Transcriptional enhancers are a key group of such elements in eukaryotes and are DNA regions that form physical contacts with gene promoters and precisely orchestrate gene expression programs. Here, we follow gradual evolution of this regulatory system and discuss its features in different organisms. In eubacteria, an enhancer-like element is often a single regulatory element, is usually proximal to the core promoter, and is occupied by one or a few activators. Activation of gene expression in archaea is accompanied by the recruitment of an activator to several enhancer-like sites in the upstream promoter region. In eukaryotes, activation of expression is accompanied by the recruitment of activators to multiple enhancers, which may be distant from the core promoter, and the activators act through coactivators. The role of the general DNA architecture in transcription control increases in evolution. As a whole, it can be seen that enhancers of multicellular eukaryotes evolved from the corresponding prototypic enhancer-like regulatory elements with the gradually increasing genome size of organisms.The genomes of all organisms abound with various  The cis-regulatory elements are noncoding DNA sequences distributed throughout the genome [Gene expression is a complicated multistep process that requires tight control. Eukaryotes have the corresponding regulatory machinery containing multiple e genome . EnhanceThe origin of transcription regulation mechanisms of eukaryotes lies in the world of prokaryotes. Therefore, it is not surprising that many features of eukaryotic regulation are common between these two groups of organisms. Thus, it is of great interest to follow evolution of the mechanism whereby transcription is activated step by step. The evolutionary approach obviously allows us to better understand the origin of the transcription regulatory complexity of higher eukaryotes, to clarify the general details of regulation, and to highlight the specific mechanisms and features that are found in the regulation of eukaryotes and are shared with prokaryotes. Here we provide an overview of enhancer-like mechanisms in prokaryotes, describe similarities in these mechanisms shared by both eukaryotes and prokaryotes, and discuss the gradual increase in the complexity of regulation by enhancers in evolution from eubacteria and archaea to unicellular eukaryotes, further to multicellular invertebrates, and finally to vertebrates.For multicellular eukaryotes, the broadly accepted concept is that a transcriptional enhancer is an independent DNA sequence that is separated from the promoter by a distance in 1D (but not in 3D) and combines sites for positive DNA-binding transcriptional regulators. Classically, a transcription enhancer is understood as a DNA sequence that is able to activate transcription over long distances regardless of its orientation with respect to the core promoter . In thisIn unicellular eukaryotes such as yeasts, an upstream regulatory region serving to activate transcription is carefully termed upstream activating sequence (UAS) or sometimes the enhancer-like sequence, meaning that a UAS possesses most, but not all, of the properties characteristic of enhancers of multicellular eukaryotes. UASs cannot activate transcription when located in a gene intron or downstream of the gene in contrast to enhancers of multicellular eukaryotes and are similar to them in being capable of activating transcription when inverted ,4.In prokaryotes, the region harboring both binding site for RNA polymerase (RNAP) and binding sites for transcriptional regulators is usually not subdivided into a promoter and bacterial enhancer(s) and is defined as a promoter. However, the term UAS ,6,7,8, oIt is clear from the brief explanations above that a united block of regulatory DNA was obviously subdivided during evolution into two independent sections, which were assumed in the recent past to have different functional properties. However, recent works provided evidence that promoters and enhancers of multicellular eukaryotes have much in common and share several properties and functions ,17,18. SCommon architecture and functional features (chromatin structure and transcription machinery recruitment) of eukaryotic enhancers and promoters reflect the conversion of one type of elements into the other during evolution of eukaryotes. Indeed, it was found that several hundreds of ancestral enhancers were converted to promoters in primate and rodent lineages of mammals during evolution ,21.An interesting hypothesis on the origin of enhancers in eukaryotes has recently appeared to suggest that developmental enhancers of multicellular eukaryotes evolved from unicellular eukaryotic inducible promoters . Here weThe architecture of genomes reflects the principles of gene expression regulation. Jacob and Monod’s operon concept , couplinTwo systems were developed by prokaryotes to regulate gene expression in response to stimuli. The first and the simplest one is the so-called one-component system. Transcriptional regulators of these systems are the most abundant and demonstrate remarkable structural similarity, containing both DNA-binding domain and a sensor domain, which can bind small-molecule ligands or metabolites. One-component systems are the most ancient signal-processing systems and are extremely diverse in relation to their function .Two-component systems originate from one-component ones and evolved to respond to environmental stimuli . These sEukaryotes developed several more systems with complicated cascades of signal transduction from membrane to DNA in addition to the two above signal transduction systems. However, both simple systems of prokaryotes and complex systems of eukaryotes converge on transcriptional regulators or TFs, which can act as activators and repressors. TFs convert incoming internal or environmental information into gene expression output, allowing the cell to adapt to changing conditions.Promoters act as integrators of different regulative signals provided by transcriptional regulators and are absolutely essential for activation of transcription. Recruitment or driving away RNAP from a promoter is, by and large, a key and unified principle underlying the regulation of transcription from unicellular prokaryotes to vertebrates.TFs are mostly homodimers in prokaryotes and heterodimers in eukaryotes. Both homo- and heterodimeric TFs occupy distinct and often single sites in a promoter, and each subunit recognizes a DNA sequence of 8–10 base pairs. A couple of sites is known as an operator or a box in prokaryotes. In multicellular eukaryotes, the sites of TFs often occur in many copies over extended DNA regions and are found in enhancers, locus control regions (LCRs), developmental enhancers, and superenhancers and may be tens, hundreds, or even thousands of kilobases away from the transcription start site (TSS), depending on how advanced the organism is evolutionarily.lac operon. The lacZ promoter harbors two binding sites for a Lac repressor, which is a product of the lacI gene and acts to prevent lacZ transcription in the absence of lactose. In addition to LacI binding site, the lacZ promoter has one binding site for the transcriptional activator known as CAP or CRP (catabolite activator protein or cAMP receptor protein), which acts to stimulate lacZ transcription at lower glucose concentrations [HOX genes, harbor many sites for different repressors and activators [Another common feature of both simple and complex systems is that binding sites for both activators and repressors may occur together in one regulatory DNA sequence. In prokaryotes, a good example is provided by the system of the trations . Complextivators . Embryontivators .Escherichia coli consists of 4.6 Mb of DNA, and the binding sites for the PhoB protein, the master regulator of the Pho regulon, which is responsible for phosphorus assimilation in bacteria, are in the immediate vicinity of the −10 and −35 core promoter elements or even overlap them [70 complex [Binding sites for regulatory proteins evolved to be farther away from the TSS as genomes grew in size and complexity. For example, the genome of lap them . A direc complex .Saccharomyces cerevisiae has a 12.1-Mb genome, and the binding sites of regulators in the enhancer-like UAS [The unicellular eukaryote like UAS  are usualike UAS ,31. In tlike UAS  and the like UAS . An incrlike UAS . The obslike UAS ,35.Drosophila, whose genome is 175 Mb and is about 20 times smaller than the human genome. At the same time, in this organism, up to 20% of all enhancers are long-range enhancers, which may jump over several genes to reach their target promoters [Drosophila, like in humans [svb and cut Drosophila genes provide well-characterized examples of the loci where long-distance interactions between enhancers and promoters are involved in the regulation. Each of the genes has at least five proven enhancers, the farthest of which are approximately 80 kb away from the TSS [Drosophila was found between the regulatory regions of scyl and chrb [Drosophila BX-C locus, which is approximately 320 kb in total size and regulates the development of the posterior half of the fly body. Both short- and long-range enhancer–promoter interactions were detected in this locus. These interactions are often mediated by boundary elements and insulators, which are located between BX-C domains [Drosophila.The majority of known enhancers are within 10 kb of the promoter in invertebrates, such as romoters ,37. The n humans . The svb the TSS ,40. This the TSS . Anotherand chrb . An extr domains . An addi domains ,43,44. HShh promoter [Long-range interactions reach a huge scale in vertebrates and, in particular, in humans, whose genome is 3300 Mb. For example, the human ZRS enhancer acts over 850 kb to regulate the promoter , and enhpromoter . In addipromoter ,48,49,50promoter  were detThus, mammals have the largest genomes, the most complex regulation, and the most distant enhancers among all organisms. The estimated number of enhancers is up to ten actual enhancers per one gene in mammals .cis regulatory elements requires the development of special complex mechanisms that ensure the convergence of these elements in space (discussed in part 9). From this point of view, one could speculate that the increase might be a consequence of Kimura’s neutral evolution rather than adaptive evolution [cis regulatory elements in evolution, leading to a duplication of the DNA between cis regulatory elements [While the distances between regulatory regions obviously grew over the course of evolution, it is not quite clear if this change provides an advantage to the organism. Moreover, an increase in distance between volution . The appvolution . The inselements .On the other hand, one could speculate that a bacterial promoter-proximal upstream regulatory region is a tool with a very limited regulating capacity. Gene expression requires much more complicated control in Metazoa . A separThe origin of transcription regulation is in the prokaryotic world. In order to see whether parallels could be drawn between enhancer-like mechanisms found in prokaryotes and in higher eukaryotes, it is required first to consider the general principles of transcription activation in eubacteria.tac [Bacterial promoters have a modular structure the same as that of eukaryotic promoters and appear to have evolved by a pick-and-mix mechanism, which produced