Pseudomonas aeruginosa M18, a rhizosphere-isolated bacterial strain showing strong antifungal activity, can produce secondary metabolites such as phenazine-1-carboxylic acid and pyoluteorin (Plt). The LysR-type transcriptional regulator PltR activates the Plt biosynthesis operon pltLABCDEFG, the expression of which is induced by Plt. Here, we identified and characterized the non-conserved pltL promoter (pltLp) specifically activated by PltR and its upstream neighboring lys box from the complicated pltR–pltL intergenic sequence. The 22 bp palindromic lys box, which consists of two 9 bp complementary inverted repeats interrupted by 4 bp, was found to contain the conserved, GC-rich LysR-binding motif (T-N11-A). Evidence obtained in vivo from mutational and lacZ report analyses and in vitro from electrophoretic mobility shift assays reveals that the PltR protein directly bound to the pltLp region as the indispensable binding motif “lys box”, thereby transcriptionally activating the pltLp-driven plt operon expression. Plt, as a potential non-essential coinducer of PltR, specifically induced the pltLp expression and thus strengthened its biosynthetic plt operon expression. Pseudomonas-produced antifungal compound with a resorcinol ring linked to a bichlorinated pyrrole moiety produced by a polyketide synthase–non-ribosomal peptide synthetase (PKS/NRPS) hybrid biosynthetic pathway Pseudomonas fluorescens Pf-5 P. aeruginosa M18 P. aeruginosa LESB58 plt locus shows a high level of conservation in gene organization and size among these three Pseudomonas strains. P. aeruginosa M18 shares 99% nucleotide sequence identity in the plt gene locus with LESB58 pltB gene plt gene cluster is composed of two pairs of divergently transcribed operons: one pair is the pltLABCDEFG and pltRM operons responsible for Plt biosynthesis and its activation, the other pair is the pltHIJKNO and pltZ operons involved in Plt export and its repression. However, the plt gene cluster has not yet been experimentally dissected for the pltR and pltL intergenic non-coding region (485 bp for Pf-5 and 652 bp for M18) containing a number of promoters and cis-acting elements in two opposite directions Pyoluteorin (Plt) is a P. aeruginosa M18 pltLABCDEFG biosynthetic operon plt operon is induced by Plt pltHIJKNO ABC (ATP-binding cassette) export operon and, thus, indirectly downregulates Plt production plt biosynthetic operon, for example, PltR's target DNA motif, Plt's receptor protein, and the effect of Plt on PltR–DNA interaction, remains unknown.Plt biosynthesis is regulated by the Gac/Rsm regulatory cascade, a ubiquitous global regulatory pathway that consists of the two-component signal transduction system GacS/GacA, the sRNAs RsmY and RsmZ, and the RNA-binding repressor protein RsmA repressing access to the ribosome binding site (RBS) Pseudomonas quinoline signal)-mediated QS system P. aeruginosa M18 plt operon, PltR and PqsR, likely differ in molecular regulatory mechanisms. The mechanisms of both remain uncharacterized.The LTTR family represents one of the largest classes of bacterial transcriptional regulatory proteins N-acyl homoserine lactones (AHLs) in Gram-negative bacteria typically induce the expression of their own LuxI-type biosynthetic genes in collaboration with the LuxR-type response regulators Pseudomonas spp., including 2,4-diacetylphloroglucinol Autoinduction of microbiological metabolism, including the QS signal and antibiotic biosynthesis, is widespread in the microbial community. The pltL direction, including the non-conserved pltL promoter (pltLp) specifically activated by PltR, and one pltR promoter in the pltR direction within the intergenic region between the divergently transcribed pltL and pltR genes. The LTTR PltR directly binds to the pltLp region at the indispensable and intact 22 bp palindromic lys box and, thus, specifically activating plt operon expression. Plt, as a potential and non-essential cofactor of PltR, specifically induces the pltLp and thus strengthens the plt operon expression.The current study identifies three promoters in the P. aeruginosa M18 (Genbank accession number AY394844), the divergently transcribed pltL and pltR genes were separated by a large and complex intergenic region (652 bp) that likely contains a number of promoters and regulatory elements. Generally, putative promoters predicted by the NNPP software (P>0.8) need further experimental confirmation through lacZ reporter fusion analysis. The NNPP analysis showed five putative promoters in both the pltR and pltL directions. These predicted promoter fragments were respectively amplified and fused with the promoterless lacZ gene in the plasmid pME6522 (lacZ fusion plasmids were assayed in E. coli DH5α and P. aeruginosa M18. As shown in R1 to R4–lacZ fusion plasmids that carried five putative promoters in the pltR direction (the R3 fragment contains two putative promoters), only the R4–lacZ fusion expressed a significantly higher β-galactosidase activity in both DH5α and M18. The R1 to R3–lacZ fusions did not display noticeable differences in β-galactosidase expression from the empty plasmid pME6522 as the control. The result indicates that the R4 fragment, spanning from−116 bp to +10 bp relative to the putative TSS  of pltR, harbors an actual pltR promoter that also shows a certain level of basal expression activity in E. coli DH5α.In the Plt biosynthetic structural, regulatory, and transport gene cluster of  pME6522 . β-GalacpltL direction were respectively fused with the lacZ gene of pME6522  in E. coli DH5α, but was almost entirely inhibited in P. aeruginosa M18. This occurrence is probably due to the presence of negative specific repressors in M18. The 164 bp L1 fragment was partially overlapped with the pltR promoter (R4) and showed similar levels of promoter activity, implying that this region likely contains a bidirectional promoter.Similarly, five predicted promoter fragments (L1 to L5) in the  pME6522 . Two of  control . HoweverpltL promoter. However, no promoter activity was detected in the L3 fragment by the above lacZ fusion analysis (lacZ fusion reporter plasmids (L3–2–lacZ fusion displayed stronger expression (approximately 900 Miller units) in the presence of the fragment ranging from +66 bp to +124 bp relative to the putative +1 site  in all three strains. This inhibited expression in the L6-3-1 to L6-3-3–lacZ fusion was reversed to the greatest extent only in M18 when the L6-3-4 fragment extended to +205   for the upstream promoter contained in the L6–3 fragment covering from +66 bp to +124 bp relative to the putative pltL +1 site.Based on the differential expression between the  M18pltR , as well in DH5α , the fra M18pltR . Interes L6-3-4) . In addi M18pltR . These rcis-elements responsible for the regulation of plt operon expression, the TSS (+1) of the pltL and pltR genes were mapped using 5′RACE (5′Rapid amplification of cDNA end). The results are shown in pltR, which is identical to the putative +1 site, was located at 63 bp upstream from the pltR translational start codon ATG  contained in the R4 fragment . The putative −10 and −35 regions in these promoters differ substantially from each other and are also markedly different from the canonical −10 (TTGACA) and −35 (TATAAT) sequences. As positively activated genes or operons generally have poor −35 and/or −10 regions their transcription mostly relies on the assistance of an activator protein to stabilize promoter-RNAP interaction. The fact that PltR regulates the pltL promoter but not the Px promoter is consistent with the low level of conservation between the −10 and −35 regions of the two promoters. The Px promoter was located at the −143 bp to −86 bp relative to the pltL TSS  lys box hereafter, was supposed to function as the target site of the LTTR protein PltR, which was verified by the following experiments.Relative to ATG site . The L6 ATG site , which wpltL TSS , which cpltL TSS . Howeverromoters . The prepromoter , L6-3-1.pltLp and Px) in the pltL direction, Px was not controlled by PltR  containing the pltLp and its upstream palindromic lys box (−84 bp to +1), was constructed and transformed into E. coli DH5α, P. aeruginosa M18, the pltR mutant