Heterologous expression of bacterial biosynthetic gene clusters is currently an indispensable tool for characterizing biosynthetic pathways. Development of an effective, general heterologous expression system that can be applied to bioprospecting from metagenomic DNA will enable the discovery of a wealth of new natural products.Escherichia coli-based heterologous expression system for polyketide biosynthetic gene clusters. We have demonstrated the over-expression of the alternative sigma factor σ54 directly and positively regulates heterologous expression of the oxytetracycline biosynthetic gene cluster in E. coli. Bioinformatics analysis indicates that σ54 promoters are present in nearly 70% of polyketide and non-ribosomal peptide biosynthetic pathways.We have developed a new 54 may be a viable method for the production of additional polyketide products.We have demonstrated a new mechanism for heterologous expression of the oxytetracycline polyketide biosynthetic pathway, where high-level pleiotropic sigma factors from the heterologous host directly and positively regulate transcription of the non-native biosynthetic gene cluster. Our bioinformatics analysis is consistent with the hypothesis that heterologous expression mediated by the alternative sigma factor σ Actinomycetes and δ-proteobacteria, have shown that there are many more polyketide biosynthetic pathways than polyketides isolated from standard cultivation techniques Bacterial polyketides possess an enormous range of chemical diversity and biological function. Many polyketides such as tetracycline Streptomyces coelicolorStreptomyces lividansMyxococcus xanthusTo access this untapped diversity of polyketide products, a general method for heterologous expression of these pathways is needed. The selection of a heterologous host is contingent upon its ability to efficiently transcribe non-native pathways, translate the often GC rich transcripts, and possess all the starter and extender units necessary for polyketide production Escherichia coli has a number of advantages that make it an appealing host for a heterologous expression system. The ease of genetic manipulation and culturing as compared to other heterologous hosts makes E. coli far more useful for bioprospecting from genomic DNA libraries. Codon usage as well as starter and extender unit availability have proven not to be obstacles for heterologous production in E. coliE. coli as a heterologous host is its inability to effectively transcribe heterologous pathways. In all examples of heterologous expression of polyketides in E. coli, all of the biosynthetic genes have been placed under the control of the T7 promoter E. coli culture conditions.E. coli into a general heterologous expression system, a mechanism for ensuring transcription of all the biosynthetic genes in a foreign pathway is required. We hypothesized that a general transcriptional regulator of polyketide biosynthesis should be present in bacteria because horizontal transfer of biosynthetic pathways is a key mechanism for their proliferation among species Streptomyces strains which produce the tetracycline family of antibiotics, the presence of yersiniabactin-like gene clusters in diverse ActinomycetesPaederus fuscipes and the sponges Theonella swinhoei and Psammocinia aff. bulbosa respectively To convert E. coli can selectively, directly, and positively regulate heterologous expression from the Streptomyces rimosus oxytetracycline biosynthetic gene cluster E. coli. This is the first successful heterologous production of an aromatic polyketide in E. coliHerein we show that this new mechanism for heterologous expression of the oxytetracycline polyketide biosynthetic pathway, where a high-level pleiotropic sigma factor from the heterologous host is used to positively regulate transcription of the non-native biosynthetic gene cluster, is highly effective. We demonstrate that one of the six alternative sigma factors in E. coli, we investigated the ability of E. coli to heterologously express the oxytetracycline biosynthetic pathway from S. rimosusTo test our hypothesis that an alternative sigma factor downstream of the stringent response could positively regulate transcription of polyketide biosynthetic pathways in E. coli could produce oxytetracycline under standard culture conditions, E. coli BAP1 S. rimosus oxytetracycline pathway Bacillus subtillis, sfp, which ensures that polyketide synthase acyl carrier proteins (ACPs) are post-translationally modified E. coli, under standard culture conditions, cannot heterologously produce oxytetracycline from the native S. rimosus gene cluster.To determine if E. coli, we quantified mRNA levels using quantitative PCR (qPCR) for five genes in the pathway. The five genes oxyB, oxyF, oxyK, oxyP, and oxyT, one per putative operon, were selected. oxyB encodes the β-ketosynthase, oxyF and oxyT encode methyltransferases and oxyK encodes an aromatase all of which are required for oxytetracycline biosynthesis oxyP encodes a malonyl-ACP transferase, which may not be required for biosynthesis of oxytetracycline oxyF, oxyK, oxyP and oxyT could be easily detected and quantified  and recruiting it to promoter sequences upstream of the gene to be transcribed 54,σH, σS, σF, σE, and FecI could positively regulate polyketide biosynthesis in E. coli, we quantified mRNA levels using quantitative PCR for oxyB. Six E. coli BAP1 strains possessing the oxytetracycline biosynthetic gene cluster and over-expressing either σ54,σH, σS, σF, σE, or FecI were investigated. No oxyB transcripts could be detected when σH, σF, and σE were over-expressed. Over-expression of σS and FecI led to detectable but low levels of oxyB transcription. When σ54 was over-expressed substantial levels of oxyB transcripts could be detected  and CLF (OxyB) must be expressed. To evaluate if σ54 over-expression constituted an effective method to produce soluble KS-CLF heterodimer, we examined the soluble fraction of oxytetracycline producing strains by SDS-PAGE (54 (54 kDa) could be easily detected, there was no evidence for high-level production of OxyA (45 kDa) or OxyB (44 kDa). Thus while σ54 over-expression must lead to some functional soluble KS-CLF heterodimer, there is not substantial accumulation of these key proteins.While most classes of bacterial natural products have been heterologously expressed in SDS-PAGE . While a54 can positively regulate transcription directly or indirectly. For direct regulation, the σ54-RNAP complex binds to a σ54 consensus promoter upstream of the operon under σ54 control. The σ54 consensus promoter is unique among bacterial promoters with highly conserved residues −12 and −24 from the transcriptional start site (TGGCACG-N4-TTGC(T/A)) 54 is directly regulating transcription of oxyB, a σ54 promoter consensus sequence must be present upstream of the operon containing oxyB. To determine if σ54 promoters were present in the oxytetracycline biosynthetic pathway the entire gene cluster was examined for σ54 promoter consensus sequences using the web-based tool PromScan (http://molbiol-tools.ca/promscan/) oxyB in a location that would allow direct transcription of the putative operon . Our 5′ RACE data identified a transcriptional start site consistent with the proposed σ54 promoter, confirming that this promoter is functional and that oxyB transcription is directly regulated by σ54.To demonstrate that the predicted σild type . To iden54 promoters are commonly found in polyketide and non-ribosomal peptide biosynthetic pathways, σ54 over-expression may be a general route to heterologous expression of polyketides and non-ribosomal peptides. To evaluate this possibility, we examined the genomes of 58 sequenced bacteria for polyketide and non-ribosomal peptide biosynthetic gene clusters and σ54 promoters. The 58 species included major secondary metabolite producers such as 10 Actinobacteria, 20 Proteobacteria, 9 Firmicutes, as well as 19 species from diverse phyla. This selection of species contains representative examples of most of the sequenced bacterial phylogenic diversity.If σManual examination of all genes annotated as polyketide synthase, 3-oxoacyl acyl carrier protein synthase, and non-ribosomal peptide synthetase  54 promoters. To identify σ54 promoters, a positional weighted matrix (PWM) describing the σ54 promoter consensus sequence 54 promoters. All experimentally validated σ54 promoters in the E. coli genome had scores ranging from the 80 s to high 90 s M. xanthus a number of previously identified σ54 promoters had scores in the low to mid 70 s Each bacterial genome was analyzed to identify all putative σE. coli genome was analyzed E. coli genome and 18.4% of the genes in the sequence randomized genome were predicted to have σ54 promoters. 18.4% thus provides a baseline for the level false positive predictions. This high false positive rate is typical of PWM based promoter prediction tools To evaluate the level of false positives, a sequence randomized glnA ortholog (COG0174), the archetypical gene under direct σ54 transcriptional control, had one or more predicted σ54 promoters upstream of this gene. In comparison only 10% of the genomes examined had predicted σ54 promoters upstream of the well-characterized non-σ54 regulated ribosomal proteins encoding genes  54 promoters.As false positives are not expected to be conserved across species, a comparative genomic approach that examines the predicted promoters across a wide range of bacterial genomes should decrease the false positive rate and increases the specificity 54 promoters appropriately positioned to regulate transcription  clusters had a putative σ54 promoter appropriately placed to directly regulate either a polyketide synthase or a non-ribosomal peptide synthetase encoding ortholog. These results demonstrate that a majority (69%) of polyketide and non-ribosomal peptide biosynthetic gene clusters possess putative σ54 promoters and that these promoters are appropriately positioned to regulate transcription of at least one operon in these gene clusters. These results are consistent with the hypothesis that σ54 over-expression may be a general method for ensuring transcription of polyketide and non-ribosomal peptide biosynthetic gene clusters in heterologous hosts, such as E. coli.Of the 180 polyketide and non-ribosomal peptide biosynthetic gene clusters identified, 124 (69%) contained one or more σcription . 