the hierarchies of promoter activities that permit a 1000-fold dynamic range in transcript initiation frequencies ,57,58. Atac ), the in70 interaction is altered by activators that redirect the complex to a certain class of promoters, the process being known as the RNAP appropriation [Bacteria use two distinct sets of mechanisms for transcription activation focusing either on the promoter or on RNAP. In RNAP-centric mechanisms, some factors interact with RNAP to alter its promoter preference. For example, alternative σ factors do this ,61. In opriation .70 with the −35 element. An activator interacts with the σ4 domain and promotes its binding to the “non-perfect” −35 hexamer. Only one of the two subunits of the activator on DNA makes contact with the σ4 domain of σ70 upon activation because RNAP carries only one σ factor. To make contact with σ4, activators have to be precisely positioned on DNA and bind to the same face of the DNA helix as the σ4 domain does. The binding of an activator to a site adjacent to or overlapping the upstream end of the −35 hexamer is often triggered by phosphorylation of a receiver domain of the activator or by binding to an activatory ligand, for example, a sugar. This is the case with the AraC regulator at the PBAD promoter when arabinose binding changes the AraC properties and converts AraC from a repressor to an activator  of the α subunit of RNAP with the UP element located upstream of the core promoter. The second one involves the interaction of the σ4 domain of σ70 subunit are hindered. This hindrance is overcome by the activator MerR, which causes a localized distortion to align the −10 element with the −35 element and thus triggers transcription activation [One more fundamental form of regulation is based on changing the DNA topology and is widespread from bacteria to human. In bacteria, activation of expression by changing the promoter conformation is a special case of this regulation. Experiments with synthetic promoters that form an angle of 90° in vitro showed that this sharp DNA bend may facilitate the binding of upstream DNA to the backside of RNAP . In vivotivation .Binding of one activator to DNA and subsequent DNA bending may facilitate a direct interaction of another activator with RNAP. For example, the CAP activator-induced bending was shown to modulate the location of binding of the activator MalT and to trigger transcription activation from the malK promoter .Altering the promoter conformation in bacteria may involve nucleoid-associated proteins (NAPs), which are evolutionary predecessors of eukaryotic histones. Their binding to a distal promoter results in translocation of superhelical energy from upstream binding sites (supercoiling-induced DNA duplex destabilized region) to the promoter-proximal region, which is unwound within the open complex, and may change the promoter activity by several dozens of times ,15,68,69NAPs, H-NS, and FIS may interplay with special shape-sensitive activators containing winged helix-turn-helix (wHTH) motifs. wHTHs use an alpha helix to read the base sequence in the major groove while inserting a beta sheet «wing» into the adjacent minor groove ,71. wHTHIn this part we provide specific examples related to bacterial UAS-like regulation. These include direct stimulation of the RNAP complex by an activator, activation loops, repression loops, activator tracing/scanning from a landing enhancer-like site to a core promoter, and rings that mediate interactions between distant DNA regions.70 complex is provided by inducible genes of the Pho regulon, which is involved in the cell response to deprivation of phosphorus , for the AraC activator-repressor. The half-sites are centered at position −52.5 bp  and partly overlap the −35 region. One more AraC half-site, O2, is 211 bp upstream (−265 to −294 from TSS). When arabinose is absent, the subunits of the AraC interact with two nonadjacent DNA sites and a repression loop forms between them [The een them ; i.e., ABAD and specifies the activator–RNAP contacts. This mechanism reminds the mechanism of loop formation by a cohesion complex in eukaryotes, when the orientation of two CTCF protein binding sites specifies the direction of loop formation. Changing the polarity of one CTCF site compromises enhancer–promoter interactions [BAD [Thus, the reorientation of one subunit of AraC to another site regulates the DNA looping in Practions ,77,78,79ons [BAD ,81.BAD, the activator CAP, is upstream of the AraC site and is centered at position −93.5. Although there is no detectable interaction between AraC and CAP upon their binding to DNA [BAD when acting alone, in the absence of AraC [BAD in vitro [BAD, enhancing the action of AraC, but is capable of binding to DNA unlike eukaryotic coactivators.The site for the other regulator of Pg to DNA , experimg to DNA . CAP doe of AraC . Convers of AraC ,85 and cWhat mechanistic consequences arise when two different activators interact at once with RNAP upon activation? One scenario is that each activator induces its own specific rearrangement of RNAP and each of these rearrangements contributes to the final organization of the complex and transcription output. Another scenario is that both activators do the same job, that is, each of them repeats the other. Experiments testify in favor of the latter; i.e., both CAP and AraC stimulate the recruitment of RNAP as well as the transition to the open complex .70 presumably contacts the AR3 region of CAP [70 interact with the −35 element, but AraC interacts with σ70 very slightly [Upon activation, the CAP–AraC–RNAP complex substantially bends the DNA, and it is very likely that CAP is positioned close to RNAP despite the fact that the AraC binding site lies between the binding sites for CAP and RNAP B. One α n of CAP . Both Arslightly . In contslightly .Thus, the example above demonstrates that bacteria already display complex interplay of different regulators on the enhancer-promoter region.Regulation of gene expression by looping is common in eukaryotes during development ,89,90, baraBAD promoter was described in BAD using microscopic [Repression loop formation was studied in bacteria in detail using several examples. The mechanism of how repression loops form in the roscopic  and biocroscopic ,92. Reprroscopic ,94. It wlac promoter [lac, when loops between operator sites are stabilized by the Lac repressor, RNAP appears not to be trapped or occluded in these loops [Using microscopy, repression loop formation was also demonstrated for the promoter  . Activators of this class bind remotely upstream of the TSS and induce the formation of large activation loops. bEBPs are special hexameric ATPases that use ATP hydrolysis to remodel theRNAP–σ complex . In addi complex ,104. Thi70 and σ54. Upon binding to the promoter, the RNAP–σ70 holoenzyme forms a closed complex, which can spontaneously isomerize to form an open promoter complex [70 is involved in the bulk of cell transcription during active growth, including all essential genes in E. coli. Unlike σ70, σ54 directs transcription of tightly regulated genes involved in the cell response to stress, including nitrogen and carbon starvation, antibiotics, and loss of membrane integrity [54 binds to RNAP to form a stable closed complex, which rarely spontaneously converts to the open one [54 and the AAA+ activation domain (AD) of the bEBP [There are two major families of σ factors in bacteria, based on sequence homology and the mechanism of action: σ complex ,61. Due ntegrity . σ54 binopen one ,106. bEBopen one , resultiopen one ,109. Enethe bEBP ,111. Thithe bEBP ,113.glnALG operon  complex. Interestingly, the distance from the NtrC binding site to the TSS should be no less than 70 bp to allow activation. The distance can be increased up to 1000 bp or more without impairing the activation appreciably [The best-studied example of activation loop formation by bEBPs is provided by the system of the G operon D. When nreciably . Recentlreciably .in cis, with the length of the loop being consistent with the spacing between the NtrC binding sites and the core promoter. Once the closed complex is converted to the open one, the interaction of a bacterial enhancer with the promoter is terminated, the DNA loop is opened, and σ54 dissociates after promoter clearance by RNAP [Electron microscopy demonstrated the presence of looped DNA  by RNAP ,117.in cis, but also in trans, and that the in trans interaction was enhanced with the increasing NRI concentration [Studies with a system of two plasmids in which one plasmid carried the promoter and the other one had activator binding sites showed that activation is possible not only ntration . A similntration . These e54 complex occurs on the promoter of the glnALG operon even in the absence of nitrogen deficiency and in the absence of the NRI activator [glnALG operon similar to the eukaryotic genes that are regulated by stalling RNAP and are constitutively associated with the RNAP–TFIID complex even when these genes are inactive [The most interesting finding is that the closed RNAP–σctivator . This feinactive .glnALG operon. Binding sites of the NRI activator are in the DNA major groove on one side of the DNA helix, while σ54 binding sites are in the major groove on the other side of the DNA helix in the glnALG promoter [glnALG promoter because the promoter has three activator binding sites and monomers bound at each site possess flexible activation domains pointing in opposite directions [54–NRI-regulated system with two NtrC binding sites was indeed face-of-the-helix dependent in another organism [The relationship between activation and helical phasing was demonstrated in the case of the activation loop as well, although not for the promoter . This isrections . This maorganism . Thus, p54 is present in an estimated 60% of bacterial genomes [E.coli, σ54 was experimentally shown to regulate 135 of 4 288 protein-coding genes [54-dependent promoters have been predicted to date [How frequent may such type of eukaryotic-like regulation be in prokaryotes? σ genomes . In E.cong genes . A numbe to date . These p to date ,124.Thus, the activation loop mechanism in bacteria is closest to what we expect from eukaryotic enhancer-dependent regulation. At the same time, the formation of an activation loop is not the only mechanism that spatially associates distant DNA regions in prokaryotes.70) [70, and the σ4 domain recognizes the -35 element. Thus, gp33 mimics the σ4 domain of σ70. The promoters of late T4 genes have a −10 element only. Thus, a blocking of the σ70-subunit binding site on RNAP by gp33 allows transcription to be activated from late T4 promoters lacking the –35 element. Interactions of the gp45 trimer with RNAP allows the transition of the σ55 (gp55)-RNAP complex from the closed state to the open one and activation of transcription [Ring complexes that encompass the DNA molecule and slide along it are widely used in bacteria and eukaryotes . The role of a sliding ring in regulating gene expression by a scanning/tracking mechanism was discovered and studied on late genes of the bacteriophage T4. A sliding clamp comprised of a trimer of gp45 polypeptides is loaded to a distal enhancer-like site by the gp44–gp62 clamp loader complex E. This s70) . On the 70) . The β-fcription .B. subtilis was shown to proceed with the help of the loop extrusion mechanism. Upon replication, two large separate hairpins form and condensin rings