M18pltR and its complementary strain M18pltR/pBBR–pltR. The β-galactosidase activity was measured in KMB to compare pltLp–lacZ fusion expression between the presence and absence of pltR. As shown in pltLp–lacZ fusion was completely inhibited in M18pltR when compared with that (approximately 200 Miller units) in the wild-type strain M18. With the introduction of the pltR overexpression plasmid pBBR–pltR, the pltLp–lacZ expression in the pltR mutant was restored and even significantly increased, compared with that in the M18 strain. The result clearly demonstrates that the 85 bp promoter region comprising its neighboring lys box needed to be activated by PltR , indicating that the pltLp expression requires the participation of other activation factors aside from the indispensable activator PltR.Among the two neighboring promoters ( by PltR . The nex by PltR . In addioli DH5α . The pltpltLp region. The pltLp fragment (−84 bp to +1) was chosen to assess its binding affinity with PltR. As presented in pltLp fragments were incubated with increasing amounts of PltR protein (0 to 70 μM) in a 10 μl reaction system, the PltR protein showed specific binding activity with pltLp. Accompanied by the increasing concentration of PltR, the lagged bands representing the PltR-pltLp complex gradually strengthened while the biotin-labled free DNA probes significantly declined and was almost entirely bound by 20 μM PltR. The Hfq protein, as negative control, showed no binding activity with pltLp. A 1000-fold molar excess of unlabeled pltLp probes entirely displaced the biotin-labeled probes from PltR. Based on these data, it can be concluded that PltR directly binds the pltLp, thereby activating plt operon expression. This observation deserves further investigation to define the key sequence responsible for the binding of PltR to pltLp.EMSA was further designed to detect whether PltR directly binds to the pltLp and its upstream palindromic box (−84 bp to +1) by Mfold 3.2 is shown in lys box (−74 bp to −53 bp) constituting a typical T-N11-A LTTR binding site was supposed to form into a stem loop structure for binding and activation by PltR. Another two stem loops located from −47 bp to −4 bp were assumed as the cis-elements (−10 and −35 sequences) for RNA polymerase. Two pltLp–lacZ derivative plasmids, p6522–pltLp-2 and p6522–pltLp-3, which respectively carry a deletion of half or the entire lys box, were constructed to assess the effect of the lys box on pltLp activity  were respectively utilized to carry out the EMSA analysis with increasing PltR concentrations (0 to 60 μM). The results are shown in pltLp fragment with the intact lys box. As expected, half or whole deletion of the lys box completely abolished the binding of pltLp with PltR. No evident binding complexes were observed in the EMSA between the biotin-labeled pltLp-2 or pltLp-3 probe and the PltR protein  and its four derived mutant plasmids were assayed in P. aeruginosa M18. As depicted in pltLp–lacZ fusion expression was significantly reduced, even entirely abolished, by the four lys-box mutants  complex bands, compared with those of the PltR–pltLp–wt complex bands. With the number of substituted bases increasing from 2 bp to 6 bp, the stem-loop structure formed by the lys box was opened step by step and thus the binding between the DNA probes and PltR gradually declined. Although the stem-loop structure was reshaped by a 4 bp complementary substitution in pltLp–M4-M, its binding with PltR was not restored to levels exceeding that of pltLp-M4 with PltR. This experiment confirms that the secondary structure and the primary sequence of the lys box are crucial for the PltR-activated expression of the plt operon.Biotin-labeled DNA probes corresponding to the wild-type and mutagenized fragments of pltLABCDEFG, has been reported in P. fluorescens Pf-5 P. aeruginosa M18 pltLp is specifically induced by Plt, two lacZ fusion plasmids, p6522–pltLp and p6522–L6-1, which respectively carry the pltLp and its upstream Px promoter, were transformed into M18pltB mutant not producing Plt. β-Galactosidase activity was assayed in KMB media amended without or with 10 µg ml−1 exogenous Plt. As depicted in pltLp–lacZ fusion expression was significantly enhanced by the addition of exogenous Plt, whereas the x–lacZP fusion expression did not reveal any noticeable difference between the KMB with and without 10 µg ml−1 exogenous Plt. The data clearly suggests that the PltR-activated pltLp was specifically induced by the secondary metabolite Plt. Moreover, the qRT-PCR result also shows that the transcript accumulation of pltA, which is the second gene in the pltLABCDEFG biosynthetic structural operon, in the M18pltB strain was strongly increased in KMB with 10 µg ml−1 exogenous Plt compared with that in KMB without the addition of Plt  that is specifically activated by PltR and its upstream closely neighboring promoter (Px) that is not controlled by PltR, and one promoter (pltR promoter) in the pltR direction from the complex intergenic region between the divergently transcribed pltR and pltLABCDEFG operons  palindromic sequence, was localized from −53 bp to −84 bp upstream of the pltL gene TSS. Moreover, it also carries the classic T-N11-A sequence. Interestingly, this 22 bp palindromic sequence was shown  to be the only homologous sequence commonly shared by two pltR–pltL intergenic regions from P. aeruginosa M18 and P. fluorescens Pf-5 (data not shown). This phenomenon suggests that the common mechanisms involved in PltR activation and Plt autoinduction may exist between both these strains.LTTRs are known to bind mostly at the palindromic e of Plt . The tarpltLp activation by PltR may not comply with the “sliding dimer” model because of the presence of the Px promoter closely neighboring upstream of the pltLp. Intriguingly, the gradual opening of the lys box formed stem-loop through a 2 bp to 6 bp substitution gave rise to a significant decrease and even complete inhibition in both the pltLp activity and the PltR–pltLp binding. This decrease was not restored by the reformation of similar stem-loops via a complementary mutation of a 4 bp replacement  in P. fluorescens CHA0 phlA transcription by binding to the phlA operator in P. fluorescens F113 phlA operator by 2,4-DAPG P. fluorescens Pf-5 pltLp at the lys box and transcriptional activation efficiency of PltR. However, further research is needed to confirm the direct binding of Plt to PltR and to assess whether the Plt binding causes the binding enhancement and allosteric transition of the PltR–DNA complex.Autoinduction is widespread in gene regulation involved in bacterial secondary metabolism, such as QS signal and antibiotic biosynthesis pltLp and Px  in the pltL direction is far from the neighboring pltLp and Px promoters. The PY promoter was shown to partly overlap with the pltR promoter (PR)  ; they dicoli DH5α. HoweverpltLp and its upstream neighboring lys box have been identified and characterized for PltR binding from the complicated pltR–pltL intergenic region. Plt, a potential and non-essential cofactor of PltR, specifically induces the pltLp expression and thus strengthens its own plt biosynthetic operon. Further work is needed to assess the possibility for the direct binding of Plt by PltR and ascertain whether the Plt binding causes the enhancement and conformation transition of PltR-pltLp binding and thereby promotes the transcriptional activation efficiency of PltR. In addition, we will focus on elaborating the complicated regulatory mechanisms hidden within the 652 bp pltR–pltL intergenic sequence, including the promoters, cis-elements, and potential sRNAs involved in the regulation of Plt biosynthesis in P. aeruginosa M18.In summary, Escherichia coli was cultured in Luria–Bertani (LB) broth at 37°C P. aeruginosa M18 and its derivatives were grown in King's medium B (KMB) at 28°C −1 X-Gal were used for blue/white screening, and 4 mg ml−1 ortho-nitrophenyl-β-d-galactopyranoside (ONPG) in 100 mM phosphate buffer (pH 7.0) were used for β-galactosidase assays. Antibiotics were added in the following final concentrations (μg ml−1): for Pseudomonas, spectinomycin (Sp) 100, tetracycline (Tc) 120, gentamicin (Gm) 45, kanamycin (Km) 50 and chloramphenicol (Cm) 200 for Pseudomonas; Tc 15, Gm 10, Km 50 and Cm 40 for E. coli.The bacterial strains, plasmids, and primers used in the present study are listed in plus DNA polymerase (Toyobo), restriction endonucleases, DNA ligase, DNA and protein markers, plasmid DNA (TaKaRa or BioDev) and genomic DNA extraction kits (BBI), and DNA gel recovery kits (Qiagen or Axygen). DNA was synthesized by Shanghai Sangon Biological Engineering Technology and Services Co., Ltd., and sequenced by the Invitrogen Biotechnology Corporation and the Beijing HuaDa Genomics Institute.All molecular biology methods not depicted in detail were carried out using standard procedures pltR–pltL intergenic region, with a minimum promoter score of 0.8 pltLp sequence was predicted by Mfold 3.2 at 28°C. The most stable structure was chosen when multiple structures were generated Neural Network Promoter Prediction (NNPP) software (v. 2.2) was used to identify putative promoter elements of the pltR and pltL were respectively synthesized from total RNA templates using DNA specific primers Ptsr1 and Ptsl1, and purified with a S.N.A.P column. A homopolymeric C tail was then added to the 3′-end of cDNAs using deoxynucleotidyl transferase (TdT) and dCTP. PCR amplification of the dC-tailed cDNA was carried out using the novel deoxyinosine-containing abridged anchor primer (AAP) and the nested DNA specific primers Ptsr2 or Ptsl2. The PCR products were purified and cloned into the pMD18-T vectors (TaKaRa). The TSSs were identified through the sequencing of multiple independent clones.5′RACE was performed using an Invitrogen 5′RACE system. Total RNAs were isolated using the NucleoSpin® RNA II kit (Macherey–Nagel). The first strand cDNAs of E. coli lacZ gene downstream from a multiple cloning site and encodes Tc resistance, was used to probe unknown promoter activity or construct transcriptional fusion plasmid. As shown in P>0.8) at the 5′-terminals of pltR and pltL operons were respectively amplified and cloned into the EcoRI/PstI-digested pME6522 to identify actual promoter activity. Four fragments (L3–1 to L3–4), which extend continuously from the right of the L3 fragment, were cloned into pME6522 to probe for potential promoter activity and location within the L6 fragment not predicted to carry any promoters of the pltLABCDEFG operon  was constructed by introducing a 85 bp DNA fragment, which ranges from −84 bp to the actual TSS (+1) and harbors the pltLp and its upstream neighboring palindromic lys box . Total RNA was isolated with NucleoSpin RNA II kit (Macherey–Nagel) and treated with RQ1 RNase-free DNase (Promega). First strand cDNA was synthesized using RevertAid™ First Strand cDNA Synthesis Kit (MBI Fermentas) and quantified using a RealMasterMix (SYBR Green) RT-PCR Kit (Tiangen). The qRT-PCR amplicons of pltA (157 bp) and the reference gene rpoD (173 bp) were synthesized from their first strand cDNA templates with the corresponding gene-specific primers. The results were normalized using the constitutively expressed gene rpoD as the internal control. Experiments were carried out in triplicate.pltR gene (1032 bp) and its operator/promoter region (146 bp), was amplified and cloned into the E. coli–Pseudomonas shuttle vector pBBR1MCS at the BamHI and HindIII sites, thus constructing the pltR overexpression plasmid pBBR–pltR.An 1178 bp fragment, including the entire pltR ORF (1032 bp) was amplified and inserted into the NdeI/BamHI-digested pET28a plasmid. The resulting plasmid, pET–pltR, was transformed into E. coli BL21 for overexpression and purification of the C-terminus His6–tagged PltR protein. E. coli BL21 carrying the pET28a–pltR plasmid was grown at 37°C in 1000 ml LB media, with shaking to an OD600 of 0.8, and induced with 0.5 mM IPTG. Subsequently, cell cultures were incubated at 16°C for 16 h. The cells were harvested by centrifugation and resuspended in 10 ml nickel A buffer . The resuspending solution was added 1 μg ml−1 leupeptin, 1 μg ml−1 aprotinin, and 50 μM phenylmethylsulfonyl fluoride (PMSF) and shaken at 4°C for 30 min. The cells were lysed in ultrasonic cell disruptor. The protein extract was loaded onto a Ni–NTA agarose column  and washed twice with 5 ml nickel buffer. The PltR protein was eluted with the elution buffer containing 500 mM imidazole. The purified PltR protein was stored in a buffer containing 20 mM Tris–HCl (pH 8.0), 200 mM NaCl, 1 mM DTT, and 1 mM EDTA at −80°C after removing the imidazole by HPLC (AKTA).The entire DNA probes were generated by PCR with the corresponding biotin-labeled primers . EMSA wapltLp region (−84 bp to +1), which respectively carried 2 bp , 4 bp , and 6 bp replacements  in the lys box . β-Galactosidase activity was measured to confirm the specific induction of pltLp by Plt. Similarly, the pltA mRNA level of the M18pltB strain was assayed by qRT-PCR in KMB with or without 10 μg ml–1 Plt.The Table S1Primers used in this study.(DOC)Click here for additional data file.


Kinetic analysis shows that RNA polymerase elongation kinetics are not modulated by co-transcriptional splicing and that post-transcriptional splicing can proceed at the site of transcription without the presence of the polymerase. RNA processing events that take place on the transcribed pre-mRNA include capping, splicing, editing, 3′ processing, and polyadenylation. Most of these processes occur co-transcriptionally while the RNA polymerase II (Pol II) enzyme is engaged in transcriptional elongation. How Pol II elongation rates are influenced by splicing is not well understood. We generated a family of inducible gene constructs containing increasing numbers of introns and exons, which were stably integrated in human cells to serve as actively transcribing gene loci. By monitoring the association of the transcription and splicing machineries on these genes in vivo, we showed that only U1 snRNP localized to the intronless gene, consistent with a splicing-independent role for U1 snRNP in transcription. In contrast, all snRNPs accumulated on intron-containing genes, and increasing the number of introns increased the amount of spliceosome components recruited. This indicates that nascent RNA can assemble multiple spliceosomes simultaneously. Kinetic measurements of Pol II elongation in vivo, Pol II ChIP, as well as use of Spliceostatin and Meayamycin splicing inhibitors showed that polymerase elongation rates were uncoupled from ongoing splicing. This study shows that transcription elongation kinetics proceed independently of splicing at the model genes studied here. Surprisingly, retention of polyadenylated mRNA was detected at the transcription site after transcription termination. This suggests that the polymerase is released from chromatin prior to the completion of splicing, and the pre-mRNA is post-transcriptionally processed while still tethered to chromatin near the gene end. The pre-mRNA emerging from RNA polymerase II during eukaryotic transcription undergoes a series of processing events. These include 5′-capping, intron excision and exon ligation during splicing, 3′-end processing, and polyadenylation. Processing events occur co-transcriptionally, meaning that a variety of enzymes assemble on the pre-mRNA while the polymerase is still engaged in transcription. The concept of co-transcriptional mRNA processing raises questions about the possible coupling between the transcribing polymerase and the processing machineries. Here we examine how the co-transcriptional assembly of the splicing machinery (the spliceosome) might affect the elongation kinetics of the RNA polymerase. Using live-cell microscopy, we followed the kinetics of transcription of genes containing increasing numbers of introns and measured the recruitment of transcription and splicing factors. Surprisingly, a sub-set of splicing factors was recruited to an intronless gene, implying that there is a polymerase-coupled scanning mechanism for intronic sequences. There was no difference in polymerase elongation rates on genes with or without introns, suggesting that the spliceosome does not modulate elongation kinetics. Experiments including inhibition of splicing or transcription, together with stochastic computational