One member of the W family of human endogenous retroviruses (HERV) appears to have been functionally adopted by the human host. Nevertheless, a highly diversified and regulated transcription from a range of HERV-W elements has been observed in human tissues and cells. Aberrant expression of members of this family has also been associated with human disease such as multiple sclerosis (MS) and schizophrenia. It is not known whether this broad expression of HERV-W elements represents transcriptional leakage or specific transcription initiated from the retroviral promoter in the long terminal repeat (LTR) region. Therefore, potential influences of genomic context, structure and orientation on the expression levels of individual HERV-W elements in normal human tissues were systematically investigated.Whereas intronic HERV-W elements with a pseudogene structure exhibited a strong anti-sense orientation bias, intronic elements with a proviral structure and solo LTRs did not. Although a highly variable expression across tissues and elements was observed, systematic effects of context, structure and orientation were also observed. Elements located in intronic regions appeared to be expressed at higher levels than elements located in intergenic regions. Intronic elements with proviral structures were expressed at higher levels than those elements bearing hallmarks of processed pseudogenes or solo LTRs. Relative to their corresponding genes, intronic elements integrated on the sense strand appeared to be transcribed at higher levels than those integrated on the anti-sense strand. Moreover, the expression of proviral elements appeared to be independent from that of their corresponding genes.Intronic HERV-W provirus integrations on the sense strand appear to have elicited a weaker negative selection than pseudogene integrations of transcripts from such elements. Our current findings suggest that the previously observed diversified and tissue-specific expression of elements in the HERV-W family is the result of both directed transcription  and leaky transcription of HERV-W elements in normal human tissues. Recent analyses of the transcriptional landscape of human cells and tissues indicate that far more than the 2% of human genome that encodes proteins exhibit tissue-specific and regulated transcription resulting in a large number of different RNA species without apparent coding capacity . Such anERVWE1 locus on chromosome 7q. This locus harbors a member of the HERV-W family with an open reading frame in the env gene that encodes a protein denoted syncytin [Five to eight percent of the human genome consists of retroviral sequences acquired during evolution . These ssyncytin . This prsyncytin .ERVWE1, the human genome harbors hundreds of elements in the HERV-W family. The prototypical HERV-W provirus consists of an internal sequence with the gag, pol and env genes flanked by identical 5' and 3' long terminal repeats (LTRs). In addition to such proviral elements (e.g. ERVWE1), elements that bear the hallmarks of processed pseudogenes (denoted pseudoelements) and elements that cannot be classified into either of these categories (denoted truncated elements) are present in the human genome [ERVWE1 locus also indicate promoter activity in the 5'-LTR as well as strong enhancer activity in flanking DNA conferring the specific and high level expression in syncytiotrophoblasts [gag sequences using high-resolution melting temperature analysis. While a highly diversified expression across both individuals and tissues was evident, tissue-specific profiles were also observed [In addition to n genome ,7. Moreon genome ,7. Previn genome . Studieshoblasts . We receobserved . Mappingobserved -13, i.e.These observations raise questions regarding the mechanisms responsible for generating such transcripts. Do they constitute products of leaky transcription of adjacent genes, unprocessed pre-mRNAs or are they independently initiated from the retroviral LTR (or other promoters)? The purpose of the present study was therefore to systematically determine the influence of the genomic context, structure and orientation on the expression of HERV-W elements. Understanding the transcriptional regulation of HERV-W elements will contribute to a better understanding of some of the least studied regions of the human genome and can shed light on mechanisms underlying the associations between expression of HERV-W elements and complex human diseases, such as MS ,15 and sApproximately 28% of genomic loci with HERV-W elements identified here were observed within introns of annotated genes. No significant difference in this distribution was observed between the three categories investigated, i.e proviral elements, solo LTRs and pseudoelements. As can be seen from Table To investigate if the genomic context of HERV-W elements determines their expression levels, we assayed the levels of transcripts from 14 HERV-W loci located in introns of genes (i.e. intronic elements) and from 10 HERV-W loci located outside of annotated genes  elements were detected at levels comparable to those encoding syncytin and at higher levels than those from pseudoelements  were calculated for each of the gene-element pairs. These ΔCt-values were subsequently compared across such pairs grouped according to structure (i.e. presence/absence of U3 region or solo LTR) and orientation (sense or anti-sense to the coding gene). As indicated by a one-way ANOVA, ΔCt-values between these six groups differed significantly  in the sense orientation were expressed at levels exceeding those observed for the corresponding spliced protein-coding mRNA. These relative levels differed from the five other groups of elements investigated. Relative to their corresponding coding transcripts, proviral elements in the anti-sense orientation were expressed at higher relative levels than pseudoelements in the anti-sense orientation. Differences in the relative expression levels were also observed between pseudoelements in the sense and in the anti-sense orientations. Based on these observations it appeared as if intronic sense transcripts were present at approximately five cycles (i.e. ~32 times) higher levels than intronic anti-sense transcripts in the regions investigated. However, this finding was not consistent across all pairs since transcripts 3' of solo LTRs oriented sense and anti-sense were detected at similar relative levels  were calculated as described above. As can be seen from Figure +) appeared to display significantly elevated transcriptional activities following influenza A/WSN/33 infection relative to their corresponding coding transcripts as indicated by the significant increase in average ΔCt-values. A similar but non-significant finding was also observed for solo LTRs. In contrast, no relative change was observed for transcripts 3' of pseudoelements (U3-) by comparing uninfected control and infected cells.Previous studies from our laboratory indicated transcriptional activation of HERV-W elements, including l origin . To inveAs for other HERV-families, the majority of surviving elements in the W family are located outside of protein-coding genes . The perIn the present study we found that the genomic context, structure and orientation of elements in the HERV-W family appear to influence their expression. While systematic effects of these features were observed throughout a range of normal human tissues, it should also be noted that a considerable degree of variation in transcripts levels was observed across individual tissues and elements. From a methodological point, it should here be noted that the variation, evident in additional file in vivo. To avoid issues regarding specificity and sensitivity, we here assayed transcription harboring non-repetitive regions proximal to the 3' end of the HERV-W elements investigated. Genomic distances were verified by PCRs on human genomic DNA. Clearly distinguishable amplicons of expected sizes were, however, generated only after careful optimization illustrating the difficulties involved when using primers in repetitive elements. By the approach taken here, transcriptional initiation outside of the LTR cannot be distinguished from LTR-initiated transcription. Moreover termination of transcription 5' of the assayed region would lead to underestimations of transcription levels. In only 3 out of 22 cases were we unable to "link" elements with their corresponding non-repetitive 3'-regions at the level of transcription in the samples investigated. Distance of the assayed region from the site of oligo-d(T) initiated reverse transcription can also confound results in this type of study. By comparing the transcriptional activity 5' with that 3' of six intronic HERV-W loci in eight tissues we observed significantly higher expression level 3' of proviral elements but not 3' of pseudoelements. This finding further support transcription originating in the U3 region of the LTR which is expected to lead to higher transcript-levels from regions 3' than 5' of the promoter. Expression levels of the two regions would presumably be more uniform if our assays detected the same un-spliced pre-mRNA of coding genes, as for the pseudoelements investigated. We even observed lower transcript levels 3' than 5' of the pseudoelements in the anti-sense orientation which may be due to a longer distance from the site-of-initiation of reverse transcription. These findings in normal tissues are thus in agreement with previous studies of the ERVWE1 locus in placental cells and a more recent study of intergenic HERV-W loci in testicular tumors where transcription of intergenic proviral elements appeared to be initiated in the U3 region of the LTR [gag, pol and/or env) also has some impact on the levels of transcripts generated. A similar observation was also observed among elements in the HERV-K family suggesting that regulatory elements are present in the internal sequence [Based on the current data, intronic elements appear to be, in general, expressed at higher levels than their intergenic counterparts, regardless of the structure of their LTR. In their study of human-specific HERV-K promoter activities, Buzdin and coworkers, also observed a major influence of genomic context on the activity of HERV-K LTRs. A larger proportion of LTRs in gene-rich regions appeared transcriptionally active as compared to LTRs in gene-poor regions . Taken t the LTR . Functiosequence . NoticeaRegardless of U3 region, the orientation of intronic HERV-W elements appears to be an important determinant of their level of expression with elements on the sense strand being expressed at higher relative levels than those on the anti-sense strand. That this does not apply to solo LTRs, further supports the conclusion that the internal sequences present in both proviral elements and pseudoelements can modify the level of transcription.According to our linear regression analyses, transcription of intronic proviral elements and solo LTRs, in general, appeared to be independent of their corresponding genes. Expression of most, but not all, intronic elements lacking the U3 region on the other hand appeared to be dependent on expression of their corresponding genes.gag sequence following virus infection extends our previous findings in human cell-lines [gag amplicons in such cultures serve to illustrate the large number of transcribed loci in these cells and the alterations in the proportions of transcripts from these following virus infection, as we previously observed in serum-starved cells [Transcriptional regulation of HERV-W elements is further