24 geneE. coliHeterologous expression of polyketide and non-ribosomal peptide biosynthetic gene clusters will play a major role in the discovery of new natural products from metagenomic and environmental DNA samples. Currently no general heterologous expression method appropriate for screening DNA libraries exists. Herein we describe a new mechanism for heterologous expression of a polyketide biosynthetic pathway, where a high-level, pleiotropic alternative sigma factor from the heterologous host positively regulates transcription of the biosynthetic gene cluster. In contrast, known methods for heterologous expression rely on either replacing each native promoter with known, well-characterized promoter from the heterologous host, such as the T7 promoter in 54, enables efficient heterologous expression of oxytetracycline biosynthetic gene cluster in E. coli. Using qPCR, we have demonstrated that no transcript is detectable under standard culture conditions for the key oxytetracycline biosynthetic gene oxyB. LC-MS/MS analysis of these cultures showed no detectable oxytetracycline production in the absence of detectable oxyB transcripts. Over-expression of σ54 positively regulates transcription of oxyB, generating detectable levels of oxyB transcripts, enabling production of oxytetracycline. Bioinformatics analysis of the oxytetracycline biosynthetic gene cluster revealed a σ54 promoter sequence appropriately positioned to directly regulate oxyB transcription. Site directed mutagenesis and 5′ RACE strongly support that this promoter is functional. Further bioinformatic analysis of 180 polyketide and non-ribosomal peptide biosynthetic pathways shows that the majority of these pathways possess putative σ54 promoter sequences (69%), suggesting that σ54-mediated heterologous expression may be a general phenomenon.Our results demonstrate that over-expression of the sigma factor, σ54, can positively regulate diverse polyketide biosynthetic pathways. Nutrient limitation, which is one of the only known general regulators of polyketide biosynthesis relA, the gene encoding the (p)ppGpp synthetase, in S. coelicolor is required to trigger polyketide biosynthesis26. (p)ppGpp disrupts the interaction between the RNA polymerase (RNAP) and the principle sigma factor, σ70, enabling RNAP to interact with alternative sigma factors, which increases alternative sigma factor-mediated transcription Substantial evidence exists to support the hypothesis that over-expression of alternative sigma factors, such as σE. coli on the transcription of oxyB and on the production of oxytetracycline. Only σ54 over-expression led to both detectable levels of the oxyB transcript and detectable levels of oxytetracycline production. σ54 is known to play a major role in response to nitrogen limitation in most bacteria delta-proteobacteria54 may play a role in regulation of polyketide biosynthesis in some bacteria, substantial work in native hosts is required to test this hypothesis.We investigated the effect of over-expression of all the alternative sigma factors from 54-mediated transcription is that it requires co-activation to initiate transcription 54-loaded RNA polymerase to form a transcriptionally active open complex. EBPs contain a DNA binding domain that interacts with a specific DNA sequence called the upstream activating sequence (UAS), directing the EBP to a specific σ54 promoter. The ATPase domain of the EBP then catalyzes ATP hydrolysis, leading to open complex formation and transcription. Without this co-activation step, transcription cannot occur. High levels of EBPs have been shown in vivo and in vitro to enable formation of the active open complex independent of EBP binding to the UAS A unique aspect of σ54-mediated transcription of the operon containing oxyB in the heterologous host E. coli, co-activation must be occurring. σ54 over-expression has been shown to positively regulate many of the twelve EBPs native to E. coliglnG, zraR, and fhlAE. coli EBP.As our data supports direct σS and FecI showed an increase in oxyB transcription but did not lead to detectable levels of oxytetracycline production. oxyB transcript levels were at least 10 fold lower for σS and FecI over-expression than when compared to σ54 over-expression. Presumably the low levels of the oxyB transcript were insufficient to generate enough OxyB to produce detectable levels of oxytetracycline. A plausible explanation for the transcription of oxyB during σS and FecI over-expression is cross-talk between these alternative sigma factors and σ54, leading to low levels of σ54-mediated transcription. This is supported by the observation that σ54, σS, and FecI are known to be linked by the polyamine response Over-expression of σ54 promoter in the S. rimosus oxytetracycline biosynthetic gene cluster and 236 putative σ54 promoters in 61 gene clusters from seven different actinobacterial genomes is highly unexpected  and E. coli TOP10 (Invitrogen) cells were used for routine cloning and plasmid preparations. E. coli BAP1 was used in all heterologous production experiments. E. coli cells were grown in LB media (Fisher) supplemented with the appropriate antibiotics when necessary. Oxytetracyline standard and IPTG were purchased from Sigma-Aldrich. Antibiotics and media components were purchased from Fisher. All strains were chemically or electronically transformed using standard transformation protocols.E. coli MG1655 genomic DNA was used as the template. Each sigma factor was cloned into pCR-Blunt and confirmed by sequencing. Genes were sub-cloned using NdeI and EcoRI restriction sites into pET28b or pKH22 (a pET21c derivative with an AvrII site engineered after the native EcoRI site) rpoE and rpoD, where HindIII instead of EcoRI and NheI instead of NdeI were used to due presence of native restriction sites. To construct pDCS61 the oxyTA1ABCD cassette was amplified from pMRH08 5′ – TAATACGACTCACTATAGGG – 3′ and the reverse primer CTCATAGCTCCAGGCTG - 3′5′ – CAGTGAATT. The oxyTA1ABCD cassette was then digested with EcoRI and inserted into pET28b. Site directed mutagenesis was then performed on pDCS61 using forward primer AAAACGGCGGTGGC – 3′5′ – CCTGCGTCCCCT and the reverse primer TTAGGGGACGCAGG – 3′5′ – GCCACCGCCGTT following the QuikChange mutagenesis protocol to construct pDCS62. The mutant was confirmed by sequencing. To construct pLRVP09, pBR322 was digested with ScaI and SspI to remove the β-lactamase marker and ligated closed. Plasmid names and descriptions can be found in the expanded experimental procedures in the supplementary information  in which the slope was calculated by regression analysis obtained from the Ct values versus calculating log number of cells in serial dilutions  and vigorously vortexed. Cell debris was removed by centrifugation and Amberlite XAD-16 resin (0.50 g) was added to the clarified media. The resin was incubated with the media overnight and collected by filtration. The organic extracts were eluted from the resin with 20 mL MeOH over 2 hours and concentrated in vacuo. Extracts were then resuspended in 100 µL MeOH for ESI-LC-MS/MS analysis. All ESI-LC-MS/MS analysis was performed on an API2000 LC/MS/MS System equipped with a turbo-ion spray ESI probe interfaced with a Prominence UFLC. A reverse phase BDS Hypersil C18 column  was employed. Mobile phases throughout experimental were A: 5% MeCN∶95% H2O with 0.05% formic acid and B: 95% MeCN∶5% H2O with 0.05% formic acid. For initial determination of oxytetracycline production a gradient program  was developed with a flow rate of 0.250 mL/min into the mass spectrometer . For oxytetracycline MS2 fragmentation scans a gradient program  was developed and the mass spectrometer settings were optimized for oxytetracycline . For MRM quantification of oxytetracycline production, a gradient program identical to that of the MS2 program was use with the following mass spectrometer settings: Ion spray 5,500 V, collision energy 33 eV, MRM Q1 461.3 m/z, MRM Q3 483.2 m/z. Quantification was performed in triplicate using a standard curve generated from authentic oxytetracycline.The negative control strains were prepared by electroporating 600 = 0.4, cultures were induced with 1 mM IPTG and grown at 20°C for 24 hours. Cells were harvested from 5 mL of cell culture by centrifugation at 4000 g and resuspended in 200 µL of lysis buffer  glycerol, 1 mg/mL lysozyme, 1 µg/mL pepstatin A, and 1 µg/mL leupeptin). The cells were disrupted by sonication on ice, and cell debris was removed by centrifugation at 15000 g and 4°C. The resulting soluble fraction was analyzed by 4–20% gradient SDS-PAGE followed by staining with Coomassie Brilliant Blue.Phosphate pH 8.0, 300 mM NaCl, 10%  annotations ormation .54 promoter sequences in bacterial genomes the PromScan Perl script http://strawberryperl.com/). The inputs were bacterial genome DNA sequence files (FNA files from NCBI's Genome Database) and the σ54 promoter positional weighted matrix file, which is based on 186 known σ54 promoter sites and can be found in the expanded experimental procedures in the supplementary information To identify putative σhttp://www.microsoft.com/express/). PTT files (from NCBI's Genome Database) containing annotation data were uploaded to the database. Using LINQ to SQL, hits generated from the modified PromScan algorithm were linked to genes located within 500 bp upstream and in the same coding direction as the gene start site. Promoter prediction data is available on the web at http://www.sigma54.ca.The results were uploaded to an SQL database created using SQL Server 2008 Express  tails were added using terminal deoxynucleotidyl transferase (Fermentas). The tailed cDNA was amplified by PCR by PrimeStar HS DNA polymerase (Takara) using the oxyA specific primer and the abridged anchor primer 5′ – GGCCACGCGTCGACTAGTACGGGIIGGGIIGGGII – 3′. The diluted PCR reaction was used as the template for a second round of PCR amplification using the oxyA nested primer 5′ – CCGCCTCGCCCGTCACAC – 3′ and the abridged universal anchor primer 5′– GGCCACGCGTCGACTAGTAC – 3′. The PCR products were gel purified and reamplified using the oxyA nested primer and the abridged universal anchor primers. The PCR products were then cloned into pCR-Blunt and sequenced.Total RNA was isolated from BAP1 with pMRH08 and pDCS11 as described for qPCR analysis. cDNA was generated using AMV Reverse Transcriptase and the Figure S1Transcript levels, determined by qPCR, for E. coli transformed with the oxytetracycline gene cluster (pMRH08) compared to a null strain lacking the oxytetracycline gene cluster.(TIF)Click here for additional data file.Figure S2Transcript levels, determined by qPCR, for E. coli54 compared to a null strain lacking the oxytetracycline gene cluster and over-expressing σ54. transformed with the oxytetracycline gene cluster (pMRH08) and over-expressing σ(TIF)Click here for additional data file.Figure S3+ESI-LC-MS/MS Q1 ion extraction chromatograms for the oxytetracycline [M+H]m/z = 461.Standard: an oxytetracycline standard. Positive control: Organic extracts from a culture of BAP1 with the oxytetracycline biosynthetic gene cluster over-expressing σ54. Negative control: organic extracts from a culture of BAP1 with the oxytetracycline biosynthetic gene cluster. Eσ: organic extracts from a culture of BAP1 with the oxytetracycline biosynthetic gene cluster over-expressing σE. Fσ: organic extracts from a culture of BAP1 with the oxytetracycline biosynthetic gene cluster over-expressing σF. Hσ: organic extracts from a culture of BAP1 with the oxytetracycline biosynthetic gene cluster over-expressing σH. MS2 traces further confirm that no oxytetracycline can be detected in the σE, σF, and σH samples.(TIF)Click here for additional data file.Figure S4SDS-PAGE of the soluble fraction from oxytetracycline producing strains. Lane 1 is protein ladder, lane 2 is the soluble fraction from uninduced BAP1, lane 3 is the solubule fraction 24 h post induction from BAP1/pDCS11, lane 4 is the soluble fraction 24 h post induction from BAP1/pMRH08/pDCS11. A band corresponding to σ54 (54 kDa) is seen in lanes 3 and 4. The OxyA-OxyB KS-CLF heterodimer (45 kDa and 44 kDa respectively) is not obeserved in lane 4.(TIF)Click here for additional data file.File S1Expanded experimental procedures.(PDF)Click here for additional data file.Table S1Complete data set for qPCR analysis as shown in  and . Standard curve, efficiencies, R2 and Ct values are given.(PDF)Click here for additional data file.Table S254 promoters identified from a bioinformatics analysis of the oxytetracycline gene cluster.List of putative σ(TIF)Click here for additional data file.Table S354 promoters identified from our bioinformatics analysis.List of PKS, NRPS and NRPS/PKS gene clusters with the associated σ(PDF)Click here for additional data file.Table S454 promoters identified from our bioinformatics analysis at increasing cutoff stringencies.Tabulated number of PKS, NRPS and NRPS/PKS gene clusters with one of more σ(TIF)Click here for additional data file.