simulation, demonstrated that pre-mRNAs can be retained on the gene when polymerase termination precedes completion of splicing. Altogether we show that polymerase elongation kinetics are not affected by splicing events on the emerging pre-mRNA, that increased splicing leads to more splicing factors being recruited to the mRNA, and that post-transcriptional splicing can proceed at the site of transcription in the absence of the polymerase. The processes of transcription and RNA processing are co-transcriptionally integrated Fos gene promoter is not associated with SR proteins when the gene is in an uninduced state, and only after transcription had begun were SR proteins recruited A unique feature of the eukaryotic RNA Pol II is the presence of a long and highly conserved tail at the C-terminus of the large subunit (Rbp1) of the polymerase, termed the CTD . It is proposed that the CTD serves as a “jumping board” for protein factors that assemble on the nascent transcript as it emerges from the polymerase Co-transcriptional splicing implies that splicing, at least in part, should occur at the site of transcription. This is supported by kinetic studies showing that splicing is completed within a short ∼5–10 min time-frame from the time of transcription, and these times are not influenced by intron length In order to examine the opposite relationship, namely whether the kinetics of the elongating RNA Pol II can be influenced by splicing events occurring co-transcriptionally on the associated pre-mRNA, we generated a series of comparable cell lines harboring inducible gene constructs that contain increasing numbers of introns and exons. These genes were stably integrated into a human cell line and formed tandem arrays containing multiple copies of the integrated gene, thereby creating a genomic locus that upon transcriptional activation is easily monitored using live-cell imaging. Such transcription sites have been used previously to analyze the kinetics of recruitment of a variety of nuclear factors to actively transcribing genes , while most typical genes have a long first intron β-globin intron is 992 nt, which is close to the median size of internal introns  In order to compare between genes, we generated additional gene constructs : (a) E1 lacO) in order to enable the detection of the genomic locus of integration lacO repeats was verified by the expression of RFP-lacI and the detection of single genomic integration sites , E4  =  2,321 nt  . The E1 on sites . YFP-MS2on sites . SeveralStable transfection results in the integration of multiple copies of the gene construct at one genomic locus that is termed a “tandem gene array,” which is useful for live-cell imaging experiments of active transcription sites nrb, and 7SK-RNA  cap of snRNA (anti-TMG) we could detect TMG staining on the transcription site of E1  and continue to detect the gene locus using the RFP-LacI DNA label. The advantage of using the 3D-FRAP microscope is that rapid 3 dimensional (3D)-imaging over time (4D) could be performed. Thereby, the signal of the whole nuclear volume could be collected with no effect on the measurements, and motion of the transcription site was easily tracked . This isβ-globin first intron is shorter than average) might cause some effect on elongation. These experiments were performed on several microscope stations to verify reproducibility (Transcription sites were followed for extended periods (up to 30 min). The YFP-MS2 signal intensity on the transcription sites was quantified using a tracking algorithm. We measured recovery kinetics on the transcription sites of all the three gene-types in order to examine whether different elongation kinetics could be identified. We compared a polymerase passing through the MS2 region of an intronless E1 gene to a polymerase moving through the MS2 region of the intron-containing E3/E4 genes that undergo co-transcriptional splicing (after the exons and introns are transcribed). The FRAP recovery curves measured on the E1, E3, and E4 genes all showed similar recovery kinetics , indicatcibility . AltogetWe examined a similar gene construct in which additional introns and exons were very closely spaced. Thereby, we expected transcription to end before the completion of splicing of all introns, which would allow the further examination of elongation kinetics. The E6 gene was generated using the E4 construct and consisted of 6 exons and 5 introns by using duplications of intron 2 . In thisFirst, we verified that there was an increase in accumulation of introns at the transcription site of the E6 gene compared to the other genes, as expected. 3D volumes of RNA-FISH labeled cells were collected, deconvolved, and analyzed. We calculated the fluorescence ratio of intron 2 in comparison to the last exon (CFP exon), in all the cell lines. Higher levels of introns generated were detected on the E6 gene  comparedWe next asked whether there would be increased accumulation of the splicing machinery on genes containing more introns. We therefore quantified the relative amounts of the splicing machinery (U snRNAs) that accumulated on each transcript using 3D volumes of RNA-FISH labeled cells . QuantifNext, polymerase kinetics on the E6 gene were measured. Interestingly YFP-MS2 FRAP curves showed significantly slower recovery kinetics compared to the previous measurements on the E1, E3, and E4 genes . In ordeTo identify the cause for the kinetic difference between the genes, we analyzed the measured FRAP recovery curves. Two previous studies have used this MS2 FRAP approach to measure Pol II transcription rates on specific genes We first examined whether the kinetic effect was due to a change in polymerase activity. FRAP of the active polymerase transcribing on the E3 and E6 genes was analyzed using a transfected GFP-Pol II that was recruited to the active genes. Similar FRAP recovery kinetics were measured on both genes . This meTo examine whether the E6 kinetic delay was due to ongoing splicing, we used two splicing inhibitors, Spliceostatin A (SSA) The above experiments suggested that the slow recovery FRAP curves of the YFP-MS2 experiments were due to a delay of the nascent transcripts on the E6 sites caused by splicing but without connection to the polymerase itself. First, we verified that the full transcripts were 3′-end processed and were present at the site of transcription of all the genes prior to release. This was shown by the recruitment of GFP-PABP2 to the active transcription sites and by the presence of polyA tails as seen by RNA-FISH . RNA-FIS1/2  =  17 min) compared to E3 cells (t1/2  =  7 min) and E4 (t1/2  =  8.9 min)  was added to actively transcribing cells containing YFP-MS2 tagged transcription sites. The “shut-down” of the transcription sites was monitored over time under the microscope. Indeed, slower clearing of the mRNAs from the gene was observed in E6 cells (t8.9 min) . This ex8.9 min)  showing Finally, to demonstrate that only the mRNAs disconnected from the polymerase were stalled on the E6 gene, we quantified the intensity ratio of the polymerase signal and the mRNA signal on the transcription sites using anti-Pol II immunofluorescence and RNA-FISH to the first exon. If indeed each mRNA is attached to one polymerase, we would expect a constant ratio for all cell lines. However, we found that the proportion of mRNA to Pol II on E6 transcription sites was three times higher than on E3 and E4 sites . When SSTo better understand the given observed results we simulated the transcriptional process using a stochastic Monte-Carlo simulation . The simWe then searched for a reliable fit to the E6 data by keeping the kinetic parameters of E3 and changing only the time-scale of transcript release. We found that the average time for a transcript to remain at the E6 site of transcription was on average ∼10 min . This isWe then used this simulation to examine the intron/exon ratio on the different genes. In this experiment  we foundGene expression profiles are influenced by the presence or lack of an intron. It has been known for many years that the addition of an intron into the cDNA of a gene sequence enhances its expression How does intron removal affect the levels