supported by our observations in primary human fibroblasts. The overall increase in relative levels of transcripts containing ll-lines . Our quaed cells . Indepened cells ,9 and inWe observed an inverse correlation between expression of intergenic HERV-W elements and their corresponding downstream genes. These correlations appeared independent of viral structure and might therefore reflect general mechanisms such as structural changes in the chromatin  or methyIn addition to the general influences of genomic context, structure and orientation noted above, other mechanisms are also likely to affect the level of transcription of individual HERV-W elements. For example, differences in methylation levels of the individual LTRs, as reported for LTRs in the HERV-E family  and for gag sequences [gag expression, dendrograms could be constructed resembling those based on gene expression patterns. Based on the present findings we suggest that both LTR mediated and leaky transcription of the human genome contribute to such tissue-specific patterns of HERV-W transcripts. Thus, the aberrant HERV-W expression associated with complex human diseases such as MS [in vivo findings support previous findings in vitro [ERVWE1 locus, contribute functional transcripts, however, remains to be established.Earlier studies have reported that a number of endogenous proviral LTRs in the HERV-E, -I, -R and -H families have been co-opted during human evolution as alternative promoters of individual genes conferring additional transcriptional control ,33. Moreequences . This apch as MS ,15 and sch as MS ,17 may bin vitro  that thein vitro . If membWe found a broad and variable expression of HERV-W elements in normal human tissues. Previously observed diversified and regulated expression of HERV-W elements appears to be due to both directed and leaky transcription of the human genome.HERV-W loci were identified by BLAT searches  of the henv of the proviral element in the ERWE1 locus previously described [The assay for the escribed  was usedTo verify genomic locations and to link HERV-W elements and their corresponding assayed 3'-regions at the transcript level, forward primers inside 22 elements were designed -primed cDNA from spleen, thymus, prostate, testis, ovary, intestine, colon and peripheral leukocytes was purchased from Clontech . These tissue cDNA were diluted 1/10 to reach a concentration of 0.1 ng/μl and then used as templates.Fibroblast cell cultures were established as previously described , The stuRNA extraction and reverse transcription was carried out as previously described  using rePCR reactions were run on a GeneAmp PCR System 9700  using 1× TITANIUM™Taq DNA polymerase mix  and 1 μl template from in 20 μl reactions. Amplicons were separated in 1% agarose gels, stained with GelRed  and visualized on a Chemi Doc system .Real-time PCR reactions were run on an ABI Prism 7000 sequence detection system (Applied Biosystems) using Platinum SYBR Green qPCR SuperMix UDG (Invitrogen) with 0.25 μM of forward and reverse primers, and 1 μl cDNA template in triplicate 25 μl reactions. All assays consistently generated amplicons with a single melting temperature (i.e. a single peak on the derivative melting curve) as determined during an obligate post-amplification melting analysis. Efficiencies for all assays were comparable as determined from standard curves generated from amplifications of serial dilutions of a human cDNA pool . For some experiments, levels of transcript encoding β-actin were used as an endogenous control and these were quantified using the forward (5'-ATCCTAAAAGCCACCCCACT-3') and reverse (5'-CTCAAGTTGGGGGACAAAAA-3') primers.gag expression were performed as previously described [Quantitative and qualitative analyses of HERV-W escribed .-ΔΔCt [post hoc tests where appropriate. P-values < 0.05 were considered significant.Threshold cycle (Ct) values from the exponential phase of PCR amplification plots for each target transcript were exported to Excel  for further calculations of averages or Δ Ct-values. For some experiments, Ct values were normalized to that encoding β-actin. From these values, fold-differences in the levels of transcripts among groups were calculated according to the formula 2 -ΔΔCt . The PRIFL designed the experiments, performed the analyses and drafted the manuscript. CN performed statistical analyses and revised the manuscript. RY revised the manuscript. HK conceived the study and revised the manuscript. All authors have read and approved the final manuscript.Average levels of transcripts 3' of intronic/intergenic HERV-W elements across human tissues. Levels of transcripts  3' of intronic (n = 14) and intergenic (n = 10) HERV-W elements across human tissues. Levels of transcripts encoding syncytin are also shown as a point-of-reference (A). Levels of transcripts 5' and 3' of individual elements (B). Transcript-levels of elements were determined by real-time PCR using assays described in materials and methods.Click here for fileRelative expression of HERV-W elements and their corresponding genes. Relative levels of transcripts 3' of intronic (A), intergenic (B) HERV-W elements, spliced exons flanking intronic HERV-W elements (C) and spliced transcripts from genes located 3' or 5' of intergenic HERV-W elements (D) across tissues and elements/genes. Levels of transcripts encoding β-actin were here used as an endogenous control.Click here for fileAverage levels of transcripts 3' of intronic HERV-W elements and their corresponding genes across individual tissues. Levels of transcripts 3' of proviruses, pseudoelements and solitary LTRs (A). Levels of spliced transcripts using primers in exons flanking introns where such elements are integrated (B).Click here for fileAverage levels of transcripts 3' of intergenic HERV-W elements and adjacent genes. Average levels of transcripts 3' of intergenic proviral elements  and pseudoelements  (A). Linear regression analyses of levels of transcripts 3' of intergenic U3+ and U3- elements and corresponding downstream genes (B).Click here for filePrimers located inside HERV-W elements. This file describes the targets, product sizes, sequences and positions of primers used to link HERV-W elements with their corresponding assayed 3'-regions.Click here for file


E. coli dissecting and analyzing their inner workings and highlightin their differences. Then, following an integral approach, we identify their commonalities and outline some of the principles exploited by the cell to effectively and globally reprogram the transcriptional machinery. These principles provide guidelines for developing biological synthetic circuits enabling an efficient and robust response to sudden stimuli.In bacteria, transcriptional regulation is a key step in cellular gene expression. All bacteria contain a core RNA polymerase that is catalytically competent but requires an additional σ factor for specific promoter recognition and correct transcriptional initiation. The RNAP core is not able to selectively bind to a given σ factor. In contrast, different σ factors have different affinities for the RNAP core. As a consequence, the concentration of alternate σ factors requires strict regulation in order to properly control the delicate interplay among them, which favors the competence for the RNAP core. This control is archived by different σ/anti-σ controlling mechanisms that shape complex regulatory networks and cascades, and enable the response to sudden environmental cues, whose global understanding is a current challenge for systems biology. Although there have been a number of excellent studies on each of these σ/anti-σ post-transcriptional regulatory systems, no comprehensive comparison of these mechanisms in a single model organism has been conducted. Here, we survey all these systems in  However, it requires an additional dissociable σ factor for specific promoter recognition and correct transcription initiation. Most σ factors belong to the σ70family, whose members contain two domains with a high degree of sequence and structural conservation: 1) σ2, which binds the RNAP β′ subunit coiled-coil and the -10 promoter element, and 2) σ4, which binds the RNAP β subunit flap and -35 promoter elements. An additional σ family, σ54, does not share any significant sequence similarity with the σ70 family and is functionally distinct [In bacteria, the regulation of gene expression is the basis for adaptability, morphogenesis, and cellular differentiation. From all the different regulatory layers, regulation of transcription initiation is a very important step for controlling gene expression. All eubacteria contain a single ∼380-kDa multi-subunit αEscherichia coli, the vast majority of transcriptional “housekeeping” is accomplished by the RNAP core in complex with σ70. However, six other σ factors provide selectivity for transcribing different sets of genes. These genes, required for adaptive responses, have related functions and are transcribed in response to specific stimuli : σS (σ38) is the general stress factor associated with stationary phase and a variety of growth-impairing stresses; σH (σ32) controls heat shock responsive promoters; σF (σ28) is used for flagellum-related functions; σE (σ24) controls responses to extracytoplasmic or extreme heat shock stress; σFecI (σ19) is required in iron transport for transcription of the ferric citrate transporter; and σN (σ54) controls promoters for nitrogen assimilation. This repertoire of alternative σ factors reflects the lifestyle of E. coli as a gut commensal bacterium [In es; σH σ3 controlsE. coli cells, the number of RNAP holoenzymes involved in transcription is 1300 molecules per cell. There are also other 700 molecules per cell as free RNAP that has to be bound to any σ factor but is not involved in transcription [E. coli to sudden environmental changes [In growing cription . In the miliéu interieur , homeostasis mediated by feedback has been confirmed as one of the fundamental and ubiquitous mechanisms underlying life. One hundred years after Bernard’s seminal idea, cybernetics was developed by Norbert Wiener through formalizing the concept of feedback and mathematically demonstrating that negative feedback is a necessary condition to give rise to a teleological  behavior in any system. In this control scheme, robust adaptation is created by feeding the output back into the input of the system, enabling it to respond to minimal deviations from the goal [Since the great French scientist Claude Bernard 1813-1878), founder of modern experimental physiology, considered the stability of the the goal , 6. This8, foundeE. coli.Molecular studies have determined specific roles for products of individual genes, but genomics has shown the decline of the traditional roles that have been assigned to proteins, such as σ factors and regulatory proteins. Despite their different regulatory mechanisms both proteins exerttranscriptional control in terms of their causal effects and both are important to the understanding of the regulatory hierarchy governing a bacterium . From thE. coli are transcribed by the σ factor that they repress, creating a negative feedback circuit between the σ factor and its corresponding anti-σ.Adequately orchestrated gene expression is necessary for the successful adaptation and survival of cells. A strategy in bacteria to control gene expression is to displace the housekeeping σ subunit of the RNAP and replace it with an alternative σ factor. Then, with the new σ, the RNAP holoenzyme, can recognize and initiate transcription for a different type of promoter. Consequently, genes