move in an ATP-dependent manner along contiguous DNA segments, processively enlarging the DNA loops [Condensins of prokaryotes and eukaryotes and cohesins of eukaryotes are collectively called SMC proteins and are evolutionarily related conserved multi-subunit complexes that form rings of various structures. Condensins/cohesins are vitally important for cell division and DNA repair in both domains of life ,129,130.NA loops  at ratesNA loops .Although more profound exploration is required to broaden the understanding of the role that SMC complexes play in regulating gene expression in bacteria, some SMC–transcription interactions have been demonstrated ,136. ForThe examples and explanations above make it obvious that the details of molecular mechanisms of regulation of bacterial transcription have analogues in the world of eukaryotes.Current models of eukaryogenesis have undergone significant revision recently . Modern Archaea are characterized by a simplified version of eukaryote-like general transcription machinery including RNAP and general TFs (GTFs), TBP (TATA-box binding protein), TFB, and TFE . Like eubacteria, archaea have one RNAP, while eukaryotes have three. Of these, RNAP most closely resembles the archaeal enzyme. Ten of 13 subunits of RNAP are identical between eukaryotes and archaea, and only 5 are identical to bacterial RNAP ,142.tbp and tfb genes, and their multiplicity was hypothesized to have a regulatory role at a higher level, reminiscent of alternative σ factors in bacteria [Eukaryotes inherited the transcription apparatus and principles of its regulation, including differential transcription regulation by multiple GTFs, TBP, and TFB, and activators/repressors from archaea ,141,142.bacteria . In turnbacteria ,142,144.Bacterial RNAP initiates transcription without the involvement of additional factors with the exception of the σ factor, whereas archaeal RNAP requires a minimal set of GTFs, TBP, and TFB. The archaeal core promoter has minimal structural differences from the eukaryotic core promoter . The GTFTFs of archaea are much more similar to bacterial ones than to eukaryotic ones. Approximately 53% of all TFs identified in archaeal genomes have at least one homologue in bacteria, as opposed to 2% having a eukaryotic homologue . HoweverMost archaeal repressors function as in bacteria by abrogating access of one or more components of general transcription machinery to the core promoter . While aActivator binding sites in archaea are preferentially located immediately upstream of or partly overlap the BRE element and are often called UASs, as in yeasts ,159. At Distal sites are often utilized by Ribbon-Helix-Helix (RHH)-family activators . It is ud element in RNAP promoters [One more example of eukaryotic-like regulation involves a transcription activator that has binding sites not only in the proximal promoter, but also in the core promoter. The sequence in the core promoter is located immediately downstream of the TATA element , analogoromoters .Some archaeal regulators appear to be of dual nature and exert opposite regulatory effects at the same promoter in a concentration-dependent manner ,170,171.Thus, Archaea combine the principles of the regulation observed in the other two domains of life, Bacteria and Eukarya. The regulation by multiple TBP and TFB factors is an intrinsic trait of Archaea that has been fully inherited by Eukarya. The regulation in the chromatin is not considered here; however, it is a second common feature between Eukarya and Archaea. The molecular details of these systems are just emerging, and our understanding lags behind the understanding of bacterial systems. It is possible that more types of sophisticated regulation will be found in archaea in the near future.As was mentioned above, the prokaryotic mechanisms resemble or even parallel the mechanisms found in eukaryotes. However, the eukaryotic systems of regulation are generally more sophisticated and include proteins with intricate multidomain structures. Gene regulatory networks are more complex in eukaryotes as a result of several genomic duplications that occurred on the way to vertebrates. The duplications gave origin to paralogs, sometime several. For example, there is a single SMC condensin gene in eubacteria, while multicellular eukaryotes developed several similar complexes with overlapping, but not identical functions. The fixation of a large number of SMC genes in evolution of eukaryotes emphasizes their significance for the function of the eukaryotic genome. Due to huge sizes of higher eukaryotic genomes, some of these complexes, which are known as cohesion complexes, acquired a function in the regulation of gene expression by the loop extrusion mechanism in interphase; their role in enhancer–promoter interactions is now well characterized in eukaryotes ,80,81.cis regulatory elements dispersed over long distances should be brought together for proper activation or repression. On the other hand, the abundance of DNA-binding proteins subtly sensing the DNA shape begun to open up in eukaryotes. For example, recognition of the DNA shape by proteins and nucleosomes was recently found to involve the A-tracts as short as three base pairs in the DNA minor groove and received the name of indirect readout [Like in prokaryotes, the DNA topology has a fundamental control over promoter activity and the ability of TFs to access their target DNA sites in eukaryotes. On the one hand,  readout . Sensing readout ,176 and  readout .Before discussing the mechanistic ideas about how enhancers work in eukaryotes, it is necessary to consider the mechanisms whereby eukaryotic activators function because the nature of an activator generally determines the way of enhancer action.A minimal eukaryotic activator consists of an activating domain (AD) and a DNA-binding domain (DBD). When expressed in eukaryotic cells, such a fusion protein can activate any gene bearing the appropriate DNA binding site nearby. This principle works despite the fact that eukaryotic transcriptional machinery is much more complex than that of bacteria, comprising many proteins in addition to RNAP. A typical eukaryotic activator will recruit whatever factors are required to form the activation complex, and transcription will ensue .cis-mediated mechanism [The binding of an activator consists in concentration-dependent dynamic interactions, and what is an enhancer to do is simply to increase the concentration of the activator nearby the TSS. Recruitment of an activator merely increases the rate at which the binding reaction occurs. In the absence of an activator, the reaction proceeds spontaneously to some extent, generating a basal level of transcripts. A gene does not stay on once activated unless RNAP is continually recruited by the action of an activator. At abnormally high concentrations of TFs, the specificity can be lost: activators will bind and work where they should not, and enzymes will work on sites that they normally leave unmodified. RNA molecules can also act as recruiters. For example, many regulatory proteins were shown to bind to non-coding RNAs that function to activate their neighbor genes using a echanism ,180,181.When contacting enhancers, activators do it by two ways. First, as in archaea, some activators interact directly with basal factors, such as TFIID-TBP or TFIIB and others, recruiting them to the nearest promoter. Second, activators convey their message through coactivators , such as the above mentioned MBF1, the Mediator complex, and histone acetyltransferases (HATs). The spectrum of the recruited factors fully depends on of the AD. For instance, the most powerful AD of the herpes simplex virus VP16 protein recruits almost all basal factors, including several TAFs; coactivators, such as NuA4, p300/CBP, PCAF, and SAGA complexes; the SWI/SNF chromatin remodeling complex; and general cofactors, such as PC4, that are capable of interacting with both ADs and basal factors .Activators make chromatin more permissive for transcription by recruiting HATs either directly, through interaction with a specific HAT subunit, e.g., Tra1p, a shared subunit of SAGA and NuA4 complexes , or indiThe remodeling of the RNAP complex due to direct contact of the activator with RNAP is common in bacteria, but exceptional in eukaryotes. For example, it was initially shown that AD of the HBx protein of hepatitis B virus interacts with the RBP5 subunit, which is present in all three RNAP I, II, III . Later sIn eukaryotes, a remodeling of RNAP is achieved by introducing a chemical modification in its structure, for example, by phosphorylating its CTD. Activators that can interact with positive transcription elongation factor b (p-TEFb), which consists of CycT and the cyclin-dependent kinase Cdk9), simulate phosphorylation of CTD by p-TEFb at Ser2, which causes the conversion of the RNAP holoenzyme from the initiation complex into an elongation one ,191.Phosphorylation of CTD of RNAP by p-TEFb or the CDK7 subunit of THIIH is not the only way to remodel RNAP. Activators can involve external third-party cell kinases for this purpose. For example, it was shown that interactions of VP16 with cell kinases other than THIIH potentiate CTD phosphorylation . Cdc2 isThus, after binding to an enhancer, an activator presents its activation domain, which either directly or through coactivators interacts with chromatin remodelers and components of basal transcription machinery. The interactions facilitate the assembly of a complete set of GTFs on the promoter and the transition of RNAP to initial RNA synthesizes, which is accompanied by phosphorylation of Ser5 in CTD of RNAP. Subsequent interactions of the activator with p-TEBb and, possibly, CTD transfer transcription into the elongation phase.Unlike in prokaryotes, eukaryotic activators do not recognize long and easily recognizable DNA sites, but rather a family of short closely related sequences of up to 10 bp. The circumstance explains why some redundancy is usually observed. To overcome redundancy and to induce a high level of target gene transcription, activators should act in combination with each other, binding multiple regulatory elements in enhancers. In addition, they should act in a proper chromatin context (chromatin context requirements are reviewed in ). This ctrans-activating, and trans-repressing properties.Eukaryotic regulators often recognize specific DNA sequences in enhancers in a cooperative manner. Cooperativity helps to ensure the specificity of binding and is used to integrate information. Two proteins might exert no effect when present in separate cells but bind (cooperatively) to DNA when present in the same cell. A special case of this principle is the situation where the binding of one activator to one of a few adjacent sites enhances the binding of the same activator to the other sites. The principle is illustrated by the fact that many glucocorticoid response elements (GREs) present in an enhancer stimulate transcription far greater than one GRE does when present alone. The cooperativity of GREs is mediated by its DBD, but not AD . This isSequential cooperativity is another sort of cooperativity and implies interplay of different activators, at least two of them. It is best illustrated by the example where the binding of a first activator  to its site enhances/induces the binding of a subsequent activator (of non-pioneer TF nature). Another example is related to the