of transcription and mRNA production? Some level of regulation can be found on the DNA itself, for instance by intronic sequences that modulate nucleosome assembly in the vicinity of the promoter In this study we wished to examine the effects of splicing on Pol II elongation in vivo, independent of promoter influences and transcriptional initiation events. Therefore, all the gene constructs were controlled by the same inducible promoter, and elongation kinetics were examined within the gene as far as possible from the initiation site. Elongation rates were measured immediately downstream of the region transcribing the exon-intron-exon elements, or in other words we could monitor the polymerase speed as it moved along the DNA after the splicing codes in the pre-mRNA had already been synthesized. This meant that polymerase movement towards the 3′-end of the gene was occurring simultaneously with splicing events accompanying the newly synthesized pre-mRNA. We measured intron accumulation on the active transcription sites that was dependent on intron numbers.nrbThe presence of various splicing machinery factors at the transcription sites was detected only when the gene loci were actively transcribing. Furthermore, RNA-FISH to intronic and exonic sequences demonstrated the presence of the introns only at the sites of transcription and not in the nucleoplasm, signifying that this is the location of the removal of these introns. However, when splicing was inhibited with Spliceostatin or Meayamycin, intron-containing pre-mRNA could be detected also throughout the nucleus, in particular within nuclear speckles, as previously described for SSA In this study we examined spliceosomal recruitment to comparable intron-containing genes as well as an intronless gene. As anticipated, spliceosomal factors were not found on the active transcription site of an intronless gene, except for components of the U1 snRNP. These data are in agreement with a recent report that quantified the recruitment of splicing factors to an intron-containing gene in which the splice sites were mutated and therefore did not undergo splicing We quantified an increased accumulation of spliceosomal components on the pre-mRNA that was dependent on the number of introns within the gene sequence. These observations assist in understanding the dynamics of spliceosome assembly on multiple introns and would be consistent with the scenario of a step-wise model of spliceosome assembly on each intron The kinetic approach we used to measure Pol II transcription kinetics enables the quantification of elongation that is free of influences of initiation at the promoter By generating a gene with many and closely spaced introns and exons (E6), we created a situation in which the transcription machinery should have reached the end of the gene before full splicing of the transcript was completed. Although the generation of the E6 and E4 genes required the duplication of intron 2 and the flanking splice sites, we did not find that this had an effect on the splicing outcome (RT-PCR) of E6 and E4 pre-mRNAs, nor on the expression levels of the CFP-peroxisome protein product. Also, there was no indication of aberrant localization of the E6 mRNA on the transcription site or in nuclear speckles, which could be indicative of aberrant mRNA production.The FRAP recovery kinetics of the E6 gene were significantly slower than the E3 and E4 genes. In order to examine whether the polymerase was the cause of these slow kinetics, or if the splicing machinery was responsible, we conducted a series of experiments. Using splicing inhibitors we saw that the impediment of the slow kinetics was released and returned to the levels of E3/E4/E1 genes, demonstrating that the slow-down was due to an active splicing environment. On the other hand, GFP-Pol II recovery kinetics and ChIP distribution patterns demonstrated that the polymerase was not paused or hindered during elongation. Use of actinomycin D that inhibits transcription by intercalating into the DNA and obstructing the path of the polymerase demonstrated that the clearance of the mRNAs from the E6 gene was slower. These transcripts were polyadenylated as seen by recruitment of PABP2 and the presence of polyA tails. The four different transcripts showed polyA accumulation on the transcription sites implying that the transcripts remained attached to the gene until 3′-end processing occurred. The quantification of the ratio of polymerase to nascent transcripts showed that there was accumulation of pre-mRNAs on the E6 gene, but not on the other genes. This accumulation was alleviated when splicing was inhibited. Together with parameter estimation by the simulation we demonstrate that unspliced E6 transcripts are retained at the site of transcription for up to 10 min before being released.The plausibility of a co-transcriptional splicing mechanism can be imagined just by the fact that human gene exons tend to be short compared to extremely larger intronic sequences TTGATATCACCGGTGGGCCCGGAAAAGAATTCGTCCATCACTE4 generation: The E3 construct . cDNA was synthesized using the RevertAid™ First Strand cDNA Synthesis Kit and an oligo(dT) primer (Fermentas). RT-PCR and sequencing of the correctly spliced mRNAs were performed with primers to the first exon and last exon regions.AGCAACCTCAAACAGACACCA3: GGTCTTGTAGTTGCCGTCGTA4: Other plasmids used in this study were: RFP-LacI, CFP-LacI, YFP-MS2(-NLS), GFP-Pol II lacO repeat plasmid and a puromycin resistance plasmid using a 1∶10∶10 ratio . Cells were cultured for 1–2 d without selection and then selected for 2–3 wk with 100 µg/ml puromycin . Single colonies were picked and positive clones were selected after the addition of doxycycline (1 µg/ml) and identification of CFP-peroxisomes and by transient transfection of RFP-LacI and YFP-MS2. For drug treatments cells were imaged before and right after the addition of 5 mg/ml actinomycin D (Sigma). For DRB treatment, cells were treated with 20 µg/µl for 2 h before imaging. For splicing inhibition, cells were photobleached before and after incubation with 100 ng/ml of Spliceostation for 6 h, kindly provided by Minoru Yoshida , or Meayamycin (300 nM) for 6 h before imaging, kindly provided by Kazunori Koide .Human U2OS osteosarcoma Tet-On cells (Clontech) were maintained in low glucose Dulbecco's modified Eagle's medium  containing 10% fetal bovine serum . For transient transfections, cells were transfected with 1–5 µg of plasmid DNA and 40 µg of sheared salmon sperm DNA (Sigma) using electroporation  or with FuGENE 6 transfection reagent (Roche). Stable co-transfections were performed by electroporation of the E genes together with a 256 GGATCACTCTCGGCATGGAC (forward) and TGCACATACCGGAGCCATTG (reverse). Primers used for the human β-globin reference gene were CAGTGCAGGCTGCCTATCAGA (forward) and GAATCCAGATGCTCAAGGCCCTT (reverse). The reaction  contained 1× SYBR green mix , 1 µl of gDNA dilution, and 0.5 mM of each primer and water. The reaction was performed using the Chromo4™ Real-Time Detector (Biorad) following the protocol: 15 min at 95°C for enzyme activation, followed by 40 cycles consisting of 15 s denaturation at 92°C, 10 s annealing at 64°C, and 20 s extension at 72°C. Fluorescent signal was measured at the end of each cycle. A final dissociation stage was performed to generate a melting curve for verification of amplification product specificity. For calculating the gene copy number, we first calculated the reaction efficiency for each set of primers by using triplicates of 5× dilutions of gDNA, using the provided software Opticon2 (Biorad). We measured the average cycle number for each gDNA sample using both primer sets and calculated the concentration (Qc) of each reaction by: gDNA was isolated using the PUREGENE™ DNA Purification System (Gentra). For the generation of standards a 10-fold dilution was prepared. Primers used for the amplification of CFP region were β-globin gene by comparing to a non-cancerous cell line.We verified that the U2OS cell line has only two alleles of the ChIP and quantitative PCR were performed as previously described nrb (BD Bioscience). After subsequent washes the cells were incubated for 1 h with a secondary