Cnp1 is present in fission yeast cells. Our analyses show that additional CENP-ACnp1 accumulates within and close to heterochromatic centromeric outer repeats, and over regions adjacent to rDNA and telomeres. The use of minichromosome derivatives with unique DNA sequences internal to chromosome ends shows that telomeres are sufficient to direct CENP-ACnp1 deposition. However, chromosome ends are not required as CENP-ACnp1 deposition also occurs at telomere repeats inserted at an internal locus and correlates with the presence of H3K9 methylation near these repeats. The Ccq1 protein, which is known to bind telomere repeats and recruit telomerase, was found to be required to induce H3K9 methylation and thus promote the incorporation of CENP-ACnp1 near telomere repeats. These analyses demonstrate that at non-centromeric chromosomal locations the presence of heterochromatin influences the sites at which CENP-A is incorporated into chromatin and, thus, potentially the location of centromeres.The histone H3 variant, CENP-A, is normally assembled upon canonical centromeric sequences, but there is no apparent obligate coupling of sequence and assembly, suggesting that centromere location can be epigenetically determined. To explore the tolerances and constraints on CENP-A deposition we investigated whether certain locations are favoured when additional CENP-A Such analyses indicate that specific DNA sequences can direct events that promote CENP-A incorporation and kinetochore assembly. Although kinetochores are normally assembled upon these preferred, defined centromeric sequences in any particular organism, there is now an abundance of examples demonstrating that centromeric sequences are sometimes not sufficient and sometimes not necessary for kinetochore assembly. On human dicentric chromosomes, one centromere can be inactivated despite the continued presence of α-satellite sequence at both centromeric loci [Centromeres are the chromosomal locations at which kinetochores, the machinery responsible for accurate chromosome segregation, are assembled. Kinetochores are assembled on unusual chromatin composed of nucleosomes in which canonical histone H3 is replaced by a centromere specific histone H3 variant, generally known as CENP-A. The incorporation of CENP-A is critical for providing the foundation for the assembly of kinetochores and thus specifying the location of active centromeres in most eukaryotes. Kinetochores are normally assembled on specific sequences such as centromeric repeat sequences (α-satellite in human) and these DNA sequences can direct the ric loci ,2. Howevric loci . Moreoveric loci . These cCID in Drosophila promotes incorporation of endogenous CENP-ACID, even after the tethered version is removed, suggesting that CENP-A chromatin is able to direct its own propagation [Together such observations suggest that centromeres are normally assembled upon canonical centromeric sequences in any particular species, but there is no obligate coupling of sequence and assembly. These observations provide strong evidence for an epigenetic component to the regulation of centromere assembly. Several lines of evidence point to the histone H3 variant, CENP-A being the epigenetic mark that specifies centromere identity. It is found only at active centromeres, including neocentromeres and is absent from inactivated centromeres . Tetheripagation . This ispagation .The incorporation of CENP-A at novel sites could be influenced in various ways. CENP-A may be rapidly turned over to prevent its accumulation at non-centromeric locations ,15. ActiSchizosaccharomyces pombe) chromosomes are composed of a central domain (cnt plus imr) flanked by repetitive DNA elements known as the outer repeats (otr). CENP-ACnp1 chromatin and the kinetochore are assembled over the central domain (~10 kb) while heterochromatin, in which histone H3 is methylated on lysine 9 (H3K9me) that allows the binding of specific chromodomain proteins, coats the outer repeats. Central domain DNA alone is unable to direct the de novo assembly of CENP-ACnp1 chromatin on this DNA following its introduction into cells. The flanking heterochromatin is required to provide an environment that somehow allows the replacement of histone H3 with CENP-ACnp1 and kinetochore assembly over the nearby central domain DNA [Cnp1 might be attracted to non-centromeric sites by sequences possessing certain features, but also by chromatin context. The importance of chromatin context is demonstrated by the fact that heterochromatin is required for the establishment of CENP-ACnp1 chromatin on adjacent central core sequences, but once established this heterochromatin is not required for the maintenance of CENP-ACnp1 [The three centromeres on fission yeast  has been shown to allow neocentromeres to form at other chromosomal locations. At these neocentromeres CENP-ACnp1 is associated with DNA sequences that lack homology to the central domain of centromeres, where CENP-ACnp1 is normally incorporated. All neocentromeres formed were within 100 kb of either the left or right telomeres. Heterochromatin is associated with fission yeast telomeres and in the absence of heterochromatin the frequency of neocentromere formation was reduced [The engineered deletion of a fission yeast centromere ( reduced .1-4) repeats are bound by specific proteins that form a specialised structure that regulates telomerase recruitment, protects chromosomal ends from degradation and anchors them at the nuclear periphery [Fission yeast telomeres are composed of ~300 bp of terminal GT rich repeats that are added by telomerase, internal to these are telomere associated sequences (TAS) on chromosome 1 and 2 . These Teriphery . Telomereriphery , and Poteriphery . Taz1 reeriphery ,28, whereriphery . In comperiphery . Ccq1 alTaz1 has been shown to inhibit telomerase-mediated telomere elongation, by ensuring cell cycle dependent telomerase recruitment to telomeres ,32. Rap1Cnp1 deposition we investigated whether certain locations are favoured by CENP-ACnp1 when present in excess. We find that when CENP-ACnp1 is moderately overexpressed in fission yeast it accumulates in telomeric regions, the same regions that can form neocentromeres. Here we use engineered telomeres on minichromosomes, integrated arrays of telomeric repeats and cells lacking telomere and heterochromatin proteins to investigate what features promote CENP-ACnp1 incorporation in the telomeric regions of chromosomes. Our analyses show that a short array of telomere repeats, independent of a chromosome end, is sufficient to promote CENP-ACnp1 incorporation and that Ccq1 plays a specific role in promoting the formation of nearby heterochromatin and CENP-ACnp1 incorporation.To explore the tolerances and constraints on CENP-ACnp1 is normally restricted to the central domain of fission yeast centromeres. However, our previous analyses show that CENP-ACnp1 is able to assemble on non-centromeric DNA inserted within the central domain [Cnp1 causes increased repression of a marker gene inserted in the central core and increased levels of CENP-ACnp1 on that marker gene. It has also been shown that the deletion of an endogenous centromere (cen1) results in CENP-ACnp1 accumulating over an extended euchromatic region adjacent to telomeres, forming neocentromeres [Cnp1 is normally restricted to the central domain at centromeres, it can assemble upon non-centromeric sequences.CENP-Al domain . Moreovetromeres . These oCnp1 deposition we expressed additional CENP-ACnp1 at a variety of levels and conditions. The CENP-ACnp1 open reading frame was expressed from different strengths of the thiamine repressible nmt promoter  [Cnp1 was tolerated, strong overexpression had a highly deleterious effect on cell growth, especially at 36oC.In order to further investigate the constraints or promiscuity of CENP-A strong) , integra strong) ,36, FiguCnp1 at non-centromeric loci, CENP-ACnp1 localisation was examined in cells expressing additional CENP-ACnp1 from the three types of promoters. During interphase, the three fission yeast centromeres are clustered adjacent to the spindle pole body  and appear as a single dot  analysis was performed on cells with low CENP-ACnp1 overexpression (nmt81-CENP-ACnp1) and cells with wild-type CENP-ACnp1 levels  or moderate (nmt41) levels of CENP-ACnp1  have telomere-associated sequences (TAS) internal to the TTAC(A) GG(G1-4) repeats [Cnp1 levels were observed on the unique DNA immediately internal to the rDNA ) internal to which are copies of telomere associated sequence (TAS) repeat elements [otr repeats [Cnp1 to domains of telomeric chromatin, we utilised versions of a well characterised minichromosome Ch16 in which new telomeres were created adjacent to a ura4+ marker gene by telomere-mediated chromosome breakage at the m23 locus [ura4+ gene (ura4+-TAS-Tel), whereas on Chs 16–72 the terminal telomere repeat array lies directly adjacent to the ura4+ gene (ura4+-Tel)  GG relative to when TAS sequences were included (ura4+-TAS-Tel)  . MoreoveNP-ACnp1 . The facCnp1 deposition at telomeres, anti-H3K9me2 ChIP was performed. Our analyses show that H3K9me2 is enriched on ura4+-Tel, but only weakly enriched on ura4+-TAS-Tel when compared to the enrichment found at outer repeats  allows growth in the absence of uracil (–ura) and no growth on media containing counter-selective FOA  and Chs 16–76 (ura4+-TAS-Tel) on FOA plates and/or reduced growth on –uracil plates  the observed incorporation of CENP-ACnp1 on the centromeric outer repeats and the central core was unaffected  in mutant cells expressing additional CENP-ACnp1. In clr4∆, taz1∆ and ccq1∆ cells the level of CENP-ACnp1 at ura4+: Int-Telo was dramatically reduced compared to the wild type control and the reduced deposition of CENP-ACnp1 correlates with the loss of H3K9me2 at ura4+: Int-Telo observed in these mutants . Although budding yeast lacks methyl-H3K9 heterochromatin, many CLRs were found in the regions surrounding centromeres [S. cerevisiae and S. pombe [Unlike in radation ,15,42. W regions ,9. As intromeres . CENP-A S. pombe ,17,43,44Cnp1 at subtelomeric regions we utilised minichromosomes with synthetic telomeres. In cells with elevated CENP-ACnp1, CENP-ACnp1 is incorporated on ura4+ placed adjacent to a terminal array of telomeric repeats (ura4+-Tel) that lack an intervening TAS element. Thus, TAS sequences are not required to attract CENP-ACnp1 to telomeric regions. As these Ch16-based minichromosomes also lack the 100 kb sub-telomeric region where neocentromeres form, we conclude that these regions are not necessary to attract CENP-ACnp1 to telomeres (but see below). Our finding that telomere repeats inserted at an internal chromosomal location can also attract CENP-ACnp1 indicates that proximity to a chromosome end is not a requirement; thus the specialised structure that caps chromosome ends and involves many telomere associated proteins is not needed. At natural telomeres, heterochromatin formation is dependent on Clr4 methyltransferase which is recruited directly by terminal telomere repeats, and by the action of RNAi on regions of the subtelomeric repeats that are homologous to centromeric outer repeats [ura4+-Tel telomere and ura4+: Int-Telo internal telomere repeats, the heterochromatin-specific mark H3K9me2 is dependent on both Clr4 and Ccq1. However, the other components of the SHREC complex do not seem to be involved as lack of the HDAC subunit Clr3 does not affect CENP-ACnp1 deposition (1-4) telomere repeats regardless of their location or the presence of TAS elements. These analyses suggest that Ccq1 has a role in recruiting Clr4 via telomeric repeats and that it is the resulting heterochromatin that promotes the incorporation of CENP-ACnp1.To investigate the preferential incorporation of additional CENP-A repeats . At the Cnp1 deposition we also detected CENP-ACnp1 accumulation on the heterochromatic outer repeats at centromeres in cells expressing additional CENP-ACnp1. However, unlike at telomeres, when heterochromatin is lost (clr4∆), CENP-ACnp1 is still incorporated on these outer repeats. It is likely that at centromeres the presence of