not being expressed can be transcribed drastically reprogramming global gene expression and enabling a proper response to an environmental cue . To accoE. coli required for growth under optimum conditions is ensured by the σ70 factor. σ70 has two anti-σ factors, Rsd and HscC, which disable σ70-dependent transcription at the onset of stationary phase and also possibly heat shock, respectively. The latter is suggested by in silico evidence [The transcription initiation of genes in evidence , so furtrsd has been validated in vitro [38 and σ70, respectively. In the promoter region of hscC two promoters, σ70 and σ32, have been proposed by in silico methods [1).The transcriptional control of in vitro . It is t methods . For gra70 factor Rsd binds to the region 4 helix-turn-helix motif of σ70, which is responsible for -35 promoter recognition, blocking association with the RNAP core [38, which is the σ factor used upon entry into stationary phase [E. coli anti-σ factors  also bind to multiple regions of their cognate σ factors to prevent the holoenzyme formation [In stationary phase, the putative anti-σNAP core . This alry phase , 23-32. ormation .70 and may function as a negative modulator of housekeeping gene expression. This anti-σ is highly homologous to DnaK and HscA [E. coli. The overexpression of hscC causes severe inhibition of cell growth. Primer extension and beta-galactosidase assays have shown that overexpression of HscC reduces σ70-dependent promoter activity. An in vitro transcription assay revealed that HscC inhibits σ70-dependent transcription of the groESL promoter. In addition, co-purification analysis showed that σ70 co-eluted with HscC [70, as suggested by the fact that chimeric proteins carrying the ATPase domain of HscC combined with other domains of either HscC or DnaK form complexes with σ70 [38 mutant backgrounds have suggested that P2 is the primary promoter for the transcription of rsd [38 to P1 expression appears to be limited to a short period during the transition from exponential growth to stationary phase, with a maximal relative Rsd intracellular level that is only 20% of the σ70 level [70 molecules in E. coli during stationary phase are similar, and that their association is significantly higher [E. coli during stationary phase [70 highly affine Rsd mutant resulted in the up-regulation of several genes transcribed by σ38, indicating that Rsd enables the redistribution of RNAP to σ38-specific promoters by sequestering σ70 [38 transcription. In fact, the transcription mediated by other alternative σ factors, such as σ24, σ32 and σ54, was also favored by Rsd overexpression [rsd transcription is not initiated primarily by σ70 but also by σ38, the fact that σ38 is only transcribed by σ70 allows the formation of an indirect negative feedback circuit between σ70 and Rsd that could contribute to its regulation, which supports cellular homeostasis .In contrast, HscC forms a complex with σand HscA , two memith HscC . HscC poith HscC . Howeverwith σ70 . Promoten of rsd . However70 level , 37. Anoy higher . The mutry phase . Neverthpression . Future pression . Althoug38 factor is the master regulator of the general stress response, transcribing more than 140 genes that confer resistance against adverse conditions, such as stationary phase, oxidative stress, UV radiation, heat shock, hyper-osmolality, acidic pH, and ethanol [The σ ethanol .70 and therefore does not have a full unique consensus sequence. Specific promoter selectivity is achieved by a broader tolerance for deviations from 1) the optimal promoter sequence, or 2) the exact geometric alignment of the -35 and -10 regions requiring an optimal spacer length of 17 bp [38 are believed to reach a maximum of one-third of those of σ70 [38 for the RNAP core [38 can recruit enough RNAP cores to carry out its transcriptional program are needed.In evolutionary terms, this σ factor is closely related to σof 17 bp . Additioof 17 bp , that bie of σ70 , 25. GivNAP core -31, 43, 38 levels in E. coli. An rssB mutant has near constitutively high levels of σ38 but is impaired in the post-transcriptional regulation of σ38 [rssB: the first, P1, is a distal promoter transcribed by σ38, which also co-transcribes rssA, and the second, P2, is situated in the rssA-rssB intergenic region from which rssB is transcribed in a σ38-dependent fashion [RssB  is a response regulator that functions as an anti-σ factor adaptor and plays a critical role in the control of cellular σn of σ38 . Two pro fashion .38 depends on three factors: the ClpXP protease, RssB, and a “turnover element”. The interface with RssB is the lysine-173 of the σ38 amino acid sequence. Binding of RssB to σ38 is stimulated by phosphorylation of the RssB receiver domain, suggesting that environmental stress affects σ38 proteolysis by modulating RssB affinity for σ38 [The proteolysis of σ for σ38 .38 are regulated at different points  by RssB and the two-component system ArcB/ArcA in a "three-component system" that coordinates rpoS transcription and σ38 proteolysis and, thereby, maintains low σ38 levels in cells growing exponentially. In this system, it has been suggested that the sensor histidine kinase ArcB monitors both the oxygen and energy supplies [38 for proteolysis) [38 levels low and are limiting factors in σ38 proteolysis. Acetyl phosphate is an accessory component that contributes to RssB and, possibly, ArcA phosphorylation [38 to phosphorylate RssB [38 is protected against degradation when it is bound to the RNAP core, its concentration affects its own rate of degradation [38concentration is increased above that of non-phosphorylated RssB, thus allowing σ38 to bind to the RNAP core and transcribe its target genes [38 and RssB because the first factor transcribes the second, assuring the production of the anti-σ RssB and the control of σ38 when the environmental stress that triggered its liberation disappears .Therefore, the cellular levels of σsupplies . In addieolysis) . Both phrylation . ClpXP iate RssB , 49. Givradation . Once thet genes . In thisE. coli homeostasis fundamentally due to thermal denaturation of folded proteins. In reaction to an upshift in temperature, E. coli employs the heat shock response to increase the activity of transcription directed by σ32 factor, inducing the up-regulation of over 120 products of this sigmulon. Some proteins produced under heat-shock conditions act as chaperones, such as GroEL and DnaK, which prevent protein turnover by maintaining proper folding, and proteases, such as Lon and Clp, which facilitate protein turnover .A temperature of 37°C or higher compromises dnaK gene is co-transcribed with dnaJ from three heat-inducible promoters, which are recognized in vitro by a RNAP containing σ32 but not by a RNAP in complex with σ70 [dnaK does not have a deleterious effect on the chaperone system function, cell growth, survival and morphology [The with σ70 . An intrrphology , and alsrphology .32, the σ factor responsible for responding to heat shock, is maintained at low concentrations during exponential cell growth [32 (in the presence of ATP), presenting it to the ATP-bound DnaK (Hsp70 homolog). In this way, DnaK competes with the RNAP core to bind σ32. The complex of DnaJ-σ32-DnaK-ADP is formed after ATP hydrolysis. Then, the chaperone GrpE presents σ32 to the free FtsH protease for degradation [32 and allowing the transcription of heat shock genes [32 can re-associate with the RNAP core to transcribe σ32 dependent promoters. As for σ38, σ32also takes part in the transcription of the components of its anti-σ system, ensuring that it is repressed when the environmental condition inducing its liberation  disappears .σl growth . During radation , 54. Undck genes , 55. ThiE. coli are organized into a regulatory hierarchy involving three classes of promoters, which groups genes according to their corresponding hierarchical level: early, middle and late. These classes of promoters are differentially expressed just prior the flagellum assembly in discrete intervals of the cell cycle.The flagellar genes of 28 activates the late genes involved in flagellum assembly. σ28 is initially expressed by an FlhDC-controlled class 2 promoter and is responsible for class 3 transcription of the flagellar regulon. In this stage, the proteins composing the flagellar filament, motility control and chemotaxis are expressed. In the late transcription hierarchical class 3, σ28 expression is also increased by transcription at an additional class 3 promoter [flgM is transcribed from two promoters. The P1 promoter, which is regulated by σ70, is situated in the flgA-flgB intergenic region, and flgM is co-transcribed with flgA and flgN. The second promoter, P2, is situated upstream of flgM and is transcribed in a σ28-dependent fashion [σpromoter , 57. In 28 during assembly of the basal body of the flagellum, σ28activity needs to be inhibited. This process is carried out by the anti-σ factor FlgM [28-dependent transcription of genes whose products are needed late in assembly, when the flagellar basal motor structure, the hook-basal body (HBB), is constructed [28, which facilitates the secretion of FlgM through the HBB, acting as an FlgM type III secretion chaperone. This results in the release of σ28, which allows σ28 to bind to a RNAP core and initiate transcription of the structural class 3 genes necessary to complete the flagellum [To avoid the participation of σtor FlgM . In thisstructed . With thlagellum .28 is a proteolysis substrate just like other σ factors. The Lon protease is responsible for σ28 proteolysis. When σ28 is in excess, its proteolysis increases the robustness and precision of this complex control system by rapidly reestablishing a 1:1 stoichiometry between σ28 and FlgM. This contributes to limiting the up-regulation of flagellar biosynthesis, and FlgM has a protective function against σ28 degradation [28 increases and then sequesters σ28 to suppress its function and proteolysis, forming a negative feedback regulatory circuit .Free σradation . When thE. coli have developed signaling pathways that sense and respond to these envelope stresses. These pathways trigger the expression of genes encoding a wide variety of proteins that are able to repair envelope . These pathways are known as extracytoplasmic (EC) or envelope stress responses. One of the EC systems monitoring envelope stress is the σ24 sigmulon, where σ factor is induced when the stress damages the periplasmic proteins. This is the only known EC response that is essential for viability in E. coli K-12 [Under certain stresses  misfolded envelope proteins are generated. As a consequence, bacteria such as oli K-12 , whose moli K-12 .24, and it is also known as an EC anti-σ factor. In general, the EC anti-σ factors are inner membrane proteins with at least one transmembrane domain. Additionally, it has been observed that the cytoplasmic N-terminal domain of EC anti-σ factors is conserved and possibly interacts directly with their cognate σ factor. On the other hand, little homology is observed in their transmembrane C-terminal domains (except between E. coli RseA and H. influenzae MclA), which is consistent with the possibility that these anti-σ factors may respond to different extracytoplasmic signals [The EC stress signal is transduced across the inner membrane to the cytoplasm via the inner membrane protein RseA, which is the anti-σ factor responsible for inhibiting σ signals .rseA gene, which encodes the anti-σ24 factor, is transcribed from three promoters. Two of these promoters, P1 and P2, are located upstream the