myogenin enhancer, where the MyoD activator initially recognizes its binding site in a proper chromatin context. MyoD utilizes its H/C and helix III domains to interact with an adjacent protein complex containing the homeodomain protein Pbx, which appears to be constitutively bound at this site. Thus, MyoD is necessary for the initiation of chromatin remodeling, but binds second at this site .The principle of cooperativity is additionally expressed at the level of interplay between activators, where different activators recruit different coactivators, enhancing the effect of each other. For example, one activator recruits TFIID, another one recruits TFIIB or HAT, and a third one recruits a chromatin remodeling complex. A cumulative effect is created in this way by the activators, which access their own sites through different mechanisms.The specificity of an activator is determined solely by the location of the DNA site recognized by its DBD. For example, the yeast transcriptional activator Gal4 can activate essentially any gene when artificially expressed in another eukaryote, provided that the gene has Gal4 binding sites inserted nearby  Figure A. HoweveSeveral models have been presented in the literature to explain how enhancers of eukaryotes function. The key point at which all models converge is the presence of the so-called pioneer TFs  that can recognize their target sites in the context of compacted («closed») chromatin, which is covered by nucleosomes and is insensitive to DNase I and other nucleases. This is especially characteristic of developmental genes, which are typically embedded in «closed» chromatin. Thus, activators that potentiate transcription in development are inherently capable of initiating chromatin opening events. However, heterochromatin (carrying the H3K9me2/3 mark) appears to be insensitive to pTFs , and chrDrosophila embryos [One of the best-studied examples of pTFs is provided by the Zelda factor, which acts to establish (but not to maintain) active loop-domains before the midblastula transition in  embryos . Zelda p embryos . Zelda a embryos . The exaDrosophila forkhead mutation causes defects in head fold involution). Mammalian FoxA opens compacted chromatin in nucleosome arrays assembled with linker histone H1 and containing the albumin gene enhancer in vitro, acting independently of the SWI/SNF chromatin remodeling ATPases, which are frequently required for chromatin opening [Direct nucleosome- and chromatin-binding studies are useful in order to investigate how exactly the pTFs mechanistically engage chromatin to initiate reprogramming and trans-differentiation of cells . Interes opening . This ef opening ,211. Dir opening , and is  opening . Thus, pAnother example of how pTFs work is provided by MyoD, a master regulator of muscle differentiation. A cascade of chromatin events was assumed to occur upon myogenin gene activation by MyoD. MyoD is recruited to its target gene enhancers via interaction with the Pbx-Meis complex, which is constitutively bound to the genes prior to activation. p300/CBP acetylates histones H3 and H4 within the promoter region to relax chromatin and then recruits lysine acetylase PCAF. Once tethered to the promoter, PCAF acetylates MyoD to facilitate the trans-activation process . SimultaThe complex of regulatory proteins on the enhancer is called the enhanceosome. The enhanceosome was crystallized for the interferon-β (IFN-β) enhancer. Transcriptional activation of the IFN-β gene requires that a complex containing the ATF-2/c-Jun, IRF-3/IRF-7, and NFκB activators is assembled on its enhancer. The factors bind to the IFN-β enhancer cooperatively and recruit coactivators to the IFN-β promoter. A crystal structure shows the association of eight proteins with the enhancer . The actThe assembly of an enhancesome was studied in vivo with another example. It was found that assembly is hierarchically ordered with the Sox2 protein engaging the target DNA first, followed by binding of Oct4. Sox2 employs a search mode, sliding along open DNA to efficiently locate its targets. Assembly takes about 15 s, including prior three-dimensional diffusion and nonspecific collisions of the factors . The exaSeveral mechanical models were proposed to explain the action of enhancers over distance. In a tracking/scanning model  Figure C, an enhDrosophila was initially identified as a «facilitator» of long-range interactions, and it was assumed that communication between an enhancer and a promoter is mediated by a chain of Chip-containing complexes that are anchored to the chromatin fiber by interactions with homeodomain proteins (HDPs) [An alternative model exploits a hypothetical linking/chaining mechanism  Figure B, accords (HDPs) . Yeast Hs (HDPs) . It was s (HDPs) . In turns (HDPs) . Thus, ts (HDPs) . Howevers (HDPs) . At the s (HDPs) .The targeting looping model is now more or less dominating in the case of multicellular eukaryotes. Even for yeasts, activation with UAS was demonstrated to proceed through the looping mechanism .The principles of metazoan genome organization were clearly identified in recent years. It was found that the genomes of multicellular eukaryotes are organized in hierarchic layers, which represent structural and functional building blocks of the genome (reviewed in ). The lacis-regulatory elements located in one TAD [TADs and subTADs are the level at which enhancers function. TADs restrict the enhancer action to prevent cross-reactivity between enhancers and promoters from different TADs and, on the other hand, facilitate the interaction between  one TAD . The pro one TAD ,239. To  one TAD .To monitor the TAD/loop formation biochemically at high resolution, special Hi-C and micro-C methods were developed based on the principle of cross-ligation of DNA fragments after digesting formaldehyde-fixed nuclei with micrococcal nuclease (MNase) or restriction endonuclease ,240. UsiProfound explanations were recently obtained for how the polarity of CTCF binding and the CTCF protein structure determine the genomic distribution of chromatin loops, bypass of CTCF sites and probably regulatory elements, and overall changes in chromatin organization . AlthougDrosophila CTCF null mutants  survive past the larval stage and die as late pupae with visible defects associated with Hox gene misexpression [cis-regulatory elements. For example, the role might be played by other molecular machines that depend on ATP hydrolysis and are capable of DNA translocation, for example, chromatin remodeling complexes [Although lack of the CTCF protein is embryonic lethal in mammals, pression . These gpression . Collectomplexes .Drosophila is not devoid of ways to bring remote regions of the genome close together. Long-range looping interactions were initially not found in Drosophila in early genome-wide studies using the Hi-C method because of the technical imperfections of the method at early stages of its development [Drosophila domains with apical interaction hotspots were identified in early embryos [Drosophila [While lacking the loop extrusion mechanism, elopment ,246. Lat embryos  and Kc e embryos . Althoug embryos , they do embryos  and do n embryos ,249. Somosophila , as wellosophila . PRC1-deosophila .Unicellular eukaryotes (yeasts) were shown to possess two types of TADs, ~200-kbp TADs, which regulate replication timing so that origins within a domain fire synchronously , and selThus, the examples above provide evidence that the way the genome is organized may change the principles of regulation by enhancers D. VertebDrosophila from the very beginning [Drosophila genome. Second, each class of enhancers was demonstrated to have its own preferences regarding the architectural protein composition [In multicellular eukaryotes, the principles of activation inside TADs might be exactly the same for both invertebrates and vertebrates because the evolutionary distance does not allow any fundamental molecular difference to develop. Instead of CTCF, which occupies TAD borders in mammals, a lot of architectural proteins were detected on the borders between TADs in eginning ,246. Somposition . A functposition . Based oDrosophila, the enhancer–promoter specificity is achieved within a TAD with the use of a compatibility code between enhancers and classes of core promoters [Thus, in romoters ,252; speromoters ; and tetromoters ).Drosophila, possibly because vertebrates possess the cohesin/CTCF-mediated loop extrusion system. However, some examples of architectural proteins were discovered. LBD1 (Chip in flies) and YY1 [In vertebrates, the regulatory system based on architectural proteins is not as obvious as in  and YY1 , studied and YY1 , dependi and YY1 . Widespr and YY1 . YY1 bin and YY1 . YY1 may and YY1 . In addi and YY1 .Thus, in the near future, the rapidly developing field of studying loop extrusion in mammals will move from accumulating general ideas of how it works to mechanistic justifications. Some examples of such a transition are already available , but stiThe origin of the mechanisms of transcriptional regulation lies in the world of prokaryotes, and we will never know what the first mechanisms of regulation were. However, using the existing diversity of living beings, we can reconstruct the evolution of regulatory mechanisms, understand its laws, and highlight the fundamental differences. The first thing worth paying attention to is the principle of initiation of transcription by RNAP. Recent findings indicate a common origin of initiation factors: the bacterial sigma subunit of prokaryotes and TFB and TFIIB of archaea and eukaryotes, respectively . The disRegulation by multiple initiation factors is another trait used by all cellular organisms. In bacteria, such factors belong to the family of sigma subunits, while multiple TBPs and TFBs are used in archaea. This feature was inherited by eukaryotes from archaea. For example, in Metazoa, transcription is regulated by TBP and its TBP homologs, TBP-related factors ,264.One more general principle that has been preserved in evolution of all groups is compensation for weak motifs of the core promoter by activators. Activators recruit sigma-initiating subunit of bacteria and functional analogues of sigma, TBP+TFB, in archaea. In different types of eukaryotes, activators perform the same job and additionally recruit various auxiliary complexes that facilitate operations with nucleosomes.Drosophila [Although the eukaryotic system of gene expression regulation is the most sophisticated, eukaryotes use the same principle of recruitment of initiating basal factors to the promoter that they inherited from Archaea. In archaea, activators bring the TBP and TFB to a promoter and facilitate their interaction with RNAP. Such a functionality of archaeal activators resembles one-component coactivator activity of eukaryotes and appears to be very stable in evolution, being maintained from unicellular eukaryotes to invertebrates and finally to vertebrates. For example, the MBF1 factor bridges TBP and the GCN4 activator in yeasts  and nuclosophila , while iosophila ,160. ThuAt the same time, gradual complication of organisms certainly affected the transcriptional regulation and eukaryotes have already acquired one-component heterodimeric coactivators. In addition, eukaryotes acquired multi-component coactivator complexes, such as Mediator, to integrate and transmit the signals from multiple TFs to a few factors of basal transcription