CENP-ACnp1 chromatin, and associated loading factors promotes the incorporation of this additional CENP-ACnp1. Barrier function has been ascribed to tRNA genes that lie between heterochromatin and CENP-ACnp1 chromatin domains at centromeres [Cnp1 on the outer repeats in cells expressing elevated levels suggests that these barriers can be overcome so that CENP-ACnp1 can spread beyond the tRNA genes into the flanking heterochromatin region , association with the nuclear periphery, or even sequence-specific properties of these 100 kb sub-telomeric regions may be responsible for attracting CENP-ACnp1 to regions near chromosome ends.It is clear that heterochromatin and telomere-associated proteins are required to recruit additional CENP-Atromeres . AlthougS. pombe, heterochromatin is required for establishment, but not maintenance, of CENP-ACnp1 chromatin on adjacent central domain sequences [Cnp1 to other regions of the genome and find that it is sufficient but not necessary for ectopic localisation of CENP-ACnp1. In Drosophila, overproduced CENP-ACID is also attracted to heterochromatic loci [Cnp1 on adjacent/overlapping sequences? One possibility is that the chromatin environment that heterochromatin engenders – such as histone hypoacetylation or transcriptional features - influences CENP-ACnp1 deposition. Alternatively, CENP-ACnp1 incorporation might be a consequence of the compartmentalisation of heterochromatin. Centromeres are clustered at the spindle pole body (SPB), whilst telomeres are located at the nuclear periphery. This tethering constrains the nuclear volume that they explore, increasing the likelihood that they will come into contact with centromeres. It is likely that a domain around the centromeres / SPB contains high concentrations of CENP-ACnp1 and assembly factors. Thus, telomeres and other heterochromatic loci may be favoured sites for CENP-ACnp1 assembly simply because they frequently come into contact with centromeres. Both centromeres and subtelomeres interact with the same inner membrane protein, Man1, at the nuclear periphery [In equences . Here, wtic loci . How doeeriphery . These iWith the exception of organisms with holocentric chromosomes, it is essential that only one kinetochore is assembled per chromosome; dicentric chromosomes are inherently unstable. On the other hand, the existence of neocentromeres indicates that CENP-A is able to assemble on non-centromeric sequences. Indeed, the formation of neocentromeres is thought to be important in genome evolution and speciation . Thus CECnp1 open reading frame was cloned into three plasmids containing different strengths of the thiamine repressible nmt promoter, pREP81X (pREP81-cnp1+), pREP41X (pREP41-cnp1+ [MluI and integrated at ars1 locus. The Ch16 minichromosomes derivatives used in this work were described in [ura4+ gene within chromosome arm (ura4+: Int-telo) is described elsewhere [Standard genetic and molecular techniques were followed. Fission yeast methods were as described . For the41-cnp1+ , or pREPribed in . The strCnp1 from integrated pREP81-cnp1+ (nmt81-CENP-ACnp1), pREP41-cnp1+ (nmt41-CENP-ACnp1) or cells with integrated empty vector were initially grown in rich medium which contains thiamine to repress the expression of additional CENP-ACnp1. Cells were grown in PMG liquid medium (with thiamine) at 25°C and shifted to 36°C in PMG lacking thiamine for 24 h to allow CENP-ACnp1 expression before ChIP analysis. Strains carrying Ch16 minichromosome derivatives were grown in media lacking adenine to select for the minichromosome.Cells expressing additional CENP-ACnp1 antibody (10 µl of anti-CENP-ACnp1 antibody per 300 µl chromatin extract), anti-CENP-CCnp3 antibody (10 µl of anti-CENP-CCnp3 antibody per 300 µl chromatin extract) and anti-H3K9me2 antibody (1 µl of mAb 5.1.1 (a kind gift of Takeshi Urano antibody per 300 µl chromatin extract) and subsequently analyzed by quantitative PCR (qPCR) or by multiplex PCR. P, 0.01) using SigmaPlot 11 software (Systat Software Inc.)ChIP was performed as described  using anCnp1 antibody per 100 µl chromatin extract [S. pombe Tiling 1.0FR Arrays (Affymetrix) 5 mM dUTP was added to the second round of DNA amplification. Fragmentation, labelling and hybridization were performed by the Affymetrix core facility at Karolinska Institiutet (BEA) using standard protocols (http://www.affymetrix.com). Raw data from Affymetrix (.CEL format) were normalized with Affymetrix Tiling Analysis Software (TAS) v 1.1 and analyzed and visualized using Integrated Genome Browser  and Alexa-488-coupled donkey anti-sheep secondary antibody (1:1000) (Invitrogen – Life Technologies). Microscopy was performed as described [http://www.moleculardevices.com). For comparison of cells expressing CENP-ACnp1 from different promoters, identical exposures are used for imaging of all cells stained for CENP-ACnp1 in any given set. Images are not autoscaled, instead they are scaled relative to the brightest images in the set in order to reflect relative signal intensity obtained from the different strengths of promoter. Thus, images are scaled relative to the nmt81-CENP-ACnp1 cells in nmt1-CENP-ACnp1 cells in Immunolocalization was performed as described . Cells wescribed  using a Figure S1A) CENP-ACnp1 can be detected when overexpressed from the nmt1 promoter. Adapted from Castillo et al., 2007 (Cnp1 from nmt81 (81xC), nmt41 (41xC) or nmt1 (3xC) promoter to give low, medium and high expression levels compared with cells containing endogenous levels (3x). (B) Western analysis of GFP-CENP-ACnp1 levels in wt and spt16-18 cells. From Choi et al., 2012 . The intensities of GFP-CENP-ACnp1 and TAT-1  signals were measured using LICOR Odyssey Infrared Imaging System software (Li-COR Bioscience) and the relative intensities of GFP-CENP-ACnp1/TAT-1 were quantified (bottom panel). GFP-CENP-ACnp1 was expressed for 24 h at 25°C before harvest. (C) Comparison of CENP-ACnp1 localisation in cells expressing CENP-ACnp1 from different strengths of nmt promoter. Immunolocalization of CENP-ACnp1 in cells expressing endogenous levels of CENP-ACnp1 or extra CENP-ACnp1 expressed from the nmt81, nmt41 or nmt1 promoters. Cells were grown in the absence of thiamine for 24 h at 36oC and stained with anti-CENP-ACnp1 antibody (green) and DAPI (DNA: red). Images are displayed to indicate the relative signal intensity in cells expressing CENP-ACnp1 from the four different promoters. Images have not been autoscaled. The images for endogenous (Endog.) and nmt81 promoters therefore appear very faint compared to nmt1. Scale bar, 5 µm.Click here for additional data file.Figure S2A) Genome browser view showing ChIP-chip occupancy profiles for wild-type (Endg.) levels in blue or additional CENP-ACnp1 levels in orange (Cnp1nmt81-CENP-A) at centromeres, pericentromeric outer repeats  or subtelomeric / rDNA-adjacent regions. Data on the Y-axis are presented in linear scale and X-axis shows genome positions (distance from the chromosome end at telomeres and rDNA loci is indicated in kb). ChIP-PCR analysis of CENP-ACnp1 association with pericentromeric outer repeats (B) or subtelomeric / rDNA-adjacent regions (C) expressing wild-type (Endog.) or additional (nmt81) levels of CENP-ACnp1. Enrichment of pericentromeric outer repeats , telomeric  and rDNA adjacent regions (primers reside 34.5 kb from the rDNA repeats on left arm of chromosome III) was assessed compared to actin (act) in immunoprecipitated (IP) relative to total (T) extract. (D) ChIP-qPCR analysis of CENP-ACnp1 association with a subtelomeric region from the right telomere of chromosome I (53 kb from the end) expressing wild-type (Endog.) or additional (Cnp1nmt81-CENP-A) levels of CENP-ACnp1. Enrichment on the sub-telomeric region was normalized to the signal obtained for the euchromatic gene encoding actin (act1+). Error bars indicate S.D. from 3 biological replicates. Mean values marked with different letter (a or b) indicate results significantly different from each other, as established by One Way ANOVA and Holm-Sidak test for multiple comparison (P<0.01).((PDF)Click here for additional data file.Figure S3A) ChIP-qPCR of CENP-ACnp1 and CENP-CCnp3 levels at TM1 in cells containing the ura4+-Tel or ura4+-TAS-Tel Ch16 minichromosomes and expressing endogenous (Endog. CENP-ACnp1) or additional (nmt41-CENP-ACnp1) levels of CENP-ACnp1. Enrichment is reported as the percentage of immunoprecipitated chromatin (% IP). Error bars indicate S.D. from 3 biological replicates. (B) ChIP-qPCR of H3K9me2 levels on centromeric otr dgI repeats in the same cells as in A. Enrichment on dgI was normalized to the signal obtained for the gene encoding actin (act1+). Error bars indicate S.D. from 3 biological replicates. Mean values marked with the same letter (a) indicate results not significantly different from each other, as established by One Way ANOVA and Holm-Sidak test for multiple comparison (P<0.01).((PDF)Click here for additional data file.Figure S4Cnp1 (nmt41-CENP-ACnp1) grown at 36°C for three days. Cells containing the m23:ura4+ (ura4+ inserted 50 kb from the left telomere of Ch16), ura4+-Tel or ura4+-TAS-Tel Ch16 minichromosomes were plated on the indicated media: no leucine, adenine or no uracil, or with counter-selective 5-FOA added.Serial dilution growth assay of wild-type cells expressing wild-type levels (Endog.) or moderate levels of additional CENP-A(PDF)Click here for additional data file.Figure S5Cnp1 levels on centromeric otr dgI repeats (A) and at TM1 in the central region of centromere 1 (B) in wild-type (wt) and mutant cells  carrying the Chs 16–72 (ura4+-Tel) minichromosome and expressing endogenous (Endog. CENP-ACnp1) or additional (nmt41-CENP-ACnp1) CENP-ACnp1 levels. (C) ChIP-qPCR of H3K9me2 levels on centromeric otr dgI repeats in wild-type (wt) and mutant cells  with the same configuration. Enrichment on dgI was normalized the signal obtained for the gene encoding actin (act1+). Enrichment at TM1 is reported as the percentage of immunoprecipitated chromatin (% IP). Error bars indicate S.D. from 3 biological replicates. Mean values marked with different letter  indicate results significantly different from each other, as established by One Way ANOVA and Holm-Sidak test for multiple comparison (P<0.01 in (A) and (B) or P<0.05 in (C).ChIP-qPCR of CENP-A(PDF)Click here for additional data file.Figure S6Cnp1 levels at TM1 in the central region of centromere 1 (A) and centromeric otr dgI repeats (B) in cells containing an array of telomeric repeats integrated at the ura4+ locus (ura4+: Int-Telo) or control cells (ura4+) and expressing endogenous (Endog. CENP-ACnp1) or additional (Cnp1nmt41-CENP-A) levels of CENP-ACnp1. Enrichment on dgI was normalized the signal obtained for the gene encoding actin (act1+). Enrichment at TM1 is reported as the percentage of immunoprecipitated chromatin (% IP). Error bars indicate S.D. from 3 biological replicates. Mean values marked with different letter (a or b) indicate results significantly different from each other, as established by One Way ANOVA and Holm-Sidak test for multiple comparison (P<0.01).ChIP-qPCR of CENP-A(PDF)Click here for additional data file.Figure S7A) ChIP-qPCR of CENP-ACnp1 levels at TM1 in the central region of centromere 1 and centromeric otr dgI repeats in wild-type (wt) and mutant cells  which contain the internal telomere (ura4+: Int-Telo) and that express endogenous (Endog. CENP-ACnp1) or additional (Cnp1nmt41-CENP-A) levels of CENP-ACnp1. (B) ChIP-qPCR of H3K9me2 levels in the same cell backgrounds. Enrichment at TM1 is reported as the percentage of immunoprecipitated chromatin (% IP). Enrichment on dgI was normalized the background signal obtained for the gene encoding actin (act1+). Error bars indicate S.D. from 3 biological replicates. Mean values marked with different letter  indicate results significantly different from each other, as established by One Way ANOVA and Holm-Sidak test for multiple comparison (P<0.01).((PDF)Click here for additional data file.Table S1(PDF)Click here for additional data file.Table S2(PDF)Click here for additional data file.Table S3(PDF)Click here for additional data file.