gene rpoE; while the third promoter, P3, is located inside the coding region of rpoE. No obvious σ70 consensus promoter sequence is found upstream of promoter P1 [24-dependent transcription regulation [24 factor partially depend on the levels of its cognate anti-σ factor and vice-versa.The moter P1 . Howevergulation . Therefo24. Under unstressed conditions, biochemical analyses and crystallographic structures have shown that the periplasmic domain of RseA interacts with RseB (protecting RseA from proteolysis), while the RseA cytoplasmic domain is sandwiched between two domains of σ24. This interaction occludes the two main binding regions of σ24 for the RNAP core, repressing σ24 activity because it is sequestered at the inner membrane by the anti-σ factor RseA [RseA anchors to the inner membrane (single pass) and contains a binding site for σtor RseA .E. coli employs a proteolytic cascade to transduce signals related to protein folding in the periplasm to the transcriptional apparatus in the cytoplasm. RseA is degraded in response to misfolded OMPs by the sequential action of two inner membrane proteases, DegS and YaeL (RseP). The C-termini of misfolded OMPs bind to the PDZ domains  of the trimeric DegS protease, activating cleavage of the periplasmic domain of RseA [24. The cascade activates the cytoplasmic ClpXP protease and SspB chaperone, which degrades the remaining domain of RseA in the final step of the proteolytic cascade, thus allowing the release of σ24 [24 binds to the RNAP core and is ready to direct transcription of its sigmulon to cope with the environmental condition produced by the envelope stress [24-dependent transcription, once σ24 is released by RseA, through subverting the dominant housekeeping σ70. In fact, σ24 activity is induced upon entry into stationary phase in E. coli by the alarmone ppGpp and not via the RseA-dependent stress signaling pathway [24-RseA complex is restored by the RseA transcription for σ24, which replaces the anti-σ RseA that was degraded by proteolysis .During envelope stress, which interferes with the folding of outer membrane proteins (OMPs) and includes heat shock, overexpression of OMP genes, and mutations in genes encoding the chaperones required for OMPs folding,  of RseA , 67. Thee of σ24 , 69. Thee stress . The reg pathway . When pe19 (FecI) sigmulon of E. coli. Two signals are necessary to activate the expression of fecI: iron starvation and the presence of ferric-citrate in the environment. Whereas iron starvation is sensed by the Fur protein, iron citrate is bound by the FecA receptor in the outer membrane [The other EC signaling pathway is the σmembrane .19 deletion mutation is totally devoid of ferric citrate transport system expression because expression of fecIR mRNA requires σ70 and the transcription of the fecABCDE transport genes is regulated by σ19, which responds to ferric citrate via the anti-σ system FecA-FecR. fecI and fecR co-transcription is inhibited by the iron-loaded Fur repressor, which in turn transcribes the fec transport genes at a low level [A σow level .E. coli [Ferric citrate serves as an iron source for E. coli ; howeverE. coli , 75. Thifec transport gene operon, and 2) an anti-σ sensor that regulates the transcription of the ferric citrate transport genes fecABCDE in the cytoplasm. Both processes are independent and require energy transduction by the inner membrane TonB-ExbB-ExbD complex [FecA (an outer membrane protein), which recognizes ferric citrate, serves a dual role: It is 1) an iron transporter of ferric citrate across the outer membrane that operates with the  complex .FecA, the anti-σ sensor, elicits a signaling cascade from the cell surface to the cytoplasm . This si19 binds to its anti-σ factor, FecR; thus, FecR sequesters σ19 until FecR is inactivated by undergoing a structural change in responseto the induction signal of FecA (iron-citrate).It has been proposed that σ19 factor, allowing activation of the σ19 factor that directs the RNAP core to induce transcription of the fecABCDE transport genes [19 contributes to the transcription of the anti-σ sensor FecA. When ferric citrate becomes exhausted or a different iron source becomes available, FecR is reverted to its prior conformation by FecA, and FecR becomes able to sequester σ19 thus inhibiting the transcription of its corresponding genes .Activated FecA interacts in the periplasmic space with the C-terminus of FecR (anchored in the cytoplasmic membrane). Then, the signal is transferred across the cytoplasmic membrane into the cytoplasm via FecR N-terminus . Thus, Frt genes . σ19 conE. coli is exclusively represented by σ54, which directs the recognition of distinct promoter motifs located at positions -24 and -12 relative to the transcriptional start site. Although initially identified for their role in nitrogen assimilation, σ54 is utilized in different physiological processes and can remedy certain conditions that make nitrogen assimilation very difficult [54 does not have primary sequence similarity to σ70 proteins and regulates transcription by a different mechanism. σ54 can not spontaneously isomerize (melt) DNA to form open promoter complexes. This step of transcription strictly depends on mechanotranscriptional activators  that utilize ATP hydrolysis to drive conformational changes for this transition [E. coli [54 factor does not have an anti-σ factor. The σ54 impairing to transcribe genes without the help of σ54-dependent transcriptional activators (bEBPs) may explain the ostensible absence of an anti-σ factor.The second family of σ factors in ifficult . σ54 doeansition . Twelve [E. coli , 78, 79.Biological systems are, both intrinsically and extrinsically, noisy. Therefore, a mechanism enabling the cell to maintain a stable internal state is required. For maintain homeostasis, organisms need to detected changes in their environment and respond accordingly in a variety of manners. Negative feedback circuits have evolved in organisms as a common tool for maintaining homeostasis and filtering noise, thus providing a robust response. A feedback mechanism occurs when an organism senses an internal or external environmental perturbation and changes the level or activity of a substance, thus starting a regulatory cascade that eventually reverses the initial perturbation. While negative feedback circuits are ubiquitous in biological regulation, certain design principles emerge as lessons from the evolution of feedback in the anti-σ control.E. coli. Following an integrative approach, we were not only interested in describing their biological differences, but also their systems-level similarities.  shows the intricate cellular complexity of the σ/anti-σ circuitry. Despite this complexity, a small set of common mechanisms could be outlined. First, the anti-σ factor releases its cognate σ factor in response to a specific environmental signal . It has been reported that the latter causes a rapid increase in the cognate σ factor concentration [3). Third, the anti-σ factor plays one of two different roles: 1) it is kept inactive by other factors or adaptors  during the presence of the specific environmental signal, or 2) it participates actively in the response to the environmental signal . Finally, when this signal disappears, anti-σ factors efficiently repress their σ factors by sequestration . The sequestered σ factor has two possible fates: it can be protected against proteolysis or proteolyzed. The negative feedback circuits generated by these conditions in all alternative σ factors (except σ54) and their cognate anti-σ factors interactions enable the cell to return to a stable condition similar to that before the environmental signal. An interesting exception to these principles is the housekeeping σ70 factor. As all the alternative σ factors, σ70 also transcribes its anti-σ factors (rsd and hscC). Nevertheless, there are some intriguing differences. Both anti-σ70 factors seem to be also transcribed by alternative σ factors. As previously discussed, the rsd expression has been validated in vitro to be promoted by σ70 and σ38, while the promoter region of hscC has two promoters, σ70 and σ32, which have been proposed in silico. Whether these predictions are functional requires further research. However, this evidence opens the possibility that the σ70 concentration may be indirectly controlled by entry into stationary phase and heat shock, respectively, two conditions requiring alternative σ factors. Whether these are the only environmental signals controlling the availability of σ70 remains an open question.Through this review, we surveyed the different regulatory systems formed by the σ and anti-σ factors in nal step , Fig. 3.Mechanisms for coordinating the regulation of gene expression are hierarchic and complex. This coordination appears to be critical for the maintenance of a balanced gene expression profile during various environmental conditions. This is achieved by two different but complementary mechanisms. On the one hand, transcriptional regulatory protein compete with many other regulators to redirect the RNAP to transcribe a set of genes. On the other hand, σ factors show a rapid increase in their concentration and then compete with a small number of other σ factors for the limited RNAP core. The scarce RNAP core is a key aspect for the successful functioning of this regulatory mechanism through competence. Then, by activating alternative σ factors, gene expression can be efficiently and globally reprogrammed to respond to sudden environmental cues. As a consequence, the specificity that σ factors confer to the RNAP for certain promoters allows the circumvention of the transcriptional regulatory proteins currently active in a previous growth condition enabling a specific and efficient response to the new condition.E. coli σ factors is regulated at multiple levels by specific signal transduction pathways. These pathways are activated in response to specific stresses or environmental conditions. Several interactions play a prominent role in this regulatory process. These interactions operate synergistically and shape complex regulatory networks and cascades, constituting the regulatory mechanism establishing the gene expression profile. Thus, the cell can have a wide range of gene expression profiles that allow cells to cope with different requirements, such as sudden environmental changes or developmental programs. Anti-σ systems evolved in E. coli as a hierarchy of negative feedback interactions, allowing the cell to control the gene expression in response to specific environmental conditions in a way resembling compartmentalization by partitioning the gene expression states into a set of discrete functional units. When these environmental conditions disappear, the system relieves gene expression thereby completing the cycle.The availability of 70, whose promoter is present in the upstream region of each alternative σ factor . Nevertheless, σ activity has to be strictly controlled by preventing its binding to the RNAP core. This also have the desired effect of silencing the genes not required under a certain biological context. Anti-σ factors facilitate this requirement by operating as a system that senses the environment and sequesters the σ factor when the gene expression driven by them is superfluous given the biological context.To allow the efficient response of some σ factors, it is important that they are present in the cell at basal levels at all times. This may be achieved by σSupplementary material is available on the publisher’s web site along with the published article.


