenylated RNA was then dissolved and reverse transcribed using poly (T) adapter. Real-time PCR was performed with SYBR green qPCR master mix reagent  in triplicates with a miRNA specific forward primer and a universal reverse primer complementary to part of the poly (T) adapter sequence. PC gene has not been reported, we aligned the 3′ flanking sequence of this gene with the 3'UTR sequence of human PC gene to obtain the consensus sequence for miRNA prediction using an online miRNAs prediction tool, PITA algorithm, with the threshold of score set at -10 Because the 3'UTR sequence of porcine −△△Ct method was used to analyze real-time PCR data. The relative quantifications of mRNA, protein, CpG methylation, histone modifications, and miRNA were expressed as the fold change relative to the mean value of control group. All experiments were carried out in triplicate. The differences were considered statistically significant when P<0.05.Data are presented as means ± SEM. When males and females were analyzed separately, control for litter was not necessary because they were all from different litters. Since none of the detected parameters showed sex disparity, we pooled male and female together making n = 16 for each group. Comparisons were made using two-tailed Student's t test for independent data. The 2Maternal betaine supplementation had no effects on litter size or litter weight . Serum cP<0.05) as compared to their control counterparts. Body weight and liver weight did not differ, nor did the serum concentrations of glucose, insulin, glucagon or the ratio of insulin to glucagon. However, serum concentrations of lactate (P<0.05) and glucogenic amino acids (P<0.05), including serine, glutamate, methionine and histidine, were all significantly higher in piglets born to betaine-supplemented sows than that from the control group. Moreover, piglets born to betaine-supplemented sows tended to have higher (P = 0.08) serum arginine concentration (Piglets born to betaine-supplemented sows exhibited significantly higher serum betaine concentration (ntration .P<0.05) in the liver (BHMT and AHCYL1 were significantly up-regulated (P<0.05), while MAT2B tended to be higher (P = 0.06), at the level of mRNA (P<0.05) in the liver of piglets born to betaine-supplemented sows   in the piglets born to betaine-supplemented sows, at both mRNA and protein levels (P<0.05) (G6PC tended to be higher (P = 0.07) at the level of mRNA, but not protein, whereas PC was significantly higher (P<0.05) at the level of protein, but not mRNA, in the liver of piglets born to betaine-supplemented sows. PEPCK1 demonstrated reversed alterations for mRNA that was significantly lower (P<0.05), and protein that was significantly higher (P<0.05), in the liver of piglets born to betaine-supplemented sows (Hepatic glycogen content was significantly higher (P<0.01) , and hepP<0.01) . Accordin levels . PC and (P<0.05) , yet wit(P<0.05) . G6PC teted sows . The uncP<0.05) on the promoter of PEPCK2 and FBP1 genes, which was reversely correlated to the up-regulation of these two genes in mRNA expression. Interestingly, the level of CpG methylation on G6PC promoter tended to be lower (P = 0.07) in the liver of piglets born to betaine-supplemented sows corresponding to the trend of higher G6PC mRNA expression. In contrast, PEPCK1 promoter was significantly hypermethylated (P<0.05), which was in accordance with the diminished PEPCK1 mRNA expression in the liver of piglets born to betaine-supplemented sows , while hepatic activation of PEPCK2, FBP1 and G6PC (P<0.05) on the promoters.The enrichment of two histone modification marks, the activation mark histone H3 lysine 4 trimethylation H3K4me3 and the repression mark histone H3 lysine 27 trimethylation (H3K27me3) on the promoter of gluconeogenic genes was determined with ChIP assay using specific antibodies. The enrichment of histone marks is normalized with that of histone H3. As shown in and G6PC  was accoP<0.05, P = 0. 