Increasing copy number of the desired genes in these pathways is a general method to solve the problem, for example, the employment of the multi-copy plasmid-based expression system. However, this method may bring genetic instability, structural instability and metabolic burden to the host, while integrating of the desired gene into the chromosome may cause inadequate transcription or expression. In this study, we developed a strategy for obtaining gene overexpression by engineering promoter clusters consisted of multiple core-in vitro assembling process, a series of promoter clusters were constructed. The transcription strength of these promoter clusters showed a stepwise enhancement with the increase of tandem repeats number until it reached the critical value of five. Application of the MCPtacs promoter clusters in polyhydroxybutyrate (PHB) production proved that it was efficient. Integration of the phaCAB genes with the 5CPtacs promoter cluster resulted in an engineered E.coli that can accumulate 23.7% PHB of the cell dry weight in batch cultivation.Through a uniquely designed tacs promoter cluster can be greatly improved by increasing the tandem repeats number of the core-tac-promoter. By integrating the desired gene together with the MCPtacs promoter cluster into the chromosome of E. coli, we can achieve high and stale overexpression with only a small size. This strategy has an application potential in many fields and can be extended to other bacteria.The transcription strength of the MCP Howeverabolites -4. Thus,abolites . As a usabolites . Howeverabolites -9. In adabolites .cat) and a homologous region of 1 kb in the chromosome. Therefore, there would finally produce significant amounts of the superfluous DNA sequences when enough gene copies were achieved. These DNA sequences in the chromosome may also be metabolic burden for the host [With the development of molecular biotechnology, many attempts have been made to overcome these flaws. Integration of the desired genes into the chromosome of the host seems able to circumvent these problems; and therefore many chromosome integration strategies have been developed in the past few years -15. Howethe host .tac-promoters (MCPtacs) in tandem. With an ingenious design, the series MCPtacs promoter clusters were assembled via the Gibson's method [tacs promoter clusters was then analyzed employing the green fluorescence protein (GFP) as an indicator. Application of the MCPtacs promoter clusters in polyhydroxybutyrate (PHB) production proved its efficiency and simplicity.To maintain high expression of the desired genes but avoid superfluous DNA sequence, we developed a strategy to improve transcription strength by constructing the promoter clusters consisted of multiple core-s method . The tratac promoter is nearly the strongest available promoter for metabolic engineering. However, even the tac promoter cannot satisfy the need of high expression of the desired genes in the pathway engineering, especially when they should be integrated into the chromosome. Therefore, to improve the expression of the desired genes in the chromosome, we came up with the idea of constructing a series promoter clusters by tandem repeating the strong tac promoter. Our preliminary experiments showed that the core-tac-promoter (containing 41 bp) possessed complete transcription function and almost the same transcription strength as the wild type tac promoter. To minimize the size of the promoter clusters for easy-to-construct, only the constitutive core-tac-promoter was then employed.The gfp gene  as the transcription strength indicator. The series constitutive MCPtacs promoter clusters were constructed in vitro following the Gibson's assembling method (See method section) [tac-promoters aligned in tandem (The 5CPtacs promoter cluster) . Meanwhile, the 5CPtacs promoter cluster can be cut into any number, from one to four, of the core-tac-promoter under the action of T5 exonuclease and a high fidelity Phusion Host Start DNA Polymerase. Thus, plasmids with arbitrary positive integer of the core-tac-promoter were assembled [-tac-promoter were obtained, and were named p1TG, p2TG, p3TG, p4TG, p5TG, p6TG, p7TG, p8TG, p9TG and p10TG, respectively.For construction, we chose the low-copy plasmid pCL1920 as the cloning vector and the tacs promoter clusters, fluorescence intensity of each construct radiated from the green fluorescence protein (GFP) was determined . The fluorescence intensity of the 5CPtacs promoter cluster was about 4.4-fold more than that of the original core-tac-promoter. The value of fluorescence/OD600 to OD600 taken from the logarithmic phase was also analyzed to exclude effect of the cell growth on fluorescence enhancement. It also exhibited a stepwise enhancement for the same OD600 with the increase of tandem repeats. This result proved that the fluorescence/OD600 enhancement was due to the increased transcription/expression eliminating the factor of increase affected by the cell growth  [phaCAB genes into the chromosome increased the stability of the construct but caused dramatically reduced PHB accumulation. Our preliminary experiment showed that single copy integration of PHB operon into the chromosome can only accumulate 3.6% of cell dry weight PHB. Previous study also found that single copy integration of the phbCAB genes in the chromosome caused a very low PHB accumulation [phaCAB genes in the downstream of the 5CPtacs promoter cluster, which was found to have the strongest transcription strength, and integrated the whole cassette together with the flanking regions  into the chromosome of E. coli, resulting in the strain E. coli DH5α/ΔpoxB::5TPHB.The MCPht (CDW) . Integramulation . TherefoThis strain was cultivated in glucose medium and found to accumulate 23.7% PHB (wt% of CDW) after 28 h batch fermentation. The PHB accumulation was 5.6-fold more than that accumulated by the control strain E. coli DH5α/ΔpoxB::1TPHB which integrated single copy core-tac-promoter. Monitoring the fermentation process, we found that consumption of glucose in E. coli DH5α/ΔpoxB::5TPHB was apparently faster than that of the control strain Figure . The fastac-promoter arranged in tandem repeats in E.coli.In nature, the repetitive sequences including interspersed and tandem repetitive elements usually exist in eukaryotic genomes , even intacs promoter clusters with various tandem repeats of the core-tac-promoter due to its 3'-5' exonuclease activity. After digesting with restriction enzyme, there were still few unpaired residues at both 3' and 5' ends of the restriction sites at the assemble fragments. Fortunately, except for 5'-3' polymerase activity, Phusion Host Start DNA Polymerase can also remove base sequence, especially the unpaired base, from 3' end of the assemble fragment due to its high fidelity. Meanwhile, T5 exonuclease acted on the 5' end of the assemble fragments to produce partial single strand at the 3' end of the DNA molecular. Thus, the five core-tac-promoters may be also sliced one to four. Hence, a random number of the core-tac-promoter beginning from one can be constructed  which was integrated into the chromosome in E.coli, the total size of the integrated heterologous DNA reached 120 kb if 20 copies were obtained through evolution, about 2.6% of the size of E. coli chromosome. However, this strain can only accumulate about 18% PHB (wt% CDW) [ΔpoxB::5TPHB, of which only 4.3 kb heterologous DNA sequence was integrated into the chromosome, can produce 23.7% PHB (wt% CDW). Although they found no growth difference between the engineered strain and the control, the extra synthesis of large amount of DNA should consume abundant nucleotides and much energy [phaCAB genes together with the 5CPtacs promoter cluster into the chromosome were also proved to be stable in our strain E. coli DH5α, in which recA was deleted.Due to the weak promoter strength, the existing gene overexpression methods, including plasmids and repeated integration, intended to realize this goal by increasing the copy number of the desired gene . The genwt% CDW) . While oh energy . The inttacs promoter cluster and integrating them into the chromosome of E. coli. Then, the desired genes can be used to replace the reverse selection maker through one-step homologous recombination. In addition, two or more different inducible promoters may be fused in tandem; therefore time-dependent expression can be achieved by responding different circumstances or adding corresponding inducers at different times.This strategy has many potential applications in the metabolic engineering. We may construct a platform for stable gene overexpression by putting a reverse selection maker under control of the 5CP-tac-promoter. Increasing tandem repeats of the core-tac-promoter can enhance the transcription/expression strength of the MCPtacs promoter clusters. By integrating of the 5CPtacs promoter cluster into the chromosome of E. coli, we achieved high and stable expression of the target gene with insertion of a small DNA fragment . The series of MCPtacs promoter clusters can be applied in pathway engineering of E. coli and extended to other bacteria.In this study, we designed a strategy which can achieve high-level gene expression by tandem repeating the coreBacterial strains and plasmids used in this study were shown in Table tacs promoter clusters was described as follows , Phusion Hot Start DNA Polymerase (New England Biolabs (NEB)) and Taq DNA ligase (NEB) at 50°C for 15 min. The resulting constructs containing different promoters were then transformed into competent cells and were firstly screened based on the fluorescence signal and PCR detection. The finally plasmid were confirmed by double digestion and sequencing.The construction of the MCPs Figure : The destacs promoter clusters were grown in 50 ml Luria broth  at 220 rpm and 37°C. Samples for measurement were taken out every 2 h and harvested by centrifugation at 14,000 × g for 2 min. After being resuspended with PBS buffer , 200 μl of bacterial culture was transferred into a 96-well plate in which OD600 and fluorescence were read with excitation at 485 nm and emission at 528 nm using a Multi-Detection Microplate Reader, Synergy HT (BioTek). For each sample, 3 repetitions were performed with PBS as a blank. Average of three repeats for the specific fluorescence and OD600 were chosen as the reference of promoter strength.Cells harboring the MCPphaCAB genes was executed by following Kuhlman's method [poxB gene of DH5α was replaced by a tetracycline resistance gene (tet) flanked by I-SceI recognition site and 25 bp of landing pad region at each side, amplified from the plasmid pTKS/CS, and E. coli DH5α(ΔpoxB::tet) was obtained. The phaCAB genes cloned from pBHR68 was inserted into NsiI and XhoI sites of p1TG, resulting plasmid p1TPHB. Both the plasmid pTKIP and p1TPHB were digested with HindIII and EcoRI and ligated together to create the plasmid pTKIP-1TPHB. Then, the plasmid pTKIP-1TPHB was transformed in DH5α (ΔpoxB::tet)/pTKRED. In vivo recombination between landing pad regions was finished under the action of RED recombinase and the stimulation effect of I-SceI, resulting the strain DH5α/ΔpoxB::1TPHB-kan. The kanamycin resistance gene was eliminated with the help of pCP20 cultivating at 42°C as described by Datshenko [ΔpoxB::1TPHB was obtained. In the same way, the strain DH5α/ΔpoxB::5TPHB was constructed.Integration of the s method . In brieatshenko  and the 2HPO4·12H2O, 22.0 mM KH2PO4, 8.6 mM NaCl, 18.7 mM NH4Cl, 2% extract, 1 mM MgSO4, 0.1 mM CaCl2) supplemented with 1.2% glucose, 0.1 M MOPS and 25 μg/ml kanamycin antibiotic at 37°C and 250 rpm. pH was adjusted at 7.0 with NH3·H2O. Samples with a defined interval were harvested by centrifugation at 14,000 × g for 2 min. The supernatant was then diluted to an appropriate concentration to measure glucose concentration using SBA-40 C (Biology Institute of Shandong Academy of Sciences). After suspending with PBS (pH 7.4), the sample cells were read at 600 nm for the optical density (OD600).Preculture was grown overnight at 37°C, 250 rpm in Luria broth medium supplemented with 25 μg/ml kanamycin. Fermentation was carried out in 250 ml shake flask containing 50 ml modified M9 medium  using a Gas Chromatography. Cultures were collected by centrifugation  and lyophilized overnight. Mixture of 20 mg lyophilized cells mass with 1 ml chloroform, 1 ml methanol and 15% (v/v) sulfuric acid was boiled for 1 h for methylation. Then 1 ml ddHed by GC .phaCAB genes were assessed in transcription level via Quantitative Reverse Transcription PCR (QRT-PCR). Cells for mRNA preparation were cultivated for 4 h and then harvested. Total mRNA of DH5α/ΔpoxB::1TPHB and DH5α/ΔpoxB::5TPHB were extracted using the RNeasy Mini Kit (Tiangen). The cDNA was amplified through reverse transcription with the total mRNA as the templates. Quantity real-time PCR (QPCR) amplification primers were designed and were listed in Table gapA gene was chosen as the control for normalization. QPCR was performed in a 96-well plate with a reaction volume of 20 μl for each sample in MyiQ5768R Real-Time PCR detection system using a SYBRs Premix Ex Taq TM II , according to manufacturer's specification (TaKaRa). The obtained data were analyzed by using the 2-ΔΔCt method described previously [The eviously .tacs: multiple core-tac-promoters; 5CPtacs: five core-tac-promotersMCPThe authors declare that they have no competing interests.ML carried out most of the experiments and wrote the manuscript. JW and YG are responsible for performing some experiments. YL participated in the design of this study. QW and QL assisted in data analysis and revised the manuscript. QQ conceived of the study, participated in its design, coordination and helped to draft the manuscript. All authors read and approved the final manuscript.