Cor410b is induced several fold above its non-stressed levels upon exposure to stresses such as cold, drought and wounding. Deletion analysis of the TdCor410b promoter revealed a single functional C-repeat (CRT) element. Seven transcription factors (TFs) were shown to bind to this CRT element using yeast one-hybrid screens of wheat and barley cDNA libraries, of which only one belonged to the DREB class of TFs. The remaining six encoded ethylene response factors (ERFs) belong to three separate subfamilies. Analysis of binding selectivity of these TFs indicated that all seven could bind to the CRT element (GCCGAC), and that three of the six ERFs could bind both to the CRT element and the ethylene-responsive GCC-box (GCCGCC). The TaERF4 subfamily members specifically bound the CRT element, and did not bind either the GCC-box or DRE element (ACCGAC). Molecular modeling and site-directed mutagenesis identified a single residue Pro42 in the Apetala2 (AP2) domain of TaERF4-like proteins that is conserved in monocotyledonous plants and is responsible for the recognition selectivity of this subfamily. We suggest that both DREB and ERF proteins regulate expression of the Cor410b gene through a single, critical CRT element. Members of the TaERF4 subfamily are specific, positive regulators of Cor410b gene expression.Expression of the wheat dehydrin gene  Among various transcription factors (TFs) reported to be associated with abiotic and biotic stress tolerance in plants, the most widely studied are the drought-responsive element (DRE) binding proteins (DREBs) and the ethylene response factors (ERFs). The DREB proteins, known also as the C-repeat (CRT) binding factors (CBFs), regulate expression of drought/cold stress-related genes by binding to the CRT element (GCCGAC) Dehydrins, a class of Late Embryogenesis Abundant (LEA) proteins, constitute an important family of abiotic-stress-responsive genes Cor410 gene was originally identified as a gene encoding a LEA protein that accumulates to similar levels in root, crown and leaf tissues of freezing-tolerant Gramineae during cold acclimation TaCor410 transcripts are highest for low-temperature tolerant wheat genotypes and lowest for tender genotypes Triticum aestivum L.) were observed on the second day of cold acclimation TaCor410 gene in transgenic strawberry at a level comparable to that in wheat after cold acclimation resulted in some improvement in freezing tolerance, although no improvement was detected in the absence of acclimation TaCor410. The closest homologues of TaCor410 reported in other plant species are AtCOR47 from ArabidopsisHvDhn8 from barley OsDhn1 from rice AtCOR47 and HvDhn8 is strongly induced by cold, but also up-regulated by drought and abscisic acid (ABA) treatments OsDhn1 is most strongly induced by drought, although induction by cold, high salinity and ABA has also been demonstrated The Arabidopsis DREB1B/CBF1 was found to up-regulate the expression of OsDhn1 in transgenic rice plants OsDhn1 promoter through a drought-responsive element(s). Similarly, up-regulation of the Dhn8 gene was observed in transgenic bahia grass plants  transformed with a CaMV35S-HsDREB1A fusion construct containing a DREB gene from Hordeum spontaneumHvDhn8 and TaCor410 in transgenic barley and wheat plants was also seen following constitutive or drought-inducible over-expression of either TaDREB2 or TaDREB3cis-acting promoter elements responsible for the constitutive expression and stress-inducible activation of either TaCor410 or TaCor410-like genes have not been identified. Moreover, while several TFs are reported to regulate Cor410 gene expression, it is not known which specific TFs are likely to be most important for stress-inducible activation of Cor410.In a previous study, over-expression of TdCor410b stress-inducible gene was isolated from durum wheat and used for identification of functional DRE/CRT cis-elements via a transient expression assay. TFs that bind the critical functional CRT element were isolated and their ability to activate the TdCor410b promoter was evaluated. Molecular modeling was used to investigate the nature of protein-DNA binding interactions between different types of ERF/DREB TFs and promoter elements.In this work, the promoter of the TdCor410b), JN681187 (TdERF6), JN681188 (TaERF6), JN681189 (TaERF4a), JN681190 (TaERF4b), JN681191 (TaERF5a), JN681192 (TaERF5b) and JN681193 (HvERF4).Nucleotide sequences reported in this work have been deposited in GenBank under Accession numbers JN681186 (TaCor410 cDNA (GenBank accession L29152) was isolated by PCR using a cDNA library obtained from spikes of drought-stressed wheat (Triticum aestivum L cv. Chinese spring) as a template. The TaCor410 cDNA was used as a probe to screen a BAC library prepared from genomic DNA of Triticum durum cv. Langdon T. durum homolog of TaCor410 (TdCor410), with primers derived from the coding region of the TaCor410 cDNA. The TdCor410b promoter sequence was identified through sequencing of the BAC clone. A 2685 bp long promoter region containing a full-length 5′-untranslated region of TdCor410b was cloned into the pMDC164 vector TdCor410b promoter deletions were generated by PCR using AccuPrime™ Pfx DNA polymerase  and the TdCor410b promoter as a template. PLACE software  was used to predict DRE/CRT elements in the TdCor410b promoter region, and forward primers were designed so as not to interrupt potential cis-elements. Promoter deletions were cloned into the pMDC164 vector and used in transient expression assays described below.The full-length coding region of the TdCor410b promoter (263 bp), with three repeats of the GCC-box (AGCCGCC). A tandem of GCC-boxes was added to the sequence of the forward PCR primer and the artificial promoter was generated by PCR. Together with the full length TdCor410b promoter, the artificial promoter was used in transient expression assays to test activation properties of ERFs and molecular variants of TaERF4a in planta.An artificial promoter was generated by substitution of the functional CRT element in the shortest active deletion of the TaDREB2, TaDREB3, TaERF4, TaERF4a, TaERF5a, TdERF6, GFP and GUS were cloned into the pENTR-D-TOPO vector (Invitrogen). The cloned inserts were verified by sequencing, subcloned into the pUbi vector The coding regions for T. monoccocum L. initiated from roots A transient promoter activation assay, based on co-bombardment of promoter-GUS fusion constructs with pUbi-TF constructs, was performed using a suspension cell culture of HindIII and KpnII restriction sites from the pMDC32 vector TdCor410b promoter. These vectors were designated as pCor410H and pCor410H2, respectively. The coding region of TaDREB3 cDNA Hordeum vulgare L. cv. Golden Promise), using Agrobacterium-mediated transformation nos terminator. The basal level of activity of the TdCor410b promoter fragments in leaves of transgenic T0 lines was determined by northern blot hybridization analysis using coding region of TaDREB3 cDNA as a probe. To analyse activity of the long and short versions of the promoter in transgenic barley plants, seedlings of each of three selected transgenic lines and three control wild type plants were grown together in 10-inch pots containing 2.5 kg of standard coco peat potting mix in a growth chamber (24°C/50% relative humidity (day) and 18°C/80% (night), with a 12 h photoperiod). For the drought induction assay, plants were well watered for three weeks and then water was withheld. Leaf samples were collected on the last day of watering; two further samples were collected at 7 and 10 days after the cessation of watering. Relative soil water content measured using a Fieldscout spectrometer  indicated soil water contents of 48 (for well-watered), 7, and 3% (v/v), respectively. For the induction of the TdCor410b promoter by cold, plants were grown for three weeks before being transferred to a cold cabinet  maintained at 4°C. Leaf samples were collected before the cold treatment and after 2, 8 and 24 hours of incubation at 4°C. For the analysis of promoter inducibility by wounding, the leaves of 3-week-old seedlings were mechanically wounded with a fine metal brush and harvested at 0, 0.5, 1, 4, 8, and 24 hours after wounding. Leaves from three biological replicates were used for RNA isolation and Q-PCR analysis.Two vectors were generated, where the 2×35S promoter was excised using the TaDREB2 (Acc. DQ353852) and TaDREB3 (Acc. DQ353853) were previously isolated from a bread wheat (T. aestivum cv. Chinese Spring) endosperm cDNA library (WENDL) A barley cDNA library (BCG) was prepared from floral tissues/flag leaf of cold-tolerant barley (cv. Haruna Nijo) under cold/frost stress. Plants were grown to anthesis in a growth chamber set to the following conditions: four weeks at 20°C (day)/8°C (night) with a 10 h photoperiod; four weeks at 21°C (day)/10°C (night) with a 12 h photoperiod; then 22°C (day)/12°C (night) with a 14 h photoperiod. At anthesis, plants were moved to a frost chamber. Flag leaves and whole spikes were sampled when the temperature at floret height (i) fell to 4°C; (ii) had been held at the minimum temperature of −5.5 °C for 2 h; and (iii) had returned to 4°C. The RNA was pooled from each time point , so that the contribution from each time point comprised equal amounts of RNA from 12 individual heads from each of three plants.T. aestivum cv. RAC875), that had been subjected to high temperatures under both well-watered and drought stress conditions. Plants were grown in well-watered conditions to anthesis in a growth chamber 22°C (day)/10°C (night) with a 14 h photoperiod). At flowering, plants were subjected to seven days of heat stress, where on each day, the day-time temperature was gradually increased to 40°C , held at 40°C for a further 4 h, then lowered to 28°C for 2 h and returned to 22°C for the remainder of the day and overnight. Watering was withheld from the second day, and plants showed signs of water deficiency from the fourth day. On the first day of the heat stress treatment (well-watered), and again on the fourth and seventh days (drought-stressed), samples of flag leaf and spike (at different stages of development) were collected at two time points; as soon as the temperature reached 40°C, and again after a further 3.5 h at 40°C. Tissue samples were collected from five plants in total. A mixture of equal amounts of total RNA from each plant was used for cDNA library preparation.A wheat cDNA library (WHSL) was prepared from flag leaves and spikes of an Australian drought-tolerant bread wheat  The WENDL, WHSL, and BCG cDNA libraries were screened with baits constructed from five repeats of a GCCGAC (CRT1) core element, or three repeats of a 16 bp long ERFs, TaERF4a, TaERF5a and TdERF6, were amplified by PCR using primers with additional EcoRI and either BamH1 (TaERF4a and TdERF6) or PstI (TaERF5a) restriction sites. The amplified fragments were cloned into the respective restriction sites of the pGBKT7 vector and the resultant constructs were transformed into yeast (Saccharomyces cerevisiae strain AH109). Yeast transformants carrying the plasmids were selected on synthetic defined (SD) (-Trp) medium and replica-plated to SD2  medium. The ability of transformants to grow on SD2 medium suggested the presence of a native activation domain in the ERF.The coding regions of selected representatives from each of the three cloned subfamilies of via 3D-PSSM Arabidopsis thalianaGCCGCCAGC-3′5′-GCTAcis-element cis-element, these data gave us the opportunity to model the wheat AP2 domains in complex with their respective cis-elements identified in the current work. Hence, AP2 of TaERF4a was modelled with GCCGAC, AP2 TaERF5a with GCCGCC and GCCGAC, and TaDREB3 with ACCGAC and GCCGAC. The individual cis-elements were generated via the Sybyl 8.0 suite of programs  and were minimized with a Tripos force field. The aligned template and target sequences with their respective cis-elements were further used as input parameters to generate 3D models of the TaERF4a, TaERF5a and TaDREB3 AP2 domains , using Modeller 9v7 http://www.pymol.org) ‘super’ algorithm were 0. 