machinery; certain other complexes, such as chromatin remodeling complexes, to operate with nucleosome-imbedded DNA; and histone-modifying complexes, to facilitate these operations.Although examples where the DNA topology affects the activity of a promoter were described in bacteria, activation of transcription in this domain was always accompanied by direct interactions of activators with bacterial RNAP and/or the sigma-initiating subunit. Unlike in prokaryotes, eukaryotic activators convey their message by contacting RNAP through the basal factors, coactivators and the Mediator complex, which surround RNAP, but never interact with RNAP directly.Despite the advances made in recent decades in studying the regulatory mechanisms in eukaryotes and other organisms, including genome-wide studies, several evolutionary problems of molecular biology are still not resolved and, apparently, will not be resolved in the nearest future. For instance, although NGS technologies made it possible to conduct metagenomics analysis of populations of microorganisms, and representatives of a new archaeal group—direct evolutionary predecessors of eukaryotes—were identified and then cultivated in the past 5 years, accumulating knowledge is still the main feature of the research process and findings of the missing transition links between archaea and eukaryotes do not allow us to obtain comprehensive answers about evolution of specific molecular regulatory mechanisms.For example, it is still an enigma when and why archaeal activators functionally turned into coactivators of eukaryotes. This can be partially explained by the increase in the number of regulators in Eukarya, where some of the regulators assumed the functions of coactivators by creating an adapter link between the remaining activators and basal factors. Alternatively, it may be explained by multiple gene and whole genome duplications, which occurred in evolution of unicellular eukaryotes and provided greater evolutionary flexibility to regularity mechanisms. This situation is clearly evident in the yeast genome, which has more than several hundreds of duplicated genes and where duplications were accompanied by asynchronous paralog differentiation and regulatory neofunctionalization ,266,267.One more enigma is the origin of the Mediator complex. Although several attempts were made to come closer to understanding this problem ,269, theHere we described diversity and the gradual increase in complexity during evolution of the mechanisms underlying transcriptional control by enhancers and enhancer-like regulatory elements (eREs) in different organisms . A direc


Long non‐coding RNAs (lncRNAs) are key mediators in various malignancies. Linc01503 was previously elucidated to promote gastric cancer (GC) cell invasion. However, the upstream mechanism of linc01503 and its involvement in GC cell cycle, apoptosis and tumorigenesis still remain unclear.Bioinformatics analysis and quantitative reverse transcription polymerase chain reaction (qRT‐PCR) assays were implicated to detect linc01503 level in GC. The role of linc01503 was detected by in vitro functional assays and in vivo xenograft tumour models. The association between linc01503 and its upstream effector was identified by chromatin immunoprecipitation (ChIP) assays. The mechanistic model of linc01503 was clarified using subcellular separation, fluorescence in situ hybridization, RNA‐sequencing, RNA immunoprecipitation (RIP) and ChIP assays.Linc01503 was remarkably elevated in GC and tightly linked with the overall survival of patients with GC. The key transcription factor early growth response protein 1 (EGR1) critically activated the transcription of linc01503. Functionally, linc01503 knockdown resulted in the activation of apoptosis and G1/G0 phase arrest in GC. Mechanistically, linc01503 interacted with histone modification enzyme enhancer of zeste 2 (EZH2) and lysine (K)‐specific demethylase 1A (LSD1), thereby mediating the transcriptional silencing of dual‐specificity phosphatase 5 (DUSP5) and cyclin‐dependent kinase inhibitor 1A (CDKN1A) in GC.EGR1‐activated linc01503 could epigenetically silence DUSP5/CDKN1A expression to mediate cell cycle progression and tumorigenesis, implicating it as a prospective target for GC therapeutics. The EGR1‐activated lncRNA linc01503 mediated gastric carcinogenesis through the axis of EZH2/LSD1/DUSP5/CDKN1A. The mechanistic model of linc01503 is a further prerequisite for progress in translational exploitation of lncRNAs for treatment of patients with GC. Firstly, bioinformatics analysis predicted a significantly altered expression of linc01503 between GC tissues and non‐tumour tissues. This perspective was subsequently verified by quantitative reverse transcription polymerase chain reaction (qRT‐PCR) assays. Also, it was notable that increased level of linc01503 tightly associated with worse overall survival (OS) of GC patients. Intriguingly, the key transcription factor early growth response protein 1 (EGR1) critically activated the transcription of linc01503 in GC. Functional assays indicated that silencing of linc01503 markedly mediated GC cell proliferation and apoptosis both in vitro and in vivo, and induced the alteration of GC cell cycle regulation. Next, RNA‐sequencing (RNA‐seq) combined with gene set enrichment analysis (GSEA) showed that linc01503 knockdown effectively affected genes associated with cell cycle and apoptosis, potentially contributing to the promotion of GC proliferation. Mechanistically, the driving forces of linc01503 were regulated, at least partially, by interacting with enhancer of zeste 2 (EZH2) and lysine (K)‐specific demethylase 1A (LSD1). Importantly, linc01503 was elucidated to epigenetically suppress dual‐specificity phosphatase 5 (DUSP5) and cyclin‐dependent kinase inhibitor 1A (CDKN1A) expression through binding with EZH2 and LSD1 in GC. In summary, our study largely enriched the functional role and novel mechanism of linc01503 in GC and the axis of EGR1/linc01503/DUSP5/CDKN1A may be an attractive target for GC therapeutics.22.1GC specimens were collected from Peking University Cancer Hospital with informed consent policy. The research was approved by the Ethics Committee of Peking University Cancer Hospital and conducted under the guidance of the Declaration of Helsinki. The detailed information of cell lines is summarized in Appendix 2.2Total RNA was extracted in TRIzol reagent (Invitrogen) based on the descriptions of the product. The detailed information is summarized in Appendix 2.3Transfections were performed using Lipofectamine 3000 (Invitrogen) based on the instructions of the manufacturer. The detailed information is summarized in Appendix 2.4Animal experiments were approved by the Animal Ethics Committee at Peking University Cancer Hospital. The detailed information is summarized in Appendix 2.5The interactions between linc01503 and potential binding proteins were detected by RIP assays using the EZ‐Magna RNA Immunoprecipitation (RIP) Kit (Millipore) under the instructions of the manufacturer. The details are summarized in Appendix 2.6According to the protocol of the manufacturer, ChIP assays were performed with a MagnaChIP Kit (Millipore). Detailed information is summarized in Appendix 2.7Subcellular separation assays were performed with a PARIS Kit (Life Technologies) in accordance with the protocol of the manufacturer. FISH assays were performed with a Ribo™ Fluorescent In Situ Hybridization Kit (RiboBio) according to the manufacturer's instructions. The detailed information is summarized in Appendix 2.8t test, Wilcoxon's test and χ2 test, as appropriate. The association between EGR1 and linc01503 was evaluated using Spearman's correlation analysis. The data were manifested as mean ± standard deviation (SD) and analysed using SPSS 21.0 software (IBM). All tests were two‐sided, and P < .05 was considered to indicate a significant difference.The differences in groups were assessed with Student's 33.1We firstly analysed the publicly available data from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) DataSets and aimed to find candidate lncRNAs potentially correlated with GC progression. The bioinformatics analysis showed that linc01503 displayed remarkable upregulation in GC tissues compared with non‐tumour tissue samples Figure . To verihttp://kmplot.com/analysis).To further clarify the relationship between linc01503 level and survival outcome of patients with GC, the Kaplan‐Meier plotter (KmPlot), which has been used in various cancers, was implicated in this research  Figure . Meanwhi4Massive studies have elucidated that lncRNAs play important roles in the pathogenesis and progression of multiple cancers.In the present study, publicly available data from GEO and TCGA data sets were downloaded and analysed to select candidate genes potentially associated with GC progression. Notably, linc01503 was markedly increased in GC and its elevation closely associated with poor survival for patients with GC. Interestingly, some of key transcription factors are highlighted to be involved in the dysregulation of lncRNAs.Functionally, silencing of linc01503 dramatically suppressed GC cell proliferation and activated the apoptotic activities, whereas overexpression of linc01503 elicited the opposite impact. Furthermore, knockdown of linc01503 resulted in a significant elevation of G1/G0 phase population in GC cells compared with the respective control groups. Subcellular separation and FISH assays showed that linc01503 was prevalently distributed in the nucleus, implying that linc01503 may be involved in the gene regulation at the transcriptional level. Importantly, bioinformatics analysis and RIP assays confirmed that linc01503 could serve as a scaffold interacting with EZH2 and LSD1 in GC cells. RNA‐seq assays combined with GSEA demonstrated that linc01503 knockdown dramatically affected genes associated with cell cycle arrest and apoptosis, potentially contributing to the promotion of GC proliferation. Interestingly, these potential targets were identified to be co‐regulated by the alteration of both EZH2 and LSD1. It was noted that DUSP5 and CDKN1A were consistently up‐regulated and co‐regulated by linc01503, EZH2 and LSD1, highlighting that DUSP5 and CDKN1A are key targets of linc01503 in GC. Finally, ChIP assays uncovered that linc01503 mediated transcriptional repression of DUSP5 and CDKN1A through H3K27 trimethylation and H3K4 demethylation in GC cells. Above all, lncRNA linc01503, at least partially, interacted with EZH2 and LSD1 to epigenetically silence the expression level of DUSP5 and CDKN1A in GC.5The regulatory axis of EGR1/linc01503/DUSP5/CDKN1A may be an attractive target for GC therapeutics. Our research may enrich the understanding of GC biology and accelerated the progress of lncRNA‐based therapeutics in GC or other types of cancers.The authors declare no competing financial interest.ZHM conceived the study, performed the experiments and drafted the manuscript. ZHM, XYG and YS participated in the figure drawing and data analysis. YS and XLW helped to conduct bioinformatics analysis. YY, XFX and JFJ provided full guidance throughout the entire research. All authors approved the final manuscript.Fig S1Click here for additional data file.Fig S2Click here for additional data file.Fig S3Click here for additional data file.Table S1Click here for additional data file.Appendix S1Click here for additional data file.