Various approaches have thus been developed to aid in the efficient identification of genes that contribute to a variety of different phenotypes, allowing more rapid design and engineering of traits desired for industrial applications. This review will highlight recent technologies that have enhanced capabilities to map genotype-phenotype relationships on a genome wide scale and emphasize how such approaches enable more efficient design and engineering of complex phenotypes.Rewiring and optimization of metabolic networks to enable the production of commercially valuable chemicals is a central goal of metabolic engineering. This prospect is challenged by the complexity of metabolic networks, lack of  Sa. Sa12]. nditions . The autnditions . It is iMicroorganisms have evolved exquisite transcriptional regulatory networks that enable rapid changes in gene expression and allow them to adjust their metabolism for optimal performance under a wide variety of environmental conditions. The ability to track genome wide changes in mRNA expression can thus provide another useful metric for identifying functional elements that are involved in various adaptive processes. In addition, transcriptome level analyses can help define the basic organization of operons and identify non-coding regulatory networks that shape cellular metabolism  Figure . Unlike E. coli as well as defining dynamic expression patterns for many operons [thrLABC operon for example, transcription was found to initiate from the most upstream promoter during log phase, but during lag phase an alternative promoter is activated to enable expression of the distal genes thrB and thrC and alleviate attenuation caused by the 5′ untranslated region in this polycistronic mRNA [Bacillus subtilis under 104 different growth conditions allowed for comprehensive identification of regulons associated with the various transcription factors in this industrially relevant organism [Transcriptome wide analysis has provided a number of recent insights into the structure of native transcription networks. For example, integration of transcriptional profiling with chromatin immunoprecipitation (CHIP) RNA polymerase II to identify global occupancy of the transcription machinery enabled the identification of a number of 4661 transcription units in  operons . In the nic mRNA . Such aporganism .E. coli compared the transcription profile of a parental strain to multiple laboratory evolved strains and subsequently confirmed causal link between up-regulation of iron transport and amino acid biosynthesis genes [E. coli that was rationally engineered for valine production compared to the parental W3110-strain derivative [Application of comparative transcriptome profiling between parental and evolved or engineered cell lines can also provide useful insight into the genes whose altered expression contributes to production and tolerance related phenotypes. For example, one study of ethanol tolerance in is genes . Supplemrivative . The trarivative .In addition to monitoring changes in expression due to environmental perturbations transcriptional profiling has been recently employed to better understand transcriptional networks that have been rewired by synthetic means. For example, global transcription machinery engineering (gTME) involves the creation of plasmid encoded libraries of major sigma factors that have altered DNA binding preferences . Followi14 kbp per sequencing run [Despite the power to map genotype fitness genome wide, the techniques described above lack the ability to completely define the genotype of an engineered or evolved strain, leaving open the possibility that off target mutations may influence the observed trait. While whole genome sequencing (WGS) can address this challenge, it has historically been costly and labor intensive. However, advances in next generation sequencing have occurred at an astounding rate. Current technologies enable ~ 10cing run , and thecing run -82 promiE. coli clones taken from different time points during the long term evolution of the population for increased citrate utilization [citT in front of an actively transcribed promoter to enable gene expression. Another study recently performed 115 parallel selections for thermotolerance (growth at 42.2°C) followed by WGS of a single isolate from each population [Long term laboratory evolution studies now utilize WGS to map mutations onto growth phenotypes. WGS has therefore enabled exciting new insights into the molecular mechanisms of adaptation to a variety of stresses. For example, a recent study sequenced the genomes of 29 lization . The seqpulation . The stupulation . NotablyOn the other hand, although WGS provides a complete description of the genome, it is limited to relatively small sample sizes making it impossible to determine the relative fitness of different mutational combinations. To determine the fitness of combinatorial mutations requires deep sampling of the combinatorial sequence space of a directed set of mutations. As tracking technologies are expanded from single allele approaches to determining the fitness of many genetic combinations at high depth they will open the door to the evaluation of increasingly complex genotype-phenotype relationships. WGS should therefore be considered as a complementary approach to current and future tracking technologies that will aid in improving the accuracy with which relevant mutations are identified.E. coli genome and have aided in uncovering genes implicated in a variety of industrially relevant traits with rapid turnover times. Recent efforts have been made to combine TRMR with combinatorial engineering strategies similar to MAGE [To the benefit of basic and applied research alike, genome wide tracking technologies have significantly enhanced the throughput with which genotype- phenotype relationships can be investigated. Approaches such as TRMR  and SCAL to MAGE  promise  to MAGE . This stcas9 was demonstrated to provide inducible transcription repression based solely on the sequence of a synthetic guide RNA [The techniques described here will need to be improved such that multigenic traits can be characterized in parallel and the engineering cycle can be performed recursively. Recent progress towards more effective combinatorial tracking has been made using synthetic RNA based regulatory devices that enable multiplexed, sequence-specific gene control from single plasmid . Similaruide RNA . SequencGill, Lynch, and Evans declare financial interest in Opxbio.AG prepared all of the illustrations and served as lead author of the manuscript. ML, RE and RG provided supervision and editing of the manuscript. All authors read and approved the final manuscript.


Pterygium is a common ocular surface disease characterized by abnormal epithelial and fibrovascular proliferation, invasion, and matrix remodeling. This lesion, which migrates from the periphery to the center of the cornea, impairs vision and causes considerable irritation. The mechanism of pterygium formation remains ambiguous, and current treatment is solely surgical excision, with a significant risk of recurrence after surgery. Here, we investigate the role of methylation in DNA sequences that regulate matrix remodeling and cell adhesion in pterygium formation.p<0.05), whereas hypomethylation was detected at CpGs +484 and +602 bp downstream of matrix metalloproteinase 2 (MMP-2) transcription start site, and −809, −762, −631 and −629 bp upstream of the CD24 transcription start site. RT-qPCR, western blot and immunofluorescent staining showed that transcript and protein expression were reduced for TGM-2 and increased for MMP-2 and CD24. Inhibition of methylation in cultured conjunctival epithelial cells increased these transcripts.Pterygium and uninvolved conjunctiva samples were obtained from the same eye of patients undergoing surgery. The EpiTYPER Sequenom technology, based on differential base cleavage and bisulfite sequencing was used to evaluate the extent of methylation of 29 matrix and adhesion related genes. In pterygium, three CpG sites at −268, −32 and −29 bp upstream of transglutaminase 2 (TGM-2) transcription initiation were significantly hypermethylated (We found regions of aberrant DNA methylation which were consistent with alteration of TGM-2, MMP-2, and CD24 transcript and protein expression, and that inhibition of methylation in cultured cells can increase the expression of these genes. Since these genes were related to cell adhesion and matrix remodeling, dysregulation may lead to fibroblastic and neovascular changes and pterygium formation. These results have implications for the prognostication of pterygium in clinical practice, for example, detection of epigenetic changes may have a role in predicting post surgical recurrence of aggressive lesions. Pterygium is a common fibrovascular ocular surface disease characterized by migration of wedge-shaped abnormal tissue from bulbar conjunctiva onto the cornea. Morbidity incurred by pterygium includes irritation, redness, and visual impairment in instances, such as visual occlusion induced by a large pterygium that has migrated to the visual axis over the central cornea, and irregular astigmatism or impaired tear film regularity induced by pterygium. The pathogenesis of this disease, however, remains ambiguous. Compelling evidence that UV-mediated limbal damage triggers this pathogenesis has been reviewed Epigenetic modifications of gene expression are known to play an established role in the development of human cancers Epigenetic changes have been implicated in the control of wound healing Promoter methylation has been known to play important roles in genes related to wound healing. For example, transglutaminase 2 (TGM-2) is involved in corneal wound healing Wound healing processes are also mediated by the matrix metalloproteinases (MMP) such as MMP-2, which is primarily secreted by keratocytes in the cornea and mediates long-term