07, The lysine methyltransferase SETD7 which trimethylates histone H3 lysine 4 (H3K4) was significantly up-regulated P<0.05, , while tPEPCK2 and G6PC gene promoter in betaine-exposed piglet liver, but no difference was detected in PEPCK1 and FBP1  GR binding to and FBP1 .PC and PEPCK1, we further detected hepatic expression of miRNAs predicted to target these 2 genes. Piglets born to betaine-supplemented sows demonstrated a significant down-regulation in the hepatic expression of miRNA-184 (P<0.01) and miRNA-196b (P<0.01), which are predicted to target PC (P<0.01), miRNA-424-3p (P<0.01), miRNA-196b (P<0.01), miRNA-370 (P<0.01), miRNA-30b-3p (P<0.05) and miRNA-92b-5p (P<0.05), which are predicted to target PEPCK1  and hypomethylation (for PEPCK2 and FBP1) were detected on the promoter of gluconeogenic genes in the liver of piglets born to betaine-supplemented sows. In line with our results, specific CpG sites in fatty acid synthase (FASN) gene promoter was found to be hypomethylated in the liver of betaine-supplemented rats DNMT1 gene, which results in the hypermethylation of global DNA Increased supply of methyl donors and enhanced methionine metabolism generally result in global DNA hypermethylation PEPCK1 promoter together with elevated H3K4me3 in the PEPCK2, FBP1 and G6PC promoter in the present study. Moreover, it is noted that the detected CpG islands on gluconeogenic gene promoters are predicted to contain binding sites for GR and the results showed higher GR binding to hepatic PEPCK2 and G6PC gene promoters in betaine-exposed piglet. In addition, previous studies demonstrate that gene promoters of PEPCK and G6PC contain positive GRE sites PEPCK2 and G6PC promoters may increase the binding of GR, thereby up-regulating the transcriptional level of the two genes.SETD7 and EZH2 are SAM-dependent enzymes PC and PEPCK1 genes. PC was unchanged and PEPCK1 was even lower at mRNA levels, but both PC and PEPCK1 were greater at protein level. The dissociation of mRNA abundance and the protein content implies possible involvement of post-transcriptional regulation. MicroRNAs are known to participate predominantly in the post-transcriptional regulation through targeting mRNA degradation and/or translational repression. Previous studies indicate that miRNAs predominantly promote cleavage of mRNAs in plants PC and 6 out of 7 miRNAs targeting PEPCK1 were dramatically suppressed in the liver of piglets born to betaine-supplemented sows, suggesting inhibition of miRNAs-mediated translation repression for these two genes. Although this finding may hint the underlying mechanisms for the mismatched mRNA and protein expression of PC and PEPCK1 genes, the roles of these miRNAs in regulating gluconeogenic genes in porcine liver await further functional verification.Another interesting finding in the present study is the incongruity between the mRNA and protein levels for In conclusion, betaine supplementation in maternal diet during gestation affects hepatic gluconeogenic genes in newborn piglets via epigenetic regulation including DNA methylation, histone modifications and miRNAs, which is associated with enhanced methionine metabolism. Neonatal changes in hepatic gluconeogenic gene expression may cause long-term consequences in glucose homeostasis later in adult life. Long-term follow-up studies are required to understand whether such fetal programming of hepatic gluconeogenic genes caused by maternal betaine supplementation is beneficial or detrimental for adult health.Table S1Composition and nutrient content of the experimental diet.(DOC)Click here for additional data file.Table S2Reproductive performance of sows fed control or betaine supplemented diet as measured by littler size and littler weight.(DOC)Click here for additional data file.Table S3Amino acids concentration in serum of sows.(DOC)Click here for additional data file.Table S4Nucleotide sequences of specific primers.(DOC)Click here for additional data file.Table S5Antibodies for this experiment.(DOC)Click here for additional data file.Table S6miRNA and the corresponding primer sequences.(DOC)Click here for additional data file.