24 Å, 0.25 Å and 0.25 Å in the Cα positions, respectively. The electrostatic potentials were calculated with the Adaptive Poisson-Boltzmann Solver  (http://apbs.sourceforge.net/) implemented in PyMol as a plugin, and mapped onto the protein molecular surfaces that were generated with a probe radius of 1.4 Å. Molecular graphics was generated with PyMol (http://www.pymol.org).Three-dimensional models were constructed by comparative (homology) modeling that relies on applying spatial restraints derived from a structural template A. thaliana) The amino acid sequences of 32 AP2 domain-containing plant TFs including those of 13 ERFs with C-terminal repressor motifs, were aligned with AtERF1 . The stress cDNA series for Q-PCR analysis was prepared from three to four leaves that were collected from each of 2 – 4 independent 6-week-old plants of either T. aestivum (cv. RAC875) and/or T. durum (cv. Langdon), subjected to each of the following stresses: drought , and two weeks after re-watering); cold stress at 4°C ; and wounding with a fine metal brush .Q-PCR analysis of the expression of the TaCor410 gene, and regulatory sequences starting 2,685 bp upstream of the translational start codon, were isolated from a BAC library prepared from Triticum durum cv. Langdon T. durum has the greatest amino acid sequence similarity to TaCor410b , and was therefore designated as TdCor410b.A homolog of the TdCor410b using PLACE software Figure S2). No GCC-box was identified in the promoter region of TdCor410b. It has previously been demonstrated that the promoters of TaCor410-like genes from rice, barley and wheat can be activated in transgenic plants through over-expression of DREB proteins TdCor410b promoter in transient expression assays, with the aim of identifying functional cis-element(s). Mixtures of equal amounts of pUbi-GFP (negative control) or pUbi-TaDREB3 with the pTdCor410b-GUS plasmid(s), containing deletions in the TdCor410b promoter, were used to co-transform a cell suspension culture of T. monoccocum. Deletions of the promoter were generated based on putative cis-acting elements at −1872, −945, −556, −417, −299, and −230 bp . Each of these deletions, except deletion −945, decreased the number of putative DRE/CRT elements by one, thus creating the opportunity to evaluate individual elements for functionality . The GCCGAC core element is referred to as a cold-responsive element that functions in Arabidopsis as the GGCCGACAT element ACCGAC, used for the isolation of TaDREB3 via cold and drought Ten DREs/CRTs/LTREs, two ABREs, and several putative abiotic stress-related MYC and MYB responsive elements TdCor410b promoter were used in transient expression assays to verify functionality of the identified cis-element (HvDhn8 promoter sequence available from the NCBI databases (Acc. AF043093) was compared with that of TdCor410b. The position and adjacent sequences of the mapped CRT element were conserved . Co-bombardment of pHvDhn8-GUS and pUbi-TaDREB3 constructs resulted in a 6-fold activation of the promoter with TaDREB3 compared with the negative control .Several single bp mutations introduced into the core sequence of the mapped functional CRT element in a −263 bp deletion of the TaDREB3 driven by 2,685 bp and 275 bp fragments of the TdCor410b promoter revealed the presence of basal levels of promoter activity, and inducibility of both promoter fragments by cold, drought and wounding . This analysis confirmed that activation of the TdCor410b promoter by stress, and even in the absence of stress, occurred providing the CRT element immediately proximal to the TATA box was retained.Unfortunately, activation of promoter fragments by stresses such as drought and wounding cannot be tested using a transient expression assay. Analysis of transgenic barley plants expressing TdCor410b promoter containing the GCCGAC core sequence, (GCCGACACGCTTTCCG) (CRT2), were used in a yeast one-hybrid system to screen three separate prey libraries. These were WENDL, a library prepared from wheat un-stressed endosperm, WHSL, a library prepared from drought/heat-stressed wheat flag leaf and spikes, and BCG, a library prepared from cold/frost-stressed barley floral tissues and flag leaf. The WENDL cDNA library was previously used for the isolation of DREB proteins and TFs that are not induced by stress TaCor410b gene is expressed in early grain/endosperm in the absence of stress, we searched for potential up-stream activators of this gene in the WENDL library. The barley cDNA library (BCG) was used because a cDNA library from wheat tissue subjected to cold/frost treatment was not available. The amounts of RNA from various time intervals in this library reflect our attempt to enrich the library with early-responsive genes and genes responsive to temperatures below zero.The core sequence GCCGAC repeated five times (CRT1), or three repeats of a fragment of the TaERF5a, TaERF4a, TaERF5b, TaERF6 and TaDREB2; from WHSL: TaERF4a and TaERF4b; from BCG: HvERF4. All listed TFs were isolated using the CRT1 element. TaERF4a and HvERF4b were also isolated in screens with the CRT2 element, as well as clones containing partial cDNA sequences of TaERF5b and TaERF6. An Expressed Sequence Tag (EST) encoding the 5′ end of the TaERF5b cDNA was identified from the NCBI databases (Acc. CA728064), and the full-length sequence of TaERF5b cDNA was isolated from WHSL cDNA using nested PCR. No complementary ESTs have been deposited in the NCBI databases for the TaERF6 cDNA. However, the intron-less gene of the TaERF6 orthologue from T. durum was identified in BAC clone #191 I19, using a segment of the coding region of TaERF6 as a probe. The full-length coding region of this gene, designated TdERF6, was used to make a construct for transient expression assays.The coding sequences of six ERFs and one DREB were isolated in Y1H screens from WENDL: TaDREB2) belonging to the DREB family. The remaining six TFs encoded TaERF4a, TaERF4b, HvERF4, TaERF5a, TaERF5b, and TaERF6, all belonging to subfamilies of the ethylene-responsive element (GCC-box) binding TFs (EREBPs or ERFs). TaERF5a, TaERF5b, and TaERF6 had been isolated previously using the GCC-box as bait from the same cDNA libraries (unpublished data). However, no TaERF4-like TFs have been isolated with the GCC-box from any cDNA library.In total, seven different AP2-domain-containing TFs were isolated, only one of these (TaDREB3 cDNA among isolated clones can be explained by low abundance of this cDNA Absence of the Arabidopsis (in bold characters and underlined), as this protein was used as a template for molecular modeling of the AP2 domains of TaERF4a, TaERF5a and TaDREB3. The full-length sequence of the selected mono- and dicotyledonous ERF and DREB proteins clustered into four independent branches, highlighting their functional roles  core element. We could not detect differences for any of the tested factors between their binding to the CRT1 (GCCGAC) and CRT2  sequences. Thus, the core element itself may be sufficient to confer specificity of binding, and the influence of adjacent sequences may be minimal. Y1H assays also established that the DREB TFs could bind the DRE (ACCGAC) motif, but could not bind the GCC-box (GCCGCC). As expected, TaERF5a and TaERF6 could interact with the GCC-box, but could not bind the DRE motif. Surprisingly, TaERF4a could bind neither the GCC-box nor DRE, binding only to CRT . TdCor410b promoter activity was reduced to basal levels by these mutations (Mutations that were introduced into a predicted ERF-associated amphiphilic repression (EAR) motif of TaERF4a strongly decreased promoter activation. The mutations consisted of substitutions of four conserved residues in the EAR motif to Ala , as well as TaDREB3 and TaDREB2ERFs and DREBs by cold stress always preceded induction of the downstream TaCor410b gene  was used for comparative structural modelling and analysis of ERF and DREB TFs isolated in our studies, due to the presence of this domain (of approximately 62 residues) in both subfamilies of TFs. Structural alignment of 32 AP2 domain-containing sequences provided information about the conservation of the AP2 domains at the amino acid level within selected TFs. Analysis indicated that the sequences could be divided into two major groups, based on conservation of a Pro residue following Arg152 in 1 gcc:A; Arg152 makes close interactions with the coding strand of a DNA element ). Of critical importance was the observation that Pro42 found in the TaERF4a-like proteins occurred exclusively in monocotyledonous species, as confirmed by the analysis of 501 sequences (data not shown) through the ConSurf server The AP2 domain (or GCC-box binding domain) of AtERF1 from A. thaliana (designated here as 1 gcc:A) as a template for modelling was confirmed through searches using PsiPred The suitability of AtERF1 from cis-elements would be expected to bind  could be severely restricted due to the presence of neighbouring Pro132. The question then arises as to why the GCCGCC cis-element is only recognised by the AP2 domain of TaERF5a and not by TaDREB3? Our modeling studies indicated that the recognition selectivity of TaDREB3 could be decided by several structural features. Firstly, the overall length of the protein segment spanning Gly49 to Arg66  might be of importance and, secondly, the specific environment around Arg48 and Arg66 might be critical, preventing binding of the GCC-box by TaDREB3. On the other hand, the environment around Arg131 in the AP2 of TaERF5a , and a shorter β-sheet region comprising 15 residues between Pro132 and Arg148 , would allow binding of both cis-elements GCCGCC and GCCGAC. However, the length of the β-sheet segment that forms a DNA binding region in TaERFs cannot be the only structural requirement that determines binding of the GCC-box, because TaERF4a does not bind to GCC-box elements. In the AP2 domain of TaERF4a, the presence of the two relatively closely positioned Pro residues could restrict flexibility of the β-sheet, thus preventing interactions with the GCC-box. Conversely, binding of GCCGAC by the AP2 domain of TaERF4a could be favourable, because an amino group in the purine ring of adenine could mediate productive interactions with AP2 , allowed generation of variant proteins of the AP2 domain of TaERF4a with potentially modified selectivity for binding the GCC box (GCCGCC) . Through site-directed mutagenesis, we mutated each of the two conserved Pro residues to create a Pro26Arg mutant (TaERF4a m1), a Pro42Arg mutant (TaERF4a m2), and a Pro26Arg+Pro42Arg double mutant . The yeast GCC-box bait strain grew on the selective medium when TaERF4a m2 was expressed, while this was not the case for TaERF4a m1  in the promoter? Can strength and/or specificity of protein-DNA interactions be modulated by genetically engineered variants of existing TFs? These and related questions have been addressed in the current work using the TdCor410b promoter.Several important TdCor410b promoter. DRE/CRT elements are involved in abiotic-stress responses, including drought and cold, and are known to be bound mostly