stromal remodeling and basement membrane synthesis Recently, a cell adhesion molecule that has never been previously associated with pterygium, CD24, was reported to be upregulated and localized in the nuclei in pterygium epithelium which suggest that cell adhesion properties may be disrupted Despite the important findings on promoter methylation and wound healing, there has been no reported study on the aberrant methylation in pterygium. We have previously investigated the genes specifically involved in wound healing mechanisms such as TGM-2, MMP-2, and CD24 in pterygium The methylation status at the promoters of TGM-2, MMP-2, and CD24 genes was significantly different between pterygium and uninvolved conjunctiva samples in at least one CpG unit. Since DNA methylation is generally a form of negative regulation of gene transcription Figure S1 shows three specific CpG islands that were significantly hypermethylated in pterygium in comparison to conjunctiva tissue, located at the −268, −32 and −29 positions of TGM-2 transcription start site (NCBI accession no. U13920). These positions corresponded to −367, −131 and −128 bp, respectively, upstream of the translation start site. Figure S2 illustrates a differentially hypomethylated CpG unit in pterygium tissue, which was located at the +484 and +602 bp downstream of MMP-2 transcription start site (NCBI accession no. NM_004530.4). In Figure S3A, a CpG unit located −698 bp upstream of CD24 transcription start site (NCBI accession no. Y14692), corresponding to −809 bp upstream of the start codon, was shown to be significantly hypomethylated in the pterygium tissue. Other differentially hypomethylated CpG units were located −762, −631 and −629 bp upstream of the CD24 transcription start site .Supplementary In order to determine whether DNA methylation might be a general phenomenon that controls cell-specific TGM-2, MMP-2, and CD24 expression in the conjunctiva, we treated spontaneously immortalized human conjunctival epithelial cells (IOBA-NHC) with 5-aza-2′-deoxycytidine (5-aza-dC) and monitored TGM-2, MMP-2, and CD24 mRNA expression. The methyltransferase inhibitor, 5-aza-dC, stimulated the upregulation of TGM-2, MMP-2, and CD24 Figure 2p<0.05) in pterygium relative to the conjunctiva. MMP-2 and CD24 transcripts however, were upregulated 2.44±0.52 fold and 2.03±0.22 fold (p<0.05), respectively in pterygium compared to normal conjunctiva. A bar graph summarizing the fold differences of these genes in pterygium tissue compared to the conjunctiva tissue is illustrated in TGM-2, MMP-2, and CD24 gene transcripts were detected in conjunctiva and pterygium tissues. TGM-2 transcript was downregulated by 0.42±0.03 fold  islands: HTF-1 (+1 to −215 with reference to start of transcription) and HTF-2 (−1315 to −1415), has been linked to tumor cell phenotype Differential methylation of the MMP-2 promoter has been reported in the region up to +733 bp from the transcription start site, in a breast cancer cell line S2). The observed biological effects of the gene expression changes may be more than expected based on differential methylation of individual genes. For example, TGM-2 can activate MMP-2 in skin fibroblasts TGM-2, MMP-2 and CD24 are shown to be linked to one another and other important intracellular signaling molecules either indirectly, or directly, in known biological pathways Figure 4Pterygium formation has been reportedly linked to UV radiation Clinically, pterygium is primarily treated by surgical excision. However, aggressive recurrences after surgery are fairly common In conclusion, we have found aberrant DNA methylation to be present in several key genes associated with wound healing processes in pterygium. In our study, we found that critical CpG islands associated with the MMP-2 and CD24 genes were demethylated in pterygium, whereas TGM-2 gene sequences were over-methylated. These results suggest that the pathogenesis of pterygium may be related to the methylated state of pivotal wound healing and developmental genes.The following protocols conformed to the tenets of the Declaration of Helsinki and were approved by the institutional review board of the Singapore Eye Research Institute, and the written informed consent was acquired from all participating patients.The surgical excision and procurement of pterygium and conjunctiva tissues from patients was carried out as previously described IOBA-NHC were a gift from Yolanda Diebold at the University of Valladolid, Spain. These cells were cultured in a medium containing equal volumes of Dulbecco modified Eagle medium (DMEM) and F12, with an additional 2 ng/ml recombinant human EGF , 1 µg/ml bovine insulin, 0.1 µg/ml cholera toxin, 0.5 µg/ml hydrocortisone, and 10% FBS . After reaching 80–90% confluency, the cells were treated with 1, 2 or 3 mM of 5-aza-dC  for 24 hours. Some of the cells were left untreated.The surgical samples were ground in liquid nitrogen with pestle and mortar after fine chopping. Extraction and purification of DNA from tissues was performed using the Genomic DNA Extraction Kit . Quality control was ascertained by ensuring that the ratio (260 nm∶280 nm) of the absorbance of the extracted DNA to be 1.80–1.95. Three samples of pterygium tissues and three un-involved conjunctiva tissues from 3 different patients were analysed in this study.The EpiTYPER Sequenom Mass Array http://www.ncbi.nlm.nih.gov/gds/ with GEO series accession number GSE2513). The methodology of this study has been reported elsewhere Previously, we have performed a microarray analysis to examine the transcript levels of >23000 genes using the Affymetrix U133A Genechip The sequences for some of the more significant sequences are shown in http://www.ebi.ac.uk/Tools/clustalw2/index.html) was performed.To visualize the proximity of the significant CpG islands from the transcription and translation start sites, alignment of the EpiTYPER target sequence with the appropriate NCBI nucleotide sequence using ClustalW2 software (https://www.roche-applied-science.com/sis/rtpcr/upl/index.jsp?id=UP030000) design tool selecting for intron spanning assays. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the internal control. For each sample, triplicate wells were used. ΔCt was calculated by subtracting the Ct of GAPDH from the Ct of the targeted gene. The untreated IOBA-NHC cells and uninvolved conjunctiva were considered as the calibrator to compare the relative abundance of TGM-2, MMP-2, and CD24 gene transcript in IOBA-NHC cells treated with 5-aza-dC and in pterygium tissue, respectively. The fold change was determined by the formula 2(−ΔΔCt), where ΔΔCt = ΔCtsample−ΔCtcalibrator.Primers shown in Conjunctival tissues from normal conjunctival epithelium and pterygium were sectioned with a Microm HM550 cryostat  at 8 µm thickness. Sections were fixed with 4% paraformaldehyde for 10 minutes, washed with 1X PBS, and blocked with 4% BSA in 1X PBS containing 0.1% Triton X-100 for one hour. Rabbit polyclonal antibodies against TGM-2  and MMP-2 , and mouse monoclonal antibody against CD24  were diluted with the blocking buffer at a factor of 1∶150, 1∶200, and 1∶100, respectively and incubated at 4°C overnight. After washing with 1X PBS, the sections were incubated with Alexa Fluor 488-conjugated secondary antibody (Invitrogen) at room temperature for 1 hour. Slides were then mounted with UltraCruz Mounting Medium containing DAPI . For negative controls, non-immune serum was used in place of the specific primary antibody. Sections were observed under and imaged with a Zeiss Axioplan 2 fluorescence microscope .In situ zymography was performed to localize the gelatinase activity in pterygium and conjunctiva cryosections using a previous reported method Normal conjunctiva and pterygium tissue were homogenized individually into RIPA lysis buffer (Santa Cruz Biotechnology). Insoluble materials were removed by 15-minute centrifugation, 10,000 g at 4°C. An equal volume of 4x SDS loading buffer was added to each sample which was then subjected to boiling for 5 min at 99°C. The sample was then left on ice for 10 minutes before reduction on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE-gradient 10%). The amounts of protein applied was 40 µg. Resolved proteins were electrophoretically transferred onto a nitrocellulose membrane and blocked in 1X TBS containing 5% non-fat milk, followed by overnight incubation with the rabbit polyclonal antibodies against TGM-2  and MMP-2 , and mouse monoclonal antibody against CD24  in 4°C with agitation. The membranes were then washed vigorously three times each for five minutes in 1X TBS, 0.1% Tween-20. The HRP-conjugated secondary antibodies (Santa Cruz Biotechnology) were then applied at a dilution of 1∶2000. Immunoreactivity was visualized with Super Signal West Pico chemiluminescence reagent .t-test or analysis of variance (ANOVA) followed by the least significant difference (LSD) test were used to determine the difference between groups. p<0.05 was accepted as significantly different.Student's Table S1Entities that were analyzed in the pathways shown in (0.04 MB DOC)Click here for additional data file.Table S2Relationship between the entities analyzed in the pathways shown in (0.05 MB DOC)Click here for additional data file.Figure S1Hypermethylation of transglutaminase 2 (TGM-2) promoter in the pterygium. The brackets indicate hypermethylated CpG sites in pterygium shown in . CGs hig(0.19 MB TIF)Click here for additional data file.Figure S2Hypomethylation of matrix metalloproteinase 2 (MMP-2) promoter in the pterygium. The bracket indicates the hypomethylated CpG site in pterygium shown in . CGs hig(0.34 MB TIF)Click here for additional data file.Figure S3Hypomethylation of CD24 promoter in the pterygium. More than one EpiTYPER sequences were used for this promoter: (A) CD24_01. (B) CD24_02. The brackets indicate the differentially methylated CpG sites that were shown in (0.93 MB TIF)Click here for additional data file.