Microsomal epoxide hydrolase (mEH) is a bifunctional protein that plays a central role in the metabolism of numerous xenobiotics as well as mediating the sodium-dependent transport of bile acids into hepatocytes. These compounds are involved in cholesterol homeostasis, lipid digestion, excretion of xenobiotics and the regulation of several nuclear receptors and signaling transduction pathways. Previous studies have demonstrated the critical role of GATA-4, a C/EBPα-NF/Y complex and an HNF-4α/CAR/RXR/PSF complex in the transcriptional regulation of the mEH gene (EPHX1). Studies also identified heterozygous mutations in human EPHX1 that resulted in a 95% decrease in mEH expression levels which was associated with a decrease in bile acid transport and severe hypercholanemia. In the present investigation we demonstrate that EPHX1 transcription is significantly inhibited by two heterozygous mutations observed in the Old Order Amish population that present numerous hypercholanemic subjects in the absence of liver damage suggesting a defect in bile acid transport into the hepatocyte. The identity of the regulatory proteins binding to these sites, established using biotinylated oligonucleotides in conjunction with mass spectrometry was shown to be poly(ADP-ribose)polymerase-1 (PARP-1) bound to the EPHX1 proximal promoter and a linker histone complex, H1.2/Aly, bound to a regulatory intron 1 site. These sites exhibited 71% homology and may represent potential nucleosome positioning domains. The high frequency of the H1.2 site polymorphism in the Amish population results in a potential genetic predisposition to hypercholanemia and in conjunction with our previous studies, further supports the critical role of mEH in mediating bile acid transport into hepatocytes. Microsomal epoxide hydrolase (mEH) is a 48-kDa bifunctional protein that is expressed on the hepatocyte endoplasmic reticulum membrane in two distinct topological orientations  where thIn order to further explore the role of mEH in sodium-dependent hepatocyte bile acid transport we investigated the occurrence of EPHX1 mutations in the Lancaster County Old Order Amish population that exhibit numerous cases of hypercholanemia  in the aPARP-1 is a multifunctional nuclear protein that plays a critical role in numerous nuclear processes including gene regulation utilizing several mechanisms such as a) modulation of chromatin structure by binding to nucleosomes and b) functioning as a transcriptional regulator by binding to DNA through numerous related but non-identical sequences ,24 resulThe results reported in this study demonstrate that EPHX1 transcription is regulated, in part, by PARP-1 and a linker histone H1.2/Aly complex. The similar DNA sequences at their respective binding sites in the proximal promoter and in intron 1 represent possible nucleosome positioning sites. The association between the high frequency of the intron 1 polymorphism resulting in a decrease in EPHX1 expression and the numerous cases of hypercholanemia in the Amish population further suggest that mEH plays a significant role in sodium-dependent bile acid uptake where the decreased expression of mEH is one factor in the etiology of hypercholanemia.The promoter region (-784/-1), nine exons, intron 1, exon-intron boundaries and the 3’ untranslated regions were analyzed in a genomic DNA sample from a hypercholanemic subject (1d)  by seque5’ AAGCTTCCCGGGCTCGAGATCTGAATTC-3’, which contains the restriction enzyme sites HindIII/SmaI/Xho1/BgiII/EcoRI. To differentiate the effect of each mutation, the DNA regions containing each mutation were exchanged between the HindIII(-784)/ScaI(-266) fragment which contained the -733 T>C mutation, the ScaI(-266)/XhoI(-103) fragment which contained the -179 T>C mutation and the XhoI(-103)/EcoR(+25) fragment, which contained the -17 T>C mutation. The core promoter fragments of the wild type and -17 mutant (-80 to +25) were also cloned into pGL3 vector. The -1797/+3458 construct containing the WT and C>G mutation as previously described taurocholate was still observed in the liver and bile compartments after 30 min [-/-) also reported a 2–3 fold increase in mEH expression [In regard to Ntcp, initial studies using leotides  suggestileotides . Studiesr 30 min . In micer 30 min . In studr 30 min ,45. Studpression . Studiespression . There ipression –51. In cpression . This obpression . This prpression  as we prpression .A recent study  reports +-dependent bile acid transport.In conclusion, the studies reported herein have established that PARP-1 and linker histone H1.2 play a critical role in the transcriptional regulation of EPHX1. The DNA sequences in the binding sites of these regulatory factors serve to further elucidate the mechanism by which they may affect chromatin structure and gene expression by regulating nucleosome positioning. The high frequency of the EPHX1 +2557 polymorphism in the Amish population associated with hypercholanemia suggests that a polymorphism in the mEH gene (EPHX1) contributes, in part, to the etiology of this syndrome and in conjunction with our numerous previous studies –10,19 st