Ab: Cy5-labeled goat anti-mouse IgG, Cy3-labeled goat anti-mouse IgG, FITC-labeled anti-mouse IgM , Alexa488-labeled goat anti-mouse IgG, Mouse anti-rabbit Alexa488, and rabbit anti-mouse Alexa594 (Molecular Probes). Coverslips were mounted in mounting medium.Cells grown on coverslips were fixed for 20 min in 4% PFA and then permeabilized in 0.5% Triton X-100 for 3 min. After blocking with 5% BSA, cells were immunostained for 1 h with: mouse anti-RNA Pol II antibody ), mouse anti-SF2/ASF (Zymed), mouse anti-PSF , mouse anti-2,2,7-trimethylguanosine (TMG) (Mercury), rabbit anti-U1A, rabbit anti-U2AF65 (Abcam), mouse anti-SC35 (Sigma), mouse anti-9G8, and mouse anti-p54RNA FISH was performed on dox activated cells as previously described ATATAGACGTTGTGGCTGATGTAGTTGTACTCCAGCTTGTGCCCCACFP: GGATACATGAATTCTTTGCCAAAGTGATGGGCCAGCACACAGACCAGCAExon1: CGTTGCTCTCGTGACAGAGAAGGGTGTAAAAGCTTCTAGCCTTTTCTCTTAIntron1: CCTTA.TAGCAAAAGGGCCTAGCTTGGACTCAGAATAATCCAGCCTTATCIntron2: CCAACCCTAGGCAATTAGGTACCTTAGGATCTAATGAACCCGGGAATACTMS2: GCAGACPolyA: an oligo(dT) probeThe U snRNA set of probes containing 5-amino-allyl thymidines (Eurogentec) were labeled with Cy3 or Cy5 monoreactive dyes .CtGGGAAAACCACCtTCGTGATCAtGGTATCTCCCCtGCCAGGTAAGtATU1-1: CGAACGCAGtCCCCCACtACCACAAATTAtGCAGTCGAGtTTCCCACAtTU1-2: AGtGGACGGAGCAAGCtCCTATTCCAtCTCCCTGCtCCAAAAATCCATtTU2: tAGCAATAAtCGCGCCTCGGAtAAACCTCATtGGCTACGATACtGCCACTU4-1: AGACtGTCAAAAATtGCCAATGCCGACtATATTGCAAGtCGTCACGGCGGtAU4-2: GGCAAGGCTCAAAAAATTGGGTTAAGACTCAGAGTTGTTCCTCTCCACGGTAU5: CGtGTCATCCTtGCGCAGGGGCCAtGCTAATCTTCtCTGTATCGTtCCAAU6: TtGGATGTGTCTGGAGTCTtGGAAGCTTGACTACtT7SK snRNA: In some cases, an immunostaining was performed after FISH using the standard protocol.2 control .Wide-field fluorescence images were obtained using the Cell∧R system based on an Olympus IX81 fully motorized inverted microscope  fitted with an Orca-AG CCD camera (Hamamatsu), rapid wavelength switching, active focus control (ZDC), a motorized stage , and driven by the Cell∧R software. The microscope is equipped with an on-scope incubator which includes temperature and COFor time-lapse imaging of ActD treatment, several cell positions under non-treated conditions were chosen and recorded. Then 5 mg/ml of actinomycin D was added and cells were imaged every 5 min for 1.5 h.Images and videos were analyzed using self-written ImageJ software macros . The co-localization intensity profile was generated with an ImageJ macro. The channel intensity correlation was generated by Matlab. The 4D image sequences were sum Z-projections. The transcription site was tracked and the mean intensity was measured at every time-point by finding the local maxima of the red (RFP-LacI) for each time point in a specific region of interest (ROI). The background was taken from an ROI outside of the cell and subtracted from all other measurements. Photobleach correction was obtained using another area in the nucleus or in another nucleus. Images acquired before drug addition were used as the initial conditions for normalization of the data. The half-time of site inactivation was calculated for each transcription site using Matlab. Each transcription site measured intensity dataset was fitted to a bi-exponential equation f(x) = a*exp(b*x)+c*exp(d*x). We then searched for the 2 using a live-cell chamber system (Tokai). Before and after the bleaching, cells were imaged in the YFP channel for the detection of YFP-MS2 (mRNPs) and in the CFP channel for the detection of CFP-lacI (genomic locus). For each acquisition, 7 Z-slices were taken every 350 nm. The active transcription site was bleached using the 491 nm laser. Six pre-bleach images were acquired. Post-bleach images were acquired in a sequence of 3 time frequencies: 15 images every 3 s, 15 images every 6 s, and 26 (or 45 for long experiments) images every 30 s.FRAP experiments were performed using a 3D-FRAP system (Photometrics) built on an Olympus IX81 microscope  equipped with an EM-CCD , 405 & 491nm lasers, Lambda DG-4 light source (Sutter), XY&Z stages (Prior), and driven by MetaMorph (Molecular Devices). Experiments were performed at 37°C with 5% COExperiments were performed on two additional systems to verify their consistency: (1) a similar 3D FRAP system in the Darzacq laboratory under the same conditions; (2) Zeiss LSM 510 Meta laser scanning confocal microscope ). Cells were scanned at 488 nm for GFP-MS2 or 514 nm for YFP-MS2, together with a 543 nm laser for the detection of RFP-lacI at the genomic locus. Post-bleach recovery images were acquired every 1 s.The experiments were analyzed using self-written ImageJ macros that sum the projected Z-stacks at every time-point, and track and measure the mean intensity of the transcription site at every time-point by finding the local maxima in a specific region of interest (ROI). For each time-point, the background taken from a ROI outside of the cell was subtracted from all other measurements. T(t) and I(t) were measured for each time-point as the average intensity of the nucleus and the average intensity in the bleached ROI, respectively. The average of the pre-bleach images used as the initial conditions are referred to as Ti  =  nuclear intensity and Ii  =  intensity in ROI before bleaching. Ic(t) is the corrected intensity of the bleached ROI at time t Data from at least 10 experiments for each cell-line were collected and the averaged FRAP measurements were fitted to the simulation.The diffusion of the YFP-MS2 protein during the FRAP analysis was disregarded since the diffusion rate of free nucleoplasmic YFP-MS2 is very rapid, while the bound YFP-MS2 is associated with high affinity to the mRNA, and does not detach, diffuse, and bind again at the transcription site Quantitative RNA FISH was used to compare the ratio of FISH signals from two different probes in two different channels. Although each cell line had a different number of gene-copies integrated, the ratio per transcript is comparable. Z stacks  of the immunostained or RNA FISH samples were collected. Images were acquired for the quantification of the exons, introns, U snRNAs, or Pol II present on the transcription sites. Each experiment in which all E cell types were analyzed were acquired under the same conditions and on the same day. Images were deconvolved using a point spread function (PSF) based deconvolution algorithm Huygens Essential II . The sum of pixel values at the transcription site in each channel were measured using Imaris , and the ratio between the channels is presented.Monte Carlo simulations (Matlab) of the transcriptional process were based on the mechanistic models of To simulate the FRAP experiments, we tracked the total “fluorescent” signal. After the system entered steady state, we “bleached” (deleted) the fluorescent molecules and measured the buildup of “fluorescent” signal during the recovery. The signal was normalized as in the FRAP experimental procedure. The kinetic parameters, namely, the elongation speed, the termination rate, and the rates of entering and exiting the paused state, were chosen as those that best fit the experimental FRAP data. We found that the number of gene arrays and the initiation rate had no effect on the measured kinetic results, due to the normalization to the pre-bleach signal.To find the above four kinetic parameters that best fit the experimental data we had to explore a four-dimensional parameter space. For each configuration, the mean square difference (MSD) between the simulated FRAP curve and the experimental curve was calculated . For theFigure S1Transcriptional induction of the different cell lines. The cell lines containing the stably integrated E1, E3, E4, and E6 genes were transcriptionally induced by dox for 12 h. RNA-FISH with a probe to the MS2 region shows the transcription sites (yellow). The CFP protein product is targeted to peroxisomes (cyan). DIC on the right .