We spliced the promoters of the human telomerase and human survivin genes  widely used for gene therapy and known to have the broadest cancer type spectrum of activity. Two head-to-tail constructs were obtained: the PhTERT-PhSurv and PhSurv-PhTERT tandems. The splicing caused quantitative and qualitative changes in the promoter features. In both constructs, only the promoter proximal to the transcribed gene retained its ability to initiate transcription, whereas the distal promoter was silent, the phenomenon never reported before. However, the distal promoter modulated the activity of the proximal one by increasing its strength and causing an appearance of additional transcription start sites. We suggested that this suppression might be due to the presence of Sp1 transcription factor binding sites in both promoters and Sp1-bridges between these sites. Such Sp1-bridges might convert the tandem promoter linear DNA into a stem-loop structure. If localized inside the formed loop, the distal promoter could lose its ability to initiate transcription. To test this hypothesis, we constructed two modified double promoters, where the proximal PhSurv promoter was replaced either by a shortened variant of the survivin promoter (PhSurv269) or by the mouse survivin promoter. Both PhSurv substitutes were considerably shorter than PhSurv and had different numbers and/or positions of Sp1 sites. In modified tandems, transcription was initiated from both promoters. We also prepared two mutant forms of the PhSurv-PhTERT tandem with two or four Sp1 sites removed from the distal “long” PhSurv promoter. In the first case, the distal PhSurv promoter remained silent, whereas the removal of four Sp1 binding sites restored its activity. In the majority of studied cancer cell lines the efficiency of transcription from the hTERT-(shortened hSurv269) promoter tandem was markedly higher than from each constituent promoter. In normal lung fibroblast cells, the tandem promoter activity was considerably lower. Although both promoters have a rather broad activity spectrum, they are still very far from being universal: e.g. PhSurv and PhTERT are active in tumors of only about 60% of patients with non-small cell lung cancer Gene therapy represents treatment modality that offers unique opportunities for tumor targeting. To this end, cellular mechanisms of gene regulation have been successfully used to direct therapeutic gene expression preferentially to cancer cells Our preliminary data (to be published) showed that the activity profiles of the two constituent promoters were to a great extent complementary with partial overlapping. Therefore, we hypothesized that a tandem combination of the PhSurv and PhTERT promoters might represent a more universal and strong promoter.hASH1 and EZH2 genes, Poulsen at al. constructed a chimeric double promoter for efficient expression of a killer gene in cells of small cell lung cancer Tumor-specific double (tandem) promoters were used in recent works High-level expression of the tBid apoptosis activator in cells of breast cancer was achieved with a hybrid promoter constituted of PhSurv and the promoter of a gene coding for mucin and known to be upregulated in tumor cells of the mammary gland In both cases above This report is mainly focused on deciphering the mechanism of interference leading to inactivation of distal promoters in tandems and the ways to avoid this effect when designing tandem promoters for different purposes. However, it is worthy of attention that one of the investigated promoters, PhTERT-PhSurv269, was significantly more active than others. In the majority of studied cancer cell lines, the initiation level of transcription from this tandem was on the average 4.8 and 3.3 times higher than that of the PhTERT and PhSurv cancer-specific promoters, respectively. In normal lung fibroblast cells, the tandem promoter activity was considerably lower.Individual promoters used in the study are schematically shown in PhSurv. A promoter region of the human survivin (BIRC5) gene (−1456 to +42) PhSurv269, a shortened promoter region of the human survivin gene (−268 to +1) PmSurv, the minimal promoter region of the mouse survivin gene (−196 to +1) PhTERT, an hTERT −191 to +48 promoter fragment The PhSurv and PhTERT promoters include initiator elements and are transcriptionally controlled by a variety of signaling pathways that promote or suppress carcinogenic processes in humans The tandem promoter constructs used in the study are shown in Transcriptional activity of the promoters was determined in eukaryotic cells by measuring firefly luciferase activity.As controls, we used non-transfected cells and cells transfected with control plasmids, a promoterless BV-pGL3 plasmid and a PV-pGL3 plasmid containing the reporter firefly luciferase gene under control of an SV40 promoter. Relative activity of the tested promoters was measured on a panel of tumor cell lines of different origin. The cell lines were different not only in origin but also in p53 status, because the activity of the single promoters under study was reported to be dependent on the p53 status of cells A comparative analysis revealed that the activity of the PhTS and PhST double promoters in most cases exceeded that of both the PhTERT and PhSurv single promoters, whose activity levels were sharply different in five cancer cell lines of the panel . The relThe activity changes of both single promoters in different cell lines were qualitatively similar, except the Calu-1 cell line, in which the activity of PhSurv was sharply increased. This effect might be due to the lack in the Calu-1 cells of functional p53 protein known to inhibit the activity of PhSurv The activity of the double promoters in human cell lines was, as a rule, higher than that of their individual components, although slightly lower than the sum of the constituent promoters' activities (except for the A549 cell line) .The PhTS and PhST double promoters are not active in normal human lung fibroblasts thus retaining tumor specificity characteristic of the single constituent promoters . In muriIn contrast, the double promoters PhTS and PhST were characterized by multiple TSSs. We have identified 5 TSSs in PhTS and 6 in PhST in both cell lines . The resThe data obtained suggest transcriptional interference between the proximal and distal promoters of the tandems. Prior to us, only three head-to-tail tandem whole core promoter constructs were described. Two of them were mentioned in the Generally, transcriptional interference was described The PhSurv and PhTERT promoters contain seven and five Sp1 transcription factor binding sites, respectively  [12], [1Electron microscopy of Sp1/DNA complexes revealed that Sp1 factors formed multimer links (Sp1-bridges) between distant DNA regions thus forming DNA loop structures. It was suggested that transcriptional synergism mediated by Sp1 is due to DNA looping via direct protein-protein association In case of the double promoters under study, upstream promoter regions might well be linked to downstream promoter regions via Sp1 multimers to form DNA stem-loop structure, with loops that could include proximal parts of the upstream promoter . As topoTo check the hypothesis above, we have constructed two new tandem promoters – PhTSurv269 and PhTmSurv , in whicBoth promoter fragments are short enough to prevent looping because the length of the hypothetical loop would be as small as ∼120 bp , that isWe estimated the activity of the luciferase gene under transcriptional control of the new tandem promoters in 5 human cell lines, as described above for PhTERT, PhSurv, PhTS and PhST. As can be seen from To make the quantitative comparison more informative, we calculated the average activity of each promoter in all cancer cell lines tested . The valThe promoter interference effect in tandem promoter constructs with proximal survivin components was analyzed for PhTS, PhTSurv269 and PhTmSurv using a semi-quantitative RT-PCR technique described earlier A similar technique was used to estimate the proximal promoter activity. To this end, a direct UPF primer, located immediately downstream of the proximal promoter and a reverse Luc_202R primer were used. The PCR data are presented in Thus, the interference effect disappeared on decreasing the proximal promoter length to a size at which the length of hypothetical loops is smaller than the persistence length of double-stranded DNA. This confirms the hypothesis that the activity of the distal promoter is suppressed due to the formation of DNA loops involving parts of this promoter.To further confirm our hypothesis, we have constructed two other tandem promoters, PhSm2T and PhSm4T, with the following mutated survivin promoters in the distal position :a modified human survivin promoter (Sm2) that contains two mutated Sp1 sites  that contains four mutated Sp1 sites  and human survivin (PhSurv) genes widely used for gene therapy and having the broadest known cancer type spectrum of activity. Two head-to-tail constructs were made: the PhTERT-PhSurv and PhSurv-PhTERT tandems.proximal to the transcribed gene retained its ability to initiate transcription, whereas the distal promoter remained silent. This kind of promoter interference has never been reported before.We investigated the transcriptional activity of the tandem promoters formed. Unexpectedly, we discovered that in both constructs only the promoter We put forward a hypothesis explaining a novel mechanism that could operate in head-to-tail positioned closely spaced promoters. The mechanism involves the formation of stem-loop DNA structures  in whichThe hypothesized mechanism was confirmed by shortening the loop length to make the loop formation energetically unfavorable, and by removing Sp1 sites from the promoter tandems to hamper the loop formation. In both cases the activity of the distal promoter was restored in accordance with the hypothesis.Based on the data obtained, the design of new tandem promoters should take into account possible undesirable looping of the constructs. To prevent the looping, we can recommend to avoid too long constructs and choose constituent promoters with a low number of multimeric transcription factor binding sites.Finally, we found a novel and efficient tandem promoter combination PhTERT-PhSurv269 with a shortened hSurv promoter. This tandem had the highest and more uniform promoter expression level in different cancer cells among all cancer specific promoters tested. In particular, its average expression level was 4.8 times higher than that of the widely used in gene therapy PhTERT and 3.3 times higher than that of another cancer-specific promoter PhSurv. The tandem also retained cancer-specificity.We hope, that the data obtained would be useful both to researchers looking for new promoter constructs for genetic therapy and to those who study mechanisms of transcription initiation.Cancer cell lines A549 (lung carcinoma), HT1080 (fibrosarcoma), PANC-1 (carcinoma of the exocrine pancreas), A375 (human melanoma), B16F1 (murine melanoma), M3 (Claudman mouse melanoma) were obtained from American Type Culture Collection . The human lung cancer cell line Calu-1 (epidermoid lung carcinoma) was obtained from European Collection of Cell Cultures . Fibroblasts IVL-11NS (NLF) were obtained from normal lung tissue adjacent to tumor according to a standard protocol A normal lung fibroblast cell line IVL-11NS (NLF) was provided by the Vishnevsky Institute of Surgery. A surgical tumor specimen for establishing this line was obtained from a patient with diagnosed lung cancer who has undergone complete resection of tumor at the Vishnevsky Institute of Surgery. The final diagnosis was confirmed by hematoxylin-eosin staining of paraffin blocks after the operation. The patient did not receive chemo- or radiotherapy before surgery. The sample was obtained with the verbal consent of the patient. The project protocol was approved by the Institutional Review Board at the Vishnevsky Institute of Surgery.5′-AGATCTCCACGCCCACAAGGCCAGGC-3′ and 5′-AAGCTTATGATGGCGTCACCACAACC-3′ that contained BglII и HindIII sites. The amplified mSurv was cloned into the pGEM-T vector, sequenced and then cloned between BglII and HindIII sites into the pGL3-Basic vector. To construct double promoters PhST and PhTS, a 1498 bp survivin promoter DNA was obtained by hydrolysis of the PhSurv-pGL3 plasmid with BglII and HindIII restriction enzymes and then blunt ended with the Klenow fragment. The DNA fragment obtained was ligated to the PhTERT-pGL3 vector pre-linearized by cleavage with HindIII or KpnI and blunt ended with the Klenow fragment. To obtain the PhTSurv269 promoter, a 269 bp human survivin promoter DNA was isolated by hydrolysis of the PhSurv269-pGL3 plasmid with BglII and HindIII restriction enzymes, and then blunt ended with the Klenow fragment. The DNA fragment obtained was ligated to the PhTERT-pGL3 vector pre-linearized by cleavage with HindIII and blunt ended with the Klenow fragment. To obtain the PhTmSurv promoter, a 196 bp mouse survivin promoter DNA was isolated by hydrolysis of the PhSurv269-pGL3 plasmid with BglII and HindIII restriction enzymes and blunt ended with the Klenow fragment. The DNA fragment obtained was ligated to the PhTERT-pGL3 vector pre-linearized by cleavage with HindIII and blunt ended with the Klenow fragment. As a result, we obtained PhTS-pGL3, PhST-pGL3, PhTSurv269-pGL3 and PhTmSurv-pGL3 vectors with the firefly luciferase gene under control of several tandem combinations of single promoters upstream of the luciferase gene start codon  and a GENios Pro  luminometer. In parallel experiments, cells were transfected with a promoterless BV-pGL3 plasmid or a PV-pGL3 plasmid, containing only the SV40 promoter (positive control). For each construct under study, at least three independent transfections were performed.Cells were transfected in 24-well plates using Lipofectamine 2000 (Invitrogen) according to the manufacturer's recommendations. Transfection was done with 0.88 µg mixture of a reporter plasmid carrying the firefly luciferase gene and an internal control plasmid pRL-TK (Promega) in the molar ratio of 10∶1. In 48 h after transfection, the activity of firefly and The values in Calu-1 and A375 cells were transfected with the PhSurv-pGL3, PhTERT-pGL3, PhST-pGL3, or PhTS-pGL3 constructs. In 48 h after transfection, the cells were harvested, total RNA was isolated with a RNeasy Mini Kit  according to the manufacturer's protocols. To locate transcription start sites, we used a FirstChoice RLM-RACE Kit  following the manufacturer's recommendations. For nested PCR, we used gene-specific primers Luc_385R and Luc_202R, hSurv_250R, hSurv_150R . The reaTotal RNA from transfected cell lines isolated as described above was further treated with DNase I (Qiagen) to remove residual DNA. cDNA synthesis was performed according to the manufacturer's protocol using random hexamer primers (Perkin Elmer), with (+RT) or without (−RT) addition of PowerScript II reverse transcriptase (Clontech).A cDNA equivalent of 20 ng total RNA was used as template in each PCR, and the amplification was carried out using specific primers as described earlier In parallel, control tests for purity of PCR reaction mixtures and non-amplification of genomic and plasmid DNA were performed. All RT-PCR reactions were reproduced at least three times in independent experiments.Figure S1Model of initiation complex loop structure formed on a double promoter through Sp1 interaction. Grey circles denote Sp1 proteins associated with Sp1 binding sites of promoters. Due to a small size of the formed loop, the supercoiling in this system is energetically unfavorable.(TIF)Click here for additional data file.