by one class of TFs, namely, DREB/CBFs. We predicted ten potential DRE/CRT/LTR elements in the TdCor410b promoter. However, activation by the TaDREB3 TF was confirmed only with the CRT element closest to the potential TATA-box. Our results show that regulation of the stress-inducible TdCor410b promoter is complex and involves the participation of several different types of AP2 domain-containing TFs. These different TFs use a single, ‘promiscuous’ CRT element with a core sequence GCCGAC. The CRT element may be involved in cold-induced activation of TdCor410b by TaDREB3 or other DREB/CBF proteins. It is possible that other upstream CRT(s) could become functional, at least partially, if the primary element was lost or mutated. Alternatively, other DREB/CBFs may target other DRE/CRT elements within the same promoter.In this study we have focused on DRE/CRT elements in the TdCor410b promoter was mapped to a -299 bp fragment of the promoter, suggesting that the same single CRT cis-element may be responsible for both constitutive activity and inducible activation of the TdCor410b promoter. This hypothesis was confirmed when several single-base mutations were introduced into the mapped element . Furthermore, barley plants were stably transformed with TaDREB3 under the regulation of the 2,567 bp and 275 bp regions of the TdCor410b promoter. Analysis of transgenic lines demonstrated that both promoter regions drove basal levels of TaDREB3 expression, and both were activated by cold, drought and wounding . These results defined the role of the CRT element proximal to the TATA box as a universal element, which could regulate TdCor410b promoter activity under optimal growth conditions and in response to a variety of abiotic stresses.Basal activity of the TdCor410b promoter, using the GCCGAC element (CRT1) as bait in Y1H screens of cDNA libraries prepared from both un-stressed and stressed wheat or barley tissues. Seven different AP2 domain-containing TFs were isolated in the screen. Surprisingly, only one, TaDREB2, belonged to the DREB subfamily. The other six TFs belonged to the ERF subfamily of the AP2 domain family. Genes from the DREB/CBF subfamily have been reported to play a critical role in responses of plants to abiotic stress through DRE/CRT elements within the core motif (A/G)CCGAC GCCGCC (bold type indicating the core GCC element) cis-elements. The Y1H assay revealed in vivo interactions for all three types of identified wheat ERFs with the GCCGAC element. However, only two types of ERFs were able to bind the GCC-box and, as expected, neither interacted with the ACCGAC element . AtERF3, AtERF4 and the tobacco ERF3 are all believed to function as repressors, and their gene products contain a C-terminal ERF-associated amphiphilic repression (EAR) motif (L/F)DLN(L/F)(X)P, that has more recently been found in other families of TFs Arabidopsis contain Arg42 instead of Pro42 in the AP2 domain, and were shown to strongly interact with the GCC-box The most abundant independent clones isolated in the Y1H screen were homologues of TaERF4a functions as a specific regulator of the TdCor410b promoter, because transcript expression of TaERF4a and TaCor410b was highly correlated  element. Stress-responsive induction of TdCor410b indicated that a complex interplay of ERF and DREB/CBF TFs takes place, which may also involve other TFs and modifying factors. The best candidate for driving constitutive activity and drought-inducible activation of TdCor410b promoter was TaERF4a. The exact role, if any, of two other types of ERFs in TdCor410b promoter regulation requires further investigation. TaERF4a possessed properties that were atypical of other ERFs investigated in this study, including unusual DNA-binding specificity and specific transcriptional activation.We suggest that Figure S1Multiple sequence alignment of protein sequences of TdCor410b and products of homoeologous genes from bread wheat, and reported homologues from barley and rice: Wcor410 (Acc. AAA20189), Wcor410b (Acc. AAB18201), Wcor410c (Acc. AAB18202), HvDHN8 (Acc. AAD022259), OsDHN1 (Acc. AAV49032). Identical amino acid residues are in yellow boxes, conserved residues are in blue boxes, and similar residues are in green boxes.(EPS)Click here for additional data file.Figure S2TdCor410b promoter with predicted CRT/DRE/LTRE elements. The putative TATA-box is in bold and underlined, the predicted elements are in grey boxes, the functional element is in a grey box and underlined. First bp of each promoter deletion used in promoter mapping is marked with a black box. Names and sizes (bp) of promoter deletions are shown above the black boxes.The sequence of the (EPS)Click here for additional data file.Figure S3TdCor410b and HvDHN8 promoters. (A) Pair-wise alignment of nucleotide sequences of the TdCor410b and HvDHN8 promoters. Computer-predicted cis-elements common for both promoters are in transparent boxes; sequence of the functional cis-element is marked with *. The putative TATA-box and translational start are in bold. (B) Basal activity of the TdCor410b and HvDhn8 promoters (1) and activity induced by overexpression of TaDREB3 (2). The promoter-GUS construct was co-bombarded in the wheat suspension cell culture with either the pUbi-GFP (1) or pUbi-TaDREB3 (2) constructs.Comparison of the (EPS)Click here for additional data file.Figure S4Activation of a -275 bp and -2,685 bp long promoter fragments by wounding, cold and drought in transgenic barley plants detected by Q-PCR.(EPS)Click here for additional data file.Figure S5The Q-PCR analysis of HvERF4 expression in leaves and roots of barley plants subjected to cold (4oC).(EPS)Click here for additional data file.Figure S6Sequence alignment of AP2 domains and EAR motifs of TaERF4a-like proteins. (A) A multiple sequence alignment of thirteen AP2 domains of the ERF sequences using PROMALS3D (41). The positions of highly conserved Pro residues in the ERF sequences are highlighted in yellow and the positions of three Pro residues conserved in the selected cereal ERF sequences are highlighted in cyan. (B) The conserved regions of the COOH-terminal EAR sequence underlying the importance of four conserved residues Asp, Leu, Asn and Pro, are in pink.(EPS)Click here for additional data file.Table S1List of primers used for Q-PCR.(DOCX)Click here for additional data file.


Here, we provide evidence that demonstrates a 119 bp 5S rDNA can influence nucleolar association in mammals. We found that integration of transgenes with 5S rDNA significantly increases the association of the host region with the nucleolus, and their degree of association correlates strongly with repression of a linked reporter gene. We further show that this mechanism may be functional in endogenous contexts: pseudogenes derived from 5S rDNA show biased conservation of their internal transcription factor binding sites and, in some cases, are frequently associated with the nucleolus. These results demonstrate that 5S rDNA sequence can significantly contribute to the positioning of a locus and suggest a novel, endogenous mechanism for nuclear organization in mammals.Changes in the spatial positioning of genes within the mammalian nucleus have been associated with transcriptional differences and thus have been hypothesized as a mode of regulation. In particular, the localization of genes to the nuclear and nucleolar peripheries is associated with transcriptional repression. However, the mechanistic basis, including the pertinent  Eukaryotic genomes are compartmentalized within nuclei such that physiological events, including transcription and DNA replication, can efficiently occur. The mechanisms that regulate this organization represent an exciting, and equally enigmatic, subject of research. In mammals, the identification of elements that influence these associations has been impeded by the complex nature of the genomes. Here, we report the identification and characterization of such an element. We demonstrate that the integration of a 5S rDNA gene, a 119 base pair noncoding RNA transcribed by RNA polymerase III, into a new genomic location can significantly influence the association of the host region with the nucleolus. This positioning has drastic, inhibitory effects on the transcription of a neighboring protein coding gene transcribed by RNA polymerase II, demonstrating a functional relationship between localization and gene expression. We also provide data that suggest this may be an endogenous phenomenon, through a class of repetitive sequences derived from 5S rDNA. Together, our data not only demonstrate a structural role for 5S rDNA but also suggest that nuclear organization of mammalian genomes may be strongly influenced by repetitive sequences. The organization of DNA within mammalian nuclei is considered nonrandom Moreover, just as pol II genes are thought to cluster in transcription ‘factories’ Xenopus and mammalian systems de novo insertion of these sequences into new genomic contexts should recapitulate this phenomenon. To study the effect of 5S rDNA sequence on sub-nuclear localization, we generated ES cell lines with stable, multicopy insertions of a reporter construct containing a single 5S rRNA gene (Tg5S) . Furthermore, association of Tg5S was higher than that of the 5S rDNA array (∼40%). This could be due to a dominant localization pattern imparted by Tg5S even at low copy, or additional forces acting to constrain localization of the endogenous 5S rDNA locus. We observed very little co-localization of Tg5S arrays and the 5S rDNA cluster , demonstrating that these loci do not occupy the same compartment in the nucleoplasm. However, we found that the structural and functional integrity of the nucleolus was essential for localization through 5S rDNA. Reorganization of nucleolar components, through pharmocological inhibition of RNA polymerase I activity, resulted in a significant decrease of both Tg5S and 5S rDNA association with the nucleolus  have been observed to form large chromatin loops in e (Tg5S) . To deteucleolus .Thymidine kinase (Tk) gene driven by the mouse Pgk1 promoter . This relationship suggests that perinucleolar targeting of transgenes via the 5S rDNA sequence has inhibitory effects on pol II transcription.The nucleolar periphery has typically been thought of as a transcriptionally quiescent compartment, often associated with examples of constitutive promoter . Tk mRNAAscl2 promoter), in three of four Tg5S lines analyzed  of the X chromosome RAR-related orphan receptor beta (Rorb) gene  than for the wt allele (52%) in Tg5S#6 (p = 0.01), or either allele in the control cell line . The localization frequency of the wt allele in the Tg5S#6 was not significantly different from the alleles in the control line (p = 0.5). Interestingly, wt Rorb alleles were associated with the nucleolus significantly more frequently than the 5S rDNA locus . Together, our observations from two independent insertion events, in two very different genomic contexts, demonstrate that ectopic 5S rDNA can influence the position of a locus.Tg5S line #6 (Tg5S#6), contains an integration in the first intron of the silent rb) gene . The allrb) gene . NucleolTk reporter gene, we hypothesized that the transgene insertion into the Rorb locus may similarly affect transcription of this gene. Rorb is not expressed in undifferentiated ES cells, therefore we differentiated the line with the Tg5S insertion in the Rorb gene (Tg5S#6) along with Tg5S#9, where the transgene insertion is not at the Rorb locus. Although activation of Rorb was variable between biological replicates, in each case Rorb expression