(3.64 MB TIF)Click here for additional data file.Figure S2Recruitment of RNA processing factors to active transcription sites of E3 cells. The gene locus was identified with RFP-LacI (red) and the recruitment of endogenous Pol II (green) was identified by (A) 8WG16 Ab (specific to the CTD repeats) and (B) H5 Ab (CTD phosphorylated on serine 2). Active transcription sites  can be seen together with immunofluorescence (red) with (C) anti-TMG identifying the cap structure of snRNAs. (D) RNA-FISH to U4 snRNA and (E) U5 snRNA, together with a probe to the MS2 region (yellow). (F) Immunofluorescence with anti-U1A, (G) anti-U2AF65, (H) anti-SF2, (I) anti-9G8, and (J) anti-SC-35; together with the YFP-MS2 protein. Enlargements show the merged signals at the transcription sites. Plots depict the degree of co-localization of the signals at the transcription site across the depicted line .(3.54 MB TIF)Click here for additional data file.Figure S3Recruitment of transcription-related factors to active transcription sites of E3 cells. (A) Transiently expressed GFP-TLS, (B) GFP-PSF, (C) GFP-p54nrb, (D) GFP-PTB, (E) GFP cyclin T1 (pseudocolored red), imaged together with YFP-MS2. (F) RNA-FISH to 7SK RNA (red) and MS2 repeats (yellow). Enlargements show the merged signals at the transcription sites. Plots depict the degree of colocalization of the signals at the transcription site across the depicted line .(3.78 MB TIF)Click here for additional data file.Figure S4Recruitment of RNA processing factors to transcription sites of the intronless E1 gene. Immunofluorescence (red) with (A) anti-TMG that identifies the cap of snRNAs, (B) anti-U1A, and (C) anti-U2AF65, together with the mRNA seen by transfected YFP-MS2 labeling (yellow). (D) RNA-FISH to U4 snRNA and (E) U5 snRNA, together with a probe to the MS2 region (yellow). Enlargements show the signals at the transcription sites. Plots depict the degree of co-localization of the signals at the transcription site across the depicted line .(1.67 MB TIF)Click here for additional data file.Figure S5Comparison of FRAP results on E3 cells performed on different microscopes. The recovery of photobleached YFP-MS2 on actively transcribing sites was monitored in E3 cells using: (a) a Zeiss confocal microscope; (b) a 3D FRAP system in our laboratory (system 1); and (c) a 3D FRAP system in the Darzacq laboratory (system 2) (average from at least n>10 in each experiment).(0.14 MB TIF)Click here for additional data file.Figure S6Genomic positioning and gene copy number have no effect on the measured kinetics. (A) FRAP results of different E6 clones (E6-3 and E6-22-22) performed on different microscopes. (B) Real-time PCR on genomic DNA was used to quantify the number of genes integrated into each gene array gene repeats: E3  =  24, E4  =  5, E6-22-22  =  40, E6-3  =  9 with standard deviation.(0.46 MB TIF)Click here for additional data file.Figure S7Analysis of FRAP curves. FRAP recovery curves of the E3 transcription site show bi-phasic kinetics, which points to two parallel processes. The red line is the curve fit for the sum of the two-exponential equation and shows a better fit than the green line that was fitted to one exponential. The yellow line presents the fast phase (70%) and the pink line is the slow phase (30%).(0.15 MB TIF)Click here for additional data file.Figure S8The effects of splicing inhibitors on the elongation kinetics. (A) Inhibition of splicing by Meayaymycin (6 h) showed that the unspliced pre-mRNA was distributed throughout the cell and in speckles (RNA-FISH on E6 cells) and was retained in the nucleus (bottom), whereas in untreated cells pre-mRNA was detected only at the site of transcription. (B) FRAP recovery curves of untreated and Meayamycin-treated E6 cells shows similar kinetics as E3 cells. (C) The treatment of cells with SSA for 9 h followed by FRAP analysis of GFP-Pol II recruited to the transcription sites shows that splicing inhibition did not affect polymerase kinetics.(1.51 MB TIF)Click here for additional data file.Figure S9Searching the kinetic parameter space for the best fit to experimental data. (A) Simulation plots of an elongating (red) or paused (blue) polymerase population. Top part shows an example of an experiment in which the MS2 fluorescence accumulates (arrow) and is then released. Bottom part shows the accumulation of fluorescence in the MS2 region (left arrow) as well as a stochastically paused polymerase (right arrow).  The simulations rely on four kinetic parameters: the elongation speed, the termination time, and entering and exiting rates of the paused state . To find the best fit to the experimental data, the kinetic parameters were varied in a systematic way. The parameters were plotted two at a time because the parameter space is four dimensional. (A) Plot of the mean square difference (MSD) between the experimental FRAP curve and the simulated curve for each pair of elongation speed and termination time. (B) Similar analysis with the pausing transition rates. The MSD is color coded: the dark blue corresponds to parameter combinations that result in the best fit to the experimental data.(1.25 MB TIF)Click here for additional data file.Figure S10Exploring how the simulation parameters affect the kinetics. (A) Changes in the elongation speed. (B) Changes in the probability to enter or exit a paused state changed the pausing time during transcription, and the average percentage of polymerases that paused in steady state. (C) Changing only the termination time.(0.64 MB TIF)Click here for additional data file.Figure S11Low DRB does not affect kinetics on E3 and E6 genes. No change in the FRAP recovery kinetics of YFP-MS2 on cells treated with DRB (20 µg/µl for 2 h before imaging): E3 cells (brown), E6 cells (dark green), or E6 cells with SSA (cyan) SSA; compared to untreated E3 (blue) and E6 (light green) cells.(0.14 MB TIF)Click here for additional data file.Video S1Transcriptional activation of E3 (top) and E6 (bottom) cells. The DNA locus is tagged with CFP-LacI and the cytoplasmic peroxisomes are also seen in CFP. The mRNA is tagged with YFP-MS2. As transcriptional induction proceeds, the transcription sites appear and then mRNAs disperse into the nucleoplasm. CFP-peroxisomes appear at later times. Since the videos were acquired in 3D and the z-stacks covered the cell volumes, we could show the nuclear CFP-LacI locus in the beginning of the videos and then later shift to the cytoplasmic plane that best shows the CFP-peroxisomes. Cells were imaged 15 min after dox addition and the time in the videos is measured from dox addition. E3, imaged every 15 min; E6, imaged every 20 min.(1.59 MB MOV)Click here for additional data file.Video S2FRAP experiment of an E4 cell. mRNA was labeled with YFP-MS2. 2D image over time of a summed projection of a 4D video. Right: Enlarged tracking of the transcription site. Time zero indicates the bleach time.(0.28 MB MOV)Click here for additional data file.Video S3Addition of actinomycin D to transcribing E6 cells and recording transcription site inactivation over time. The mRNA was tagged with YFP-MS2 (green) and the transcription site locus with RFP-LacI (red). Time zero indicates ActD addition time.(0.94 MB MOV)Click here for additional data file.Video S4Example of a simulation run. Each dot denotes a polymerase moving along the gene: elongating  =  red dot, and stochastically pausing  =  dot becomes green. When the polymerase terminates and the transcript still remains on the transcription site, the dot becomes yellow. The bleached polymerase becomes blue. The x-axis  =  gene length of E3; in the y-axis each line represents a gene within the transcription site. Top: a single gene view. Bottom: an array of 1,000 genes running in parallel. The last frame contains a graph showing the MS2 intensity measured from the simulation.(4.30 MB MOV)Click here for additional data file.