Gene transcription occurs in short bursts interspersed with silent periods, and these kinetics can be altered by promoter structure. The effect of alternate promoter architecture on transcription bursting is not known. We studied the human prolactin (hPRL) gene that contains 2 promoters, a pituitary-specific promoter that requires the transcription factor Pit-1 and displays dramatic transcriptional bursting activity and an alternate upstream promoter that is active in nonpituitary tissues. We studied large hPRL genomic fragments with luciferase reporters, and used bacterial artificial chromosome recombineering to manipulate critical promoter regions. Stochastic switch mathematical modelling of single-cell time-lapse luminescence image data revealed that the Pit-1–dependent promoter showed longer, higher-amplitude transcriptional bursts. Knockdown studies confirmed that the presence of Pit-1 stabilized and prolonged periods of active transcription. Pit-1 therefore plays an active role in establishing the timing of transcription cycles, in addition to its cell-specific functions. Prolactin is a multifunctional mammalian hormone with a major role in lactation as well as other biological functions, including reproduction, immunomodulation and behavior ,2. This vs extra-pituitary) by influencing the stability or translational efficiency of alternatively transcribed messages (The human prolactin (hPRL) gene is located on the short arm of chromosome 6 and consists of 5 coding exons within a region of 10 kb. The gene has 2 distinct, alternative promoter elements that are 5.8 kb apart and show selective and tissue-specific activation . The promessages ,9,10.Pit-1  is a member of the POU family of transcription factors that are characterized by the presence of a bipartite DNA binding domain, known as the POU domain . It is eThe hPRL genomic locus has many conserved regions far upstream of the transcriptional start site. Outside of the genomic locus, the PRL gene is surrounded by over 1 Mb of noncoding DNA, a gene desert that could have a functional impact on hPRL regulation . It has In our earlier work, single-cell imaging of the hPRL WT BAC in pituitary cell lines and tissues showed that the hPRL gene displays dramatic pulses in transcriptional activity ,20. ThisGalK  coding sequence. A positive recombinant selected on galactose containing minimal media was subjected to a second round of recombination to remove the inserted GalK sequence and replace with the short (256 bp) immediate hPRL promoter sequence necessary for transcription initiation, which also contains the alternative RNA splice acceptor site at position −246 relative to Luc translation start site . Jurkat cells were cultured in RPMI  supplemented with glutamine and 10% FBS. Serum starving conditions were in media containing 0.25% bovine serum albumin in place of FBS.6 GH3 cells in a 10 cm dish or 2 × 106 Jurkat cells in 25 cm2 flask using ExGen500 transfection reagent. Media was changed 48 h post transfection and supplemented with 500 μg/mL G418. Media and antibiotic were refreshed every 3 to 4 days. Colonies formed 2 to 3 weeks after culturing in selective media were recloned into individual wells of a 48-well plate. The stable transfectant clones that were found positive for Luc expression were sequentially scaled up to large culture vessels as necessary.For stable transfection of cell lines, BAC DNA (hPRL WT BAC or PitProKO BAC) was prepared by maxiprep  and 3 μg was used to transfect 106) were washed once with PBS then lysed using 200 μL of lysis buffer . Cell lysis was aided by agitation at room temperature for 15 min, adenosine triphosphate added to a final concentration of 1 mM and Luc activity of samples measured using a FLUOstar Omega (BMG Labtech). Each experiment was performed in duplicate in 3 independent studies.GH3 cells (1 × 104 per well) were seeded into 96-well microplates  in serum-free media containing 1 mM luciferin (Biosynth). After 24 h, cells were stimulated as indicated and microplates sealed with Breathe-Easy sealing film (Sigma). Luciferase activity from each well was measured using FLUOstar Omega (BMG Labtech) over a period of 24 h while maintaining the cells at 37°C in the presence of 5% CO2. Photon counts from each well were integrated over 5 s after every 15 min. Results are shown as mean fold induction relative to an untreated control and represent triplicates of 3 independent experiments.Pituitary GH3 cells (1.5 × 106) were seeded in 10-cm dishes and left for 48 h. Chromatin immunoprecipitation (ChIP) assay was performed as described previously . Genomic DNA was removed using genomic DNA eliminating columns provided with the kit. First-strand complementary DNA was synthesized using random octamers in VILO Superscript II (Life technologies). Reverse transcription PCR for the detection of pituitary or extra-pituitary derived Luciferase mRNA was performed using forward primer 98  in PRL e2 in humid atmosphere. A bright-field image was taken before and after the luminescence imaging to track the localization of cells. Luminescence images were obtained using a Fluar 10×, 0.75 NA objective. Images were captured using a photon-counting charge coupled device camera . Sequential images, each integrated over 10 min, were acquired using Kinetic Imaging software AQM6 . The same software was used for the analysis of the imaging data. Regions of interest were drawn around each single cell, and total photon counts for individual cell areas were obtained from each image. Mean luminescence intensity data were collected after the average instrument dark count (corrected for number of pixels being used) was subtracted from the luminescence signal. Each imaging experiment was performed at least 3 times, with a representative experiment presented in figures.GH3 BAC transfectant cells and collagenase dispersed primary pituitary cells were cultured in 35-mm glass coverslip-based dishes  in the presence of 10% FBS. Luciferin (1 mM) was added at least 10 h before the start of the imaging. Cells were transferred to a Zeiss Axiovert 200 microscope in a dark room, equipped with an incubator maintained at 37°C, 5% CO2 in humidified conditions) in a dark room. Luminescence images were collected using a Fluar 10×, 0.5-NA objective (Zeiss), and captured using a photon-counting charge coupled device camera . Sequential images were taken with a 30-min integration period and then analyzed using Kinetic imaging software AQM6 (Andor). Lipopolysaccharide (LPS) was added directly to the dish . Imaging experiments were performed 3 times, with a representative experiment shown in The generation of hPRL-Luciferase rats has been described previously . Bone ma5 hPRL WT GH3 cells were seeded into 12 well plates with 15 pmol of Pit-1 Stealth RNAiTM (ThermoFisher Assay ID HSS108266) in the presence of Lipofectamine RNAimax reagent (ThermoFisher Scientific) according to the manufacturers’ protocol. Cells were also transfected in the same manner with Stealth RNAiTM small interfering (siRNA) negative control, medium guanine-cytosine duplex to control for sequence independent effects. Seventy two hours after transfection cells were used for live cell luminescence imaging experiments or lysed to assess the efficiency of Pit-1 knockdown.For siRNA transfections 1.5×10oC (Abcam), washed, and then incubated with horse radish peroxidase conjugated secondary antibodies and developed using enhanced chemiluminescence. Efficiency of Pit-1 knockdown was assessed in at least 3 experiments.After transfection with either Pit-1 siRNA, siRNA negative control or lipofectamine RNAimax reagent only, hPRL-WT GH3 cells were lysed with Laemlli sample buffer . Proteins were resolved by SDS-PAGE, transferred to nitrocellulose and blocked in 5% nonfat milk. Blots were incubated in primary antibodies over night at 4To quantify the duration of transcriptional phases raw single-cell luminescence data from 3 independent experiments for each imaging condition  was analyzed using a previously developed stochastic switch model (SSM) . This moPulsatile cycles of prolactin transcription have previously been shown in single cells from pituitary cell lines and in primary pituitary cells ,29,34-37The first aim was to compare the dynamic behavior of the WT hPRL promoter (hPRL WT BAC) in pituitary and nonpituitary cells, using primary pituitary cells and bone marrow myeloid cells from hPRL WT BAC transgenic rats  and 1B. To evaluate the influence of promoter architecture on transcriptional bursting the hPRL WT BAC was modified to delete the entire 5 kb proximal pituitary promoter, leaving the upstream exon 1a and the alternative promoter intact . This prThe hPRL alternative promoter is often referred to as the extra-pituitary promoter, based on the notion that its activity is confined to sites outside of the pituitary. Here, to assess the contribution the alternative promoter plays in the transcriptional regulation of the hPRL gene in the pituitary, we generated rat pituitary GH3 stable cell lines expressing hPRL WT BAC-Luc or hPRL PitProKO BAC-Luc. Incorporation of the entire BAC without truncation into the cell lines was confirmed following genomic DNA extraction, with PCR using primer sets spanning the entire BAC construct, including the deleted 5 kb region  spanning 163 Kb of the human PRL genomic locus and engineered it to insert the Luc reporter gene at the start of the hPRL exon 1b, thus preventing expression of the hPRL coding sequence . We haveAlternative promoters are a common occurrence in the mammalian genome and can allow diversity and flexibility in gene expression ,47. The The activity of Pit-1 is regulated in response to signal-transduction pathways by its interaction with co-repressor and activator complexes containing nuclear receptor co-repressor 1 and 3′,5′-cyclic adenosine 5′-monophosphate-response element binding protein, respectively . In respAn important finding of the present work was that manipulation of the promoter structure had clear effects on the timing and structure of transcriptional cycles in living, intact cells. We and others have previously found that the prolactin gene, like other genes studied, displays cycles of transcriptional activity that are likely to involve chromatin remodeling ,23,37,52An important challenge is to assess the physiological significance of pulsatile transcription and the role that its modulation may play on normal physiology. The present studies were performed in the pituitary GH3 cell line, which has significant limitations: it is an immortalized clonal cell line, and the cells lack functional dopamine receptors. Nonetheless, they have proved a valuable test-bed in which to explore how pituitary hormones may be regulated, and a system in which complex genetic manipulations may be trialed before conducting studies in living animals. In our previous work using transgenic hPRL-EGFP reporter rats, we found identical transcriptional pulses in living intact normal pituitary cells ,20,34,37In summary, Pit-1 plays an important role in the timing of transcription cycles, rather than simply being necessary to permit tissue-specific gene expression. The proximal promoter displays a binary  activation step, with the presence of Pit-1 and Pit-1 binding sites associated with prolongation of the subsequent “on-phase,” and multistep graded inactivation. In the absence of Pit-1 or Pit-1 binding, as occurs with the alternative upstream promoter in nonpituitary tissues, both the activation and inactivation steps of the transcriptional cycles have binary characteristics of a smaller amplitude. Pit-1 is localized in nuclear foci and dynamically partitioned, with a key role in interacting with the nuclear matrix . In addi