was significantly reduced in Tg5S#6  loci, TFIIIC-bound regions not associated with a pol III complex or transcription unit Collectively, these observations suggest broad and diverse roles for pol III genes and derived sequences in the organization of chromatin within the mammalian nucleus. Because of their number, pol III promoters may exert a stronger influence on structural organization than pol II-directed gene activity. As pol III activity is coupled with differentiation and cellular metabolism, association of pol III and transcription factors with elements such as the 5S pseudgoenes we have described, may provide the basis for global organizational and structural changes within the nucleus in response to external stimuli 5 ES cells were plated on 60 mm2 dishes without LIF and in the presence of 0.1 µM retinoic acid (Sigma) then cultured for 8 days, with passaging to maintain low cell density. For immunofluoresence and DNA FISH, cells were plated at low density and grown on coverslips 18–24 hours. Coverslips were permeabilized with cytoskeletal (CSK) buffer , then fixed in 4% paraformaldehyde  for 10 minutes at room temperature, washed twice for 5 minutes in 1× PBS (Cellgro), then stored in 75% ethanol at 4°C. Coverslips were re-hydrated with several washes of 1× PBS prior to DNA FISH experiments.E14 ES cells were cultured under standard conditions. To generate stable lines, ES cells were transfected with 1 µg of linearized plasmid using lipofectamine (Invitrogen) and selected in the presence of G418 for 14 days. We verified stable neomycin resistance for most lines by culturing with G418 and noted no increased levels of cell death. To induce differentiation, 2×10Thymidine kinase gene under the control of the mouse Pgk1 promoter . Tg0 was the vector without the 5S rDNA insert.To generate Tg5S, the 5S rDNA sequence  was cloned into a vector that contains the neomycin resistance gene under the control of the HSV promoter, and the Tk mRNA levels were first normalized to Gapdh levels. Real-time quantitative PCR was carried out on 25 ng of cDNA, in triplicate for each gene, on an ABI 3700 (Applied Biosystems), using the Fast SYBR Green Master Mix (Applied Biosystems). Data was analyzed in Microsoft Excel (Microsoft), and is shown as the log2-transformation of RNA levels relative to copy number. Statistical significance was determined by two-tailed t-test.RNA was isolated from cultured cells with Trizol reagent (Invitrogen), DNAsed  and 500 ng of total RNA was used for each reaction. Samples were reverse transcribed using random-hexamer primers, with Superscript II Reverse Transcriptase (Invitrogen). Primers are listed in Transgene integration sites were determined using one of two approaches. The Tg5S#6 insertion site was identified using the TAIL PCR protocol, with degenerate primers as described Coverslips were rehydrated in 1× PBS, before blocking in 10 mg/ml IgG-free BSA  and 0.2% Tween-20 (Fisher) for 20–30 minutes at room temperature. Rabbit anti-Nucleolin  was added at 1∶400 dilution into blocking buffer and incubated overnight at 4°C. Coverslips were then washed with 1× PBS, and incubated with biotinylated goat-anti-rabbit antibody, diluted in blocking buffer at 1∶400, for 2–3 hours at room temperature. Following washes with 1× PBS, cells were post-fixed with 2% PFA for 3 minutes at room temperature, washed extensively with 1× PBS, then treated with 0.01 mg/ml pepsin (Sigma) diluted in pre-warmed 0.01 N HCl for 5 minutes, and then washed extensively with 1× PBS. Following a dehydration series in ethanol, DNA was denatured in 70% formamide (Ameresco) and 2× SSC (Cellgro) for 10–20 minutes at 85°C. After several washes with cold 2× SSC, cells were incubated with prehybridized DNA FISH probes (see below) overnight at 37°C. Coverslips were washed twice with 50% formamide and 2× SSC, twice with 2× SSC (one wash had 100 ng/ml DAPI added), once with 1× SSC. To detect biotinylated secondary antibodies, coverslips were then washed once with 4× SSC for 5 minutes, incubated for 20–30 minutes in Streptavidin-647 (Invitrogen) in 2 mg/ml BSA and 4× SSC, followed by 5 minute washes of 4× SSC, 4× SSC with 0.5% Tween-20 (Fisher), and 4× SSC. All washes and incubations for biotin detection were carried out at 37°C.Mid1 (RP24-229F18); and fosmids: 6∶112 (G135P69622C7), 8∶48 , 19∶19 (G135P64778C12), PAR (G135P601180H2) . BACs and fosmids were isolated by a standard alkaline lysis protocol. Approximately 25 ng of DNA was labeled with BioPrime DNA labeling kit (Invitrogen), using FITC-conjugated dUTP (Roche), Cy3- or Cy5-conjugated dCTP , and stored in 70% ethanol at −20°C. To prepare FISH probes for hybridization, probes were precipitated with mouse Cot-1 DNA (Invitrogen), yeast tRNA (Invitrogen), and Salmon Sperm DNA (Invitrogen). After washes with 75% and 100% ethanol, probes were air-dried and denatured for 10 minutes in 50–100 µl of 100% formamide at 85°C. An equal volume of 2× hybridization buffer (25% dextran sulfate/4× SSC) was then added, and probes were pre-hybridized for 60 to 90 minutes at 37°C. Probes were stored at −20°C until use.In addition to the vector backbone, the following BAC and fosmid probes were used in this study: BACs: 5∶134 (RP24-193L24), 7∶30 (RP23-151J21), 8∶126 (RP24-372G15), 10∶27 (RP24-213F23), 11∶74 (RP23174M12), IF-DNA FISH was carried out as described in Neo gene copies relative to an endogenous locus (the Ascl2 promoter), then normalized to a genomic DNA sample containing 1 copy of Neo for each diploid genome. Primers are listed in Copy number was determined by quantitative PCR to determine the number of 7 cells/ml and fixed with 1% formaldehyde. After quenching with 0.125 M glycine, cells were pelleted, washed once with cold 1×PBS, pelleted again and used for ChIP or frozen at −80°C. Protease inhibitors (Sigma) and PMSF (Sigma) were added to all steps until washing steps. For GTF3C5 and pseudogenes, ChIP was performed as described 7 cells/ml, mixed at 15 minutes and gently pelleted at 4°C. Cell pellet was resuspended in solution L2  at 107 cells/ml, mixed at 15 minutes and gently pelleted at 4°C. Cells were lysed in solution L3  for 10 minutes at 4°C. Chromatin was sheared by sonication to generate fragments 2–600 bp. Before immunoprecipitation, 1/10th of each sample was removed as ‘input’. 5 µg of Antibody  or normal rabbit sera (Abcam) was conjugated to protein A/G beads in 0.5%BSA/1×PBS overnight at 4°C on a nutating platform. Chromatin was incubated with bead-conjugated primary antibody overnight at 4°C with gentle mixing. For GTF3C5 ChIP, beads were then washed for 5 minutes at 4°C with gentle mixing, using the following solutions: Low Salt Buffer , twice; High Salt Buffer, once; LiCl buffer , twice; and TE , twice. For histone modifications, beads were washed 4 times with RIPA buffer, and once with TE containing 50 mM NaCl. Chromatin was eluted from beads with 2, 15-minute washes at 65°C using freshly prepared Elution Buffer (1% SDS/0.1 M NaHCO3). To isolate DNA, 5 M NaCl was added to pooled eluates or input chromatin to a final concentration of 0.2 M, and incubated for at least 4 hours at 65°C, then treated with 30 µg of Proteinase K (Roche) for 2 hours at 55°C. After addition of 10 µg linear acrylamide as a carrier (Ambion), DNA was extracted with 25∶24∶1 phenol∶choloform∶isoamyl alcohol (Sigma), precipitated with 100% ethanol, and resuspended in nuclease-free ddH20 (Promega). For psuedogenes, three replicates of quantitative PCR were carried out on an ABI 3700 (Applied Biosystems), using the Fast SYBR Green Master Mix (Applied Biosystems). For transgene and 5S rDNA enrichment, 2–5 replicates were performed on Bio-Rad CDX96 instrument, a using SsoFast EvaGreen Supermix (Bio-Rad). PCR primers are listed in ES cells were trypsinized, counted, resuspended at 10Figure S1A. Combined Immunofluoresence (IF)-DNA FISH images showing localization of the 5S rRNA gene arrays in mouse ES cells. At least one allele was associated with the nucleolar periphery in ∼40% of nuclei. Examples of Tg5S nucleolar association (B) or no association (C). D. Nucleolar association of each individual Tg5S and Tg0 ES cell line. (n) is indicated below each line number. E. Relationship between association frequency (Y-axis) and copy number (X-axis). Scale bar is 2 µm.(TIF)Click here for additional data file.Figure S2Pharmacological inhibition of RNA polymerase I (pol I) activity results in nucleolar reorganization and a decrease in 5S rDNA and Tg5S nucleolar association. ES lines were treated with a low dose of Actinomycin D  for 2 hours prior to fixation to inhibit Pol I elongation. Note that ActD treatment results in redistribution of the pol I transcription factor UBF1(green) from intranucleolar foci (A) into focal concentrations at the nucleolar periphery , and restructuring of nucleoli into a more spherical morphology. We measured the size of ActD-treated nucleoli to be 51% smaller than untreated nucleoli. C. Localization of 5S rDNA (n = 112) and Tg5S after ActD treatment . To normalize for changes in nucleolar size, we calculated the ‘expected’ localization as the frequency of association in untreated cells by the relative nucleolar size in ActD treated cells. Statistical significance was determined by comparing the expected frequency to the observed frequency by chi-squared; N.S., not significant.(TIF)Click here for additional data file.Figure S3Tk shown for each individual line. Each technical replicate is shown as a different symbol.Normalized (TIF)Click here for additional data file.Figure S4Quantification of histone modification enrichment over transgenes for (A) H3K4me2, (B) H3K9me3, (C) H3K9me2, and (D) H3K27me3. Values are represented as fold-enrichment relative with a negative control region lacking that modification. Also shown is a schematic of the transgene with positions of regions assayed. 2–3 replicates of each reaction were performed for each point.(TIF)Click here for additional data file.Figure S5Rorb allele in Tg5S#6; Tg5S#4 is shown as a negative control.A. PCR assays to genotype PAR insertion in Tg5S#9; Tg5S#2 was used as a negative control. B. PCR assay to genotype insertion of the transgene into (TIF)Click here for additional data file.Figure S6http://mfold.rna.albany.eduG), suggesting these elements are likely to be pseudogenes. Predicted 5S rRNA structure is shown in (B), while the structure of the most thermostable of 5S pseudogenes in (C).A. Distribution of 5S rDNA as annotated by in Ensembl (NCBIM37) (red dots). Perfect A and C boxes are shown as blue dots; note that a number of perfect A/C boxes are found outside of annotated 5S rDNA. The 5S rDNA array is located near the telomere of chromosome 8 (bold). Since the structure of 5S rRNA is highly conserved, we hypothesized that if the single genes were truly 5S rRNA, then they should form the expected structure. Using a folding algorithm Click here for additional data file.Figure S7n = 83) and pseudogenes: 6∶112 (n = 49), 8∶48 (n = 61), and 10∶27 (n = 21). Pseudogenes are labeled by their location in the genome as chromosome∶megabase. For analysis, deconvolved Z-stacks were rendered as 3-dimensional models(see Nucleolar association of 5S rDNA (odelssee . Frequen(TIF)Click here for additional data file.Table S1Mouse 5S Pseudogenes. Positional information of 5S rDNA psuedogenes in the mouse genome (based on the NCBI m37 mouse assembly). Also included are the percent identity of the A and C boxes, as well as the entire sequence, to the 5S rDNA consensus.(DOC)Click here for additional data file.Table S2PCR primers used in this study.(DOC)Click here for additional data file.