Escherichia coli PAIusp is a small pathogenicity island encoding usp, for the uropathogenic specific protein (Usp), a genotoxin and three associated downstream imu1-3 genes that protect the producer against its own toxin. Bioinformatic analysis revealed the presence of the PAIusp also in publically available Salmonella bongori and Salmonella enterica subps. salamae genome sequences. PAIusp is in all examined sequences integrated within the aroP-pdhR chromosomal intergenic region. The focus of this work was identification of the usp promoter and regulatory elements controlling its activity. We show that, in both E. coli and S. bongori, the divergent TyrR regulated P3 promoter of the aroP gene, encoding an aromatic amino acid membrane transporter, drives usp transcription while H-NS acts antagonistically repressing expression. Our results show that the horizontally acquired PAIusp has integrated into the TyrR regulatory network and that environmental factors such as aromatic amino acids, temperature and urea induce usp expression.The  Escherichia coli and Salmonella bongori are both members of the Enterobacteriacae, (class gammaproteobacteria) and are presumed to have diverged from a common ancestor 120–150 million years ago [Salmonella consists of two species, S. enterica and S. bongori, that have diverged between 40–63.4 Myrs ago [S. enterica is divided into six subspecies, enterica, salamae, arizonae, diarizonae, houtenae and indica [Salmonella serovars belonging to S. enterica, subspecies enterica, provoke disease in humans and other warm-blooded animals, S. bongori and the remaining five S. enterica subspecies are mostly considered nonpathogenic and commensals of cold-blooded animals. Therefore, only limited research into the remaining five S. enterica subspecies and S. bongori has been conducted. Nevertheless, S. bongori serovar 48:z35 strain RKS3044 is considered endemic in Sicily, Italy and has been reported to infect humans, mainly children aged 1 month to 3 years [S. bongori strain N19-781 was recently isolated and a draft genome sequence published [enterica subspecies do cause sporadic disease in mammals, with children and immuno-compromised individuals at highest risk [Escherichia coli is a common commensal inhabitant of the human gastrointestinal tract however, strains that produce virulence factors provoke intestinal as well as extraintestinal infections.Bacterial virulence factor genes are frequently encoded by pathogenicity islands (PAIs), inserted DNA segments that can be mobile and affect host pathobiology as well as bacterial fitness ,10. GramE. coli uropathogenic specific protein (Usp) is a genotoxin active against mammalian cells and is associated with strains that provoke prostatitis, pyelonephritis and bacteraemia [usp+E. coli strains in colorectal cancer patients [usp encodes usp and three associated downstream imu1-3 genes required for protection of the producer against its own toxin.The teraemia ,13,14. Gteraemia ,16,17,18patients . The smaE. coli (UPEC), mostly belonging to the B2 phylogroup, PAIusp is located within the aroP-pdhR chromosomal intergenic region  is a nucleoid associated protein playing a key role in nucleoid organization in Gram negative bacteria. In addition, H-NS is a global regulator of gene expression involved in regulating global transcriptional responses to environmental stress as well as a silencer of xenogenic DNA acquired by horizontal gene transfer . H-NS isusp in S. bongori and S. enterica subsp. salamae strains. In all, PAIusp was found to be inserted at the same genomic location as in E. coli usp+ strains. Furthermore, we investigated and compared usp promoter regions and promoter activities from both S. bongori and E. coli, using reporter gene expression assays as well as Usp protein levels. In both species we identified the aroP P3, as the sole promoter of the usp gene. Our results also show that the horizontally acquired PAIusp has integrated into the TyrR regulatory network and that H-NS represses expression, while environmental factors such as aromatic amino acids, temperature and urea induce usp expression.In this study, we report the presence of the highly conserved PAIusp promoter regions were PCR amplified from the uropathogenic E. coli strain TA211 which was isolated at the Institute of microbiology and immunology, Medical Faculty, University of Ljubljana, Ljubljana, Slovenia and from the S. bongori strain NCTC 12419. The E. coli Keio collection strain JW1889 and the tyrR defective JW1316 strain, were used throughout the study [The strains and plasmids used in this study are listed in he study . BacteriE. coli UTI89 Usp protein (accession no. ABE05631.1) as queried against the NCBI non-redundant protein database. For phylogenetic analysis, multiple sequence alignments  were based on a ClustalW program implemented in MEGA7 software [Genome mining for Usp homologs was carried out by PSI-BLAST and tBLASTn analysis using the software , using tE. coli K-12 MG1655 (RefSeq NC_000913), E. coli UTI89 (RefSeq NC_007946.1), S. bongori NCTC 12419 (RefSeq NC_015761.1) and S. enterica subsp. salamae RKS2993 (RefSeq NZ_JXTT01000055). To compare genomic regions of the four strains, we performed nucleotide alignment using EasyFig version 2.1 [The genomic regions of interest for comparison analysis were manually extracted from genomes of sion 2.1 , with thusp were determined by cross reference of the Conserved Domain Database and Search Service version 3.16 [A basic comparative analysis of protein or nucleotide sequences was carried out using ClustalW version 2.0 . Conservion 3.16 .All cloning was performed as described previously . Plasmidusp-CAT reporter transcription fusions, the promoter region was PCR amplified from the S. bongori NCTC 12419 chromosomal DNA using the Bong_L_Pst_F and Bong_L_Bst_R primer pair  and subsequently 0.1 mL of the diluted culture was inoculated into a series of tubes with increasing concentrations of chloramphenicol and grown for 24 h. Background MIC values from cells carrying a promoterless pJCAT, were subtracted.All of the plasmids harboring promoter fragments  were traPlasmids harboring promoter fragments  were traE. coli K12 MG1655 strain, transformed with plasmids pUSP4L (containing UPEC usp gene under native P3 promoter) and pCUPBI (containing UPEC usp gene under native S. bongori P3 promoter), were grown at the designated temperatures with shaking in LB medium. After 24 h the cells, normalized to optical density, were collected by centrifugation. N-terminally His6-tagged Usp was affinity purified by Ni-NTA metal affinity chromatography  and eluted with 200 µl of buffer C . Proteins in eluate were precipitated with TCA and washed with ice-cold acetone. The pellet was dried and resuspended in SDS loading buffer, separated on 12% SDS-PAGE gel and transferred at 200 mA for 90 min onto a PVDF membrane  for immunoblotting. The membrane was blocked in Tris-Buffered saline with 0.1% Tween 20 (TBS-T) containing 5% (w/v) bovine serum albumin (BSA). The membrane was subsequently incubated for 2 h with mouse anti-penta-histidine antibodies (Thermo Fisher Scientific) for anti-His staining, followed by 1 h incubation with horseradish peroxidase-conjugated rabbit anti-mouse IgG. The protein bands were visualized by an enhanced chemiluminescence kit .E. coli BL21 (DE3) cells containing the pET8HT or pET9HT expression plasmids were grown in LB medium and induced with 0.8 mM of isopropyl β-D-thiogalactopyranoside (IPTG) for 4 h at 37 °C. N-terminally His6-tagged TyrR protein was affinity purified by Ni-NTA metal affinity chromatography (Qiagen). The native purification protocol was followed according to the manufacturer’s instructions. Protein purity was assessed on a 10% SDS-PAGE gel, followed by Coomassie blue staining and the eluate was dialyzed overnight against a buffer  at 4 °C. The concentration of the purified TyrR protein was determined using NanoDrop 1000 (Thermo Fisher Scientific) with the extinction coefficients at 280 nm of 36,245 M−1 cm−1.To isolate the purified TyrR and H-NS proteins, 6-tagged TyrR protein (~15 nM), in a total volume of 10 µl of binding buffer , 25 µg/mL bovine serum albumin (BSA)) with added phenylalanine (0.1 mM) and ATP (0.1 mM). Protein-DNA binding reactions were incubated at 37 °C for 30 min and then analyzed on 2% agarose gel . Protein-DNA complexes were resolved at 60 volts at 4 °C. The DNA fragments were stained with ethidium bromide and visualized. Binding reactions with the purified H-NS protein were performed as described previously [The reactions for EMSA assay were carried out as described previously with modifications . Bindingeviously .Usp protein levels were quantified using GeneTools .Data analyses were performed using GraphPad PRISM version 6.01 software. Statistically significant differences in gene expression from each promoter were determined by two-way analysis of variance (ANOVA) with post-hoc comparisons using the Tukey multiple comparison test.E. coli generally have a size of 30–100 kb and are associated with genes for tRNA, encode integrases and are flanked by direct repeats, usp is encoded on a small approximately 4 kb pathogenicity island, encoding only usp and the tightly associated imu genes. Our analysis showed that PAIusp is in all publicly available + E. coliusp genome sequences, inserted into the intergenic region aroP-pdhR. The aroP gene encodes the aromatic amino acid permease and pdhR, the pyruvate dehydrogenase complex regulator. Interestingly, bioinformatic analysis revealed the presence of PAIusp homologs in all publicly available Salmonella bongori genomes, including the clinical isolate N268-08 [Salmonella enterica subsp. salamae genomes, inserted at exactly the same genome position as in all usp+E. coli genomes , an extended C-terminal S-type pyocin domain (PF06958) and a colicin nuclease domain (PF12639) [E. coli and S. bongori strains, revealed ~76% sequence identity. Furthermore, the additional ORFs encoding Usp homologs in Salmonella enterica subsp. salamae strains show protein sequence identity of ~82% and ~77% compared to Usp from S. bongori strains and E. coli strains, respectively. All of the above-mentioned proteins from the various bacteria have the same domain structure as the Usp from E. coli  similar to the average G + C content of an Escherichia genome (~51%) . On the other hand, all usp and associated immunity protein genes (imu1-3) have considerably lower G + C contents  indicating acquisition via horizontal gene transfer  , in three and two bases, respectively (Herein we investigated activities of the t codon) . As in bmoter P3 , locatedectively .S. bongori NCTC 12419 aroP-usp intergenic region, was investigated using the promoter-probe pJBLC plasmid with a chloramphenicol acetyl transferase reporter gene (CAT). Fragments with promoter activity drive CAT expression, resulting in resistance to chloramphenicol , without the two (weak and strong) TyrR binding boxes, was approximately two fold lower, compared to that of the full length fragment indicating, that the TyrR protein acts as a transcription activator of the S. bongori P3 promoter. Furthermore, no significant effect on MIC of chloramphenicol was observed, if only the weak TyrR box was excluded . The smallest DNA fragment that could support significant expression was the 111 bp region harboring the strong TyrR box and the intact P3 promoter (not shown). Interestingly, when the 111 bp DNA fragment was extended for 64 bp (pJBPTD3), MIC of chloramphenicol was, in comparison to the full-length fragment (pJBLC), 4.4 fold higher. The same observation was made by Wang et al. (1997b) for the E. coli aroP promoter P3. They proposed that E. coli P3 and P1 have AT rich associated elements which could function as UP sequences and confer higher transcription levels  to CAAGCT  and 2 fold , respectively. On the other hand, modifications of the -35 regions exhibited no significant effect on promoter activity  and in the t-yrR defective strain (JW1316) in minimal medium with or without phenylalanine.In activity . To asce+tyrR strain, the aromatic amino acid phenylalanine provoked a statistically significant increase in expression from both the S. bongori and E. coli P3 promoters 2-fold  and 1.6 fold , respectively  indicating, that binding of the TyrR protein to the P3 upstream region, is critical for activation and regulation.Our results showed that in the ectively . On the aroP-pdhR in E. coli strain K-12. Here we demonstrated that the TyrR protein also specifically binds to the TyrR boxes within the S. bongori aroP-usp intergenic region. Incubation of the purified His6-tagged TyrR protein, with a DNA fragment encompassing the wild type aroP-usp intergenic region (403 bp) provoked, a mobility shift consistent with TyrR-DNA complex formation  and -hns defective E. coli strain (JW1225).To ascertain whether other transcription factors are involved in regulation of ootprint . Three pusp-lacZ fusions as well as higher Usp protein levels in the -hns defective E. coli strain compared to the wild type indicating that H-NS acts as a repressor of usp expression  and 2.1 fold , respectively . Induction of S. bongori usp promoter activity was comparable in the presence of 0.6 M urea and 0.4 M urea. Surprisingly, we observed an ~1.8– fold  reduction of E. coli P3 activity in the presence of 0.6 M urea compared to 0.4 M urea. On the other hand, 0.3 M NaCl provoked no significant increase in P3 promoter activity (data not shown).Our results showed statistically significant increases in β-galactosidase activity from ectively . Howeverusp, usp promoter activity. Point mutations and DNA rearrangements are significant for evolution and speciation of bacterial species nevertheless HGT, the ability to acquire foreign genetic material, enables much more rapid adaptation and survival in novel ecological niches [E. coli and Salmonella spp., that presumably occurred approximately 140 million years ago [Here we present a study of the dissemination and regulation of the horizontally acquired PAIl niches ,44. HGT l niches ,46. A paears ago .usp. Interestingly, S. bongori was previously shown to have effectors with virulence associated homologues in enteropathogenic E. coli and enterohemorrhagic E. coli [E. coli, S. bongori and S. enterica subsp. salamae strains indicates a common yet unknown ancient source and that acquisition via HGT occurred only once, or alternatively, that the aroP-pdhR intergenic region represents a specific integration site. Regulatory systems control gene expression and genes acquired via HGT must be in the new host appropriately expressed. While exogenous sequences can be eventually incorporated into preexisting regulatory networks, expression may initially be downregulated by specific “xenogenic silencing proteins”, such as the H-NS family of proteins and others, that bind to AT-rich DNA [E. coli and S. bongori the horizontally acquired PAIusp gene cluster appears to have evolved new regulation after transfer and integration into the aroP-pdhR intergenic region, enabling expression of an otherwise silent aroP promoter P3. Thus, while usp-lacZ fusions showed significantly higher levels of expression from the S. bongori P3 promoter compared to that of E. coli P3, higher Usp protein levels from the latter could indicate more efficient translation possibly, to fine tune usp expression in particular species and subspecies inhabiting distinct ecological niches. Furthermore, a number of differences in the E. coli and S. bongori regulatory regions, such as position of and subtle differences in H-NS binding sequences as well as possibly in DNA topology, could be responsible for the observed higher expression of the S. bongori usp-lacZ fusion.Our analysis of publicly available genome sequences, indicated limited dissemination of the PAI E. coli . The prerich DNA . Furtherusp usurped the divergent TyrR regulated aroP P3 promoter resulting in adjustment of PAIusp gene expression with amino acid availability in the environment. This process required specific promoter mutations which enabled activation of the otherwise silent promoter P3.Integration of virulence factor genes acquired by HGT often does not suffice to promote pathogenicity or to exhibit a newly acquired phenotype ,49,50 pousp expression in both bacteria, making usp a new member of the TyrR regulon. This is the first report of TyrR mediated expression of a virulence gene in E. coli and S. bongori, extending the significance of the TyrR regulon. Nonetheless, evidence indicates that TyrR [Yersinia pestis pathogenesis and extracellular survival/proliferation [aroE and aroA) play a crucial role in Y. pestis fitness in deep tissues during infection [Listeria monocytogenes and Shigella flexneri [In our study TyrR in conjunction with aromatic amino acids, induced a significant increase in hat TyrR  is direcferation . Additionfection . Aromatiflexneri ,54.E. coli and S. bongori native promoters in a hns defective strain compared to the wild type. In accordance, usp-lacZ expression from the E. coli promoter was also higher in the hns mutant but, in contrast, expression from the S. bongori promoter was lower. These results indicate that in S. bongori additional, possibly indirect posttranscriptional mechanisms involving H-NS could regulate Usp synthesis. Furthermore, deletion of the promoter region harboring the putative H-NS binding site, significantly increased usp promoter activity as demonstrated by an increase in MIC for chloramphenicol from plasmid pJBPTD3 (usp promoter activity and Usp protein levels were investigated and compared following growth at a number of temperatures. Indeed, while from the E. coli promoter the wild type strain produced approximately 5 fold higher levels of Usp at 37 °C and 42 °C than at 25 °C, in the hns mutant, Usp levels were comparable at all three temperatures indicating, that in E. coli H-NS plays a significant role in temperature dependent usp regulation. At the molecular level, temperature provokes alterations in local DNA structure [E. coli usp-lacZ fusion temperature dependent expression was observed in the hns defective strain indicating that, at the level of promoter activity, other as of yet unresolved factors, are also involved. Additional studies will be performed to further unravel the complex molecular mechanisms regulating usp expression in E. coli and S. bongori in response to environmental cues.H-NS is a repressor of a number of virulence factor genes frequently encoded on horizontally acquired pathogenicity islands, preventing their expression under non-permissive conditions, i.e., outside the host. The protein consists of an N terminal dimerization domain, a second, central dimerization domain and a C-terminal domain for DNA binding. The N terminal domain harbors four α-helices that allow self-association by contacts “head-to-head” and “tail-to-tail”. These interactions may occur simultaneously allowing H-NS oligomerization to form nucleoprotein filaments that are either bridged or linear. H-NS compacts DNA, repressing expression particularly at the level of transcription ,56,57. O pJBPTD3 A. Tempertructure  as well tructure . NeverthE. coli receptor or transcription regulator that responds to urea has been identified. As UPEC colonize the urinary tract they are exposed to high osmolality, due to significant amounts of urea and inorganic ions in urine. Previous studies have shown that H-NS plays a significant role in NaCl induced changes in osmolarity. However, our results demonstrate that urea, but not NaCl, mediate activation of P3 independently of the transcriptional regulator TyrR. Indeed, a previous gene array study also revealed that usp was upregulated in the presence of urea [E. coli and S. bongori. In UPEC strains, the presence of urea in urine could trigger upregulation of usp expression in the urinary tract.Urea freely penetrates cell membranes and destabilizes protein conformation , however of urea . We therusp gene encoded by PAIusp. We further demonstrate that usp is a novel member of the TyrR and H-NS regulons due to its integration into the aroP pdhR intergenic region where the aroP P3 promoter drives its expression. TyrR and H-NS play antagonistic roles in regulating usp expression, with usp P3 promoter activity stimulated by TyrR and repressed by H-NS. In the host, various environmental factors such as aromatic amino acids, temperature as well as urea could induce significant increases in usp expression. Following acquisition of PAIusp by a common ancestor, species divergence and subsequent mutations in the usp P3 promoter region fine-tuned expression in the bacterial hosts.In conclusion, our results highlight the complex regulation of the


RNA polymerases initiate transcription at DNA sequences called promoters. In bacteria, the best conserved promoter feature is the AT-rich -10 element; a sequence essential for DNA unwinding. Further elements, and gene regulatory proteins, are needed to recruit RNA polymerase to the -10 sequence. Hence, -10 elements cannot function in isolation. Many horizontally acquired genes also have a high AT-content. Consequently, sequences that resemble the -10 element occur frequently. As a result, foreign genes are predisposed to spurious transcription. However, it is not clear how RNA polymerase initially recognizes such sequences. Here, we identify a non-canonical promoter element that plays a key role. The sequence, itself a short AT-tract, resides 5 base pairs upstream of otherwise cryptic -10 elements. The AT-tract alters DNA conformation and enhances contacts between the DNA backbone and RNA polymerase. Escherichia coli, a multisubunit core RNA polymerase (α2ββ') binds one of seven dissociable σ factors to recognise promoter DNA  and -35 elements (5′-TTGACA-3′) (70 and the -10 element occur only in the context of single stranded DNA (All living organisms transcribe their genomes using the enzyme RNA polymerase . The proymerase αββ' bindsymerase αββ' bindsGACA-3′) . For insnded DNA ,5.E. coli genome acquired by horizontal gene transfer have an unusually high AT-content . RNA polymerase was mixed with either WT or R451A σ70 then added to reactions for 10 min at 37°C. RNA products were visualized on a 7% denaturing polyacrylamide gel. RNAI transcript was used as a loading control. Full gel images are shown in b et al.  and the   tailed with 5′-3′ exo- Klenow (NEB). NEXTflex barcoded adaptors (Bioo Scientific) were attached by ligation. Following elution, complexes were de-crosslinked by boiling. The resulting DNA libraries were amplified by PCR and quantified by Qubit analysis before pooling and sequencing using an Illumina MiSeq instrument. The raw data are available from ArrayExpress (E-MTAB-8778).Experiments were done according to the protocol of Haycocks et al.  using st38 binding were called if the average read depth was 4 or above. The same analysis was applied to results of a ChIP-seq assay of σ38 binding in the parent strain RPB104 . The different promoters are illustrated schematically alongside LacZ activity measurements in Figure lacZ expression. Addition of the partial -35 element had no impact. Conversely, addition of either AT-tract variant resurrected promoter activity. This activation increased further when a partial -35 element was also present. To confirm our observations, selected promoters were cloned upstream of the λoop terminator in plasmid pSR. Transcripts terminating at λoop can be detected following electrophoresis. The RNAI transcript is derived from the pSR replication origin and serves as an internal control. No transcripts were produced from promoters lacking AT-tracts . We examined 103 promoters containing ATR; all were transcriptionally active . The control promoter is dependent on a near consensus -35 hexamer and has a G at position -18 . We next replaced the AT-tract upstream of each -10 element with a GC-rich sequence. Only promoters requiring σ70 R451 were inactivated when the AT-tract was removed  if H-NS was absent . Conversely, σ38 binding decreased at most other genomic regions (H-NS low). Figure 38 binding within H-NS targeted genes. Multiple peaks for intragenic σ38 binding are evident in Δhns (red) but not wild type (blue) cells.Our search for promoter motifs provided evidence that AT-tracts play a key role at happenstance promoters Figure . Hence, ′ motifs . Further8 Figure , bottom 8 Figure . Hence, rivative . The pro70 and the -10 hexamer only occur after DNA unwinding (70 side chain R451 and the double helix backbone. Hence, otherwise cryptic -10 elements are able to participate in the process of transcription initiation. Many intragenic promoters also function with σ38 bound RNA polymerase. This may explain why inactivation of rpoS is a pre-requisite for deletion of hns in Salmonella spp. (We previously noted the widespread spurious transcription of AT-rich horizontally acquired genes . The phenwinding . In thisnwinding . Consistnwinding ,28–30. Tlla spp. .The simplest explanation for the abundance of promoters within AT-rich genes is chance occurrence . HoweverE. coli genome (i.e. ∼50%, Figure E. coli RNA polymerase could be hardwired to initiate transcription inefficiently at DNA sequences with an AT-content resembling coding DNA. We speculate that the AT-content threshold, above which promoters spontaneously arise, will differ depending on the genome AT-content of a given organism. Similarly, we predict that RNA polymerases isolated from bacteria with AT-rich genomes could be adapted to such templates and generate fewer spurious transcripts compared to the E. coli enzyme. In summary, we provide an explanation for the widespread occurrence of spurious promoters within horizontally acquired sections of the E. coli genome. Our data also have implications for our understanding of RNA polymerase specificity and promoter evolution.Intriguingly, the frequency of chance promoter occurrence increases only when the DNA AT-content exceeds that of the ArrayExpress accession E-MTAB-8778.gkaa244_Supplemental_FilesClick here for additional data file.