Xist gene expression. The mechanisms of Xist expression regulation have not been well understood so far. In this work, we compared Xist promoter region of vole Microtus rossiaemeridionalis and other mammalian species. We observed three conserved regions which were characterized by computational analysis, DNaseI in vitro footprinting, and reporter construct assay. Regulatory factors potentially involved in Xist activation and repression in voles were determined. The role of CpG methylation in vole Xist expression regulation was established. A CTCF binding site was found in the 5′ flanking region of the Xist promoter on the active X chromosome in both males and females. We suggest that CTCF acts as an insulator which defines an inactive Xist domain on the active X chromosome in voles.X chromosome inactivation takes place in the early development of female mammals and depends on the  Xist and Tsix. Xist is expressed from the inactive X chromosome. Further, Xist RNA spreads along the inactivating X chromosome, leading to its heterochromatinization and gene silencing Dosage compensation in female mammals is achieved by inactivation of one of the two X chromosomes. X-inactivation occurs in early embryogenesis and comprises several stages such as counting of the X-chromosome number per diploid autosome set, choice of the X-chromosome to be inactivated, initiation of inactivation, spreading of the inactive state, and its maintenance in cell lineage. X-inactivation is controlled by a locus referred to as the X chromosome inactivation center, XIC. It contains several non-coding RNA genes, most importantly Xist is expressed at a low level from both X chromosomes. Then XICs of two X chromosomes transiently associate and the mutually exclusive choice of the future active and inactive X chromosomes occurs Xist allele on one X chromosome is up-regulated, triggering the X-inactivation process, whereas Xist expression on the other X chromosome is repressed. The mechanisms underlying such expression regulation have not been well understood. In rodents, Xist expression on the active X chromosomes is repressed through antisense transcription of Tsix across the Xist promoter TSIX in the human XIC Xist expression seems to be more complicated and Xist promoter may possess some elements which can both activate and repress its transcription. To date Xist promoter region was studied only in two mammalian species, human and mouse. A number of binding sites for widespread transcription factors  and uncharacterized regulatory proteins were found within Xist minimal promoter Before the onset of X-inactivation, Microtus are characterized by interspecific differences in X chromosome morphology . Moreover, X-inactivation in these hybrid females is skewed. The M. arvalis X chromosome remains active in 80% of the cells in the M. arvalis×M. rossiaemeridionalis, M. arvalis×M. transcaspicus, and M. arvalis×M. kirgisorum hybrids Xist alleles may be involved Common voles of the genus In the four closely related common vole species the sequences of XIC elements and their expression pattern were previously analyzed Xist promoter region of M. rossiaemeridionalis and searching for common and species-specific regulatory proteins which could influence Xist expression. We identified factors being potential activators and repressors of Xist expression at different stages of X-inactivation. CpG methylation of the promoter region demonstrated playing an important role in Xist regulation. In addition, we were not able to detect a CTCF binding site in the vole Xist minimal promoter which is well known in human and mouse. CTCF binding was found in the 5′ flanking region of the Xist promoter on the active X chromosome in both males and females, allowing us to suggest that CTCF is an insulator which defines an inactive Xist domain on the active X chromosome in voles.This work was focused on studying the Xist were compared in different mammalian lineages. A comparison of Xist 5′ regions between M. rossiaemeridionalis and other mammals revealed a homology of the sequences in the −1 kb to +1 bp region. The homology was then interrupted with species-specific SINE and LINE mobile elements  is located 25 bp upstream of the transcription start site and is likely to interact with TBP-like protein Xist transcription start site and seems to bind to the initiator protein YY1 CNS1 was found in the Xist transcription start site . Note that CNS3 contains a potential CTCF binding site detected by MatInspector with high expected value.In addition, using multiple alignment of on, CNS3 . Its locin vitro footprinting. Two Xist promoter regions [−267/+54 bp] and [−551/−372 bp] were labeled with [γ_32P]ATP and used in footprinting reactions. The nuclear extracts from female vole fibroblast cell culture were added to binding reactions.To identify regions interacting with regulatory proteins CNS1, CNS2, and the adjacent nucleotide sequences were analyzed by DNase I Nine protected regions on the coding DNA strand  and six Xist promoter in vitro , pCx9 , and pCx12  and a decrease in the reporter gene activity for the constructs pCx4 , pCx7 , and pCx11  (Xist transcription. Decreasing in luciferase activity for the construct pCx14 [−1453/−843 bp] can be explained by the presence of CTCF binding site in CNS3 [−964/−944 bp] (Among the constructs contained more than 100 bp of the vole P≥0.999) . This su−944 bp] .The construct pCx20 comprising the region [−1453/−4 bp] in an inverse orientation failed to demonstrate any significant luciferase activity.Xist expression we treated the constructs pCx5 and pCx14 with M.Sss I which methylates cytosines in 5′-CG-3′ sequences. The luciferase activity of the methylated constructs (pCx5-Me and pCx14-Me) was assessed 48 h after their transient transfection into female vole fibroblasts (Sd10). Both constructs failed to display any significant luciferase activity  with adenine (A) at position −43 bp was found in the M. rossiaemeridionalis and M. arvalis males and females as well as in primary female mouse fibroblasts. The data on the M. rossiaemeridionalis female fibroblasts (Sd10) are given in To determine the functional significance of the substitution the constructs pCx14 G/A [−1453/+67 bp] and pCx5G/A [−190/+67 bp] bearing G or A at position −43 bp were used. Their activity was analyzed in the primary fibroblasts of Xist promoter revealed a DNA motif with a weak similarity to the known CTCF consensus   . Thus, CMicrotus ochrogaster (closely related vole species) transcriptome published in databases (SRP002127). A comparison of the vole and human CTCF by FASTA has demonstrated 98.2% identity and 99.2% similarity at the amino acid level. In addition, Western-blot confirmed that the antibodies for the human CTCF  recognized a protein of 140 kDa in protein extracts from both human and vole fibroblasts  was hypermethylated . At the same time the active Xist allele (the inactive X chromosome) was hypomethylated . Thus, the results of EMSA and CpG methylation assays imply that CTCF can not bind directly to the Xist minimal promoter on the active vole X-chromosome.CTCF binding is known to be almost completely inhibited by CpG methylation ll lines . The M. http://genome.ucsc.edu.) in tissues and cell cultures of different mouse lines, we found CTCF binding to the mouse Xist promoter around [−1026/−1006 bp], corresponding to CNS3  (The third conserved region (CNS3) [−964/−944 bp] also comprises a potential CTCF binding site . When an to CNS3 , Fig. 2Acontrol) . Taking Xist is expressed on both X chromosomes at a low level. During the initiation stage the Xist allele on the future inactive X chromosome is up-regulated whereas on the active X chromosome it is repressed. Thus, the Xist promoter has to comprise some elements activating Xist transcription and those responsible for its silencing in each female somatic cell. In this work, we attempted to shed more light on general principles of Xist expression regulation. We indentified the most conserved regions of the Xist promoter between vole and other mammalian species and characterized them by DNase I footprinting, luciferase reporter analysis, EMSA, and methylation assay.X-inactivation occurs in early embryogenesis of female mammals, before initiation of X-inactivation, Xist minimal promoter spans the region [−100/+67 bp] and contains INR and binding sites for the transcription factors YY1, TBP, SP1, and AP2. In the EMSA experiments, the oligonucleotides with TBP («Promega») и YY1 (SRE-element) Xist promoter contributes significantly to increasing luciferase activity. It comprises CNS1 [−73/−44 bp] which overlaps the protected regions V, IV, and 2 indentified in the footprinting experiments with nuclear extract from Sd10 cells and Sp1 protein. The oligonucleotides contained Sp1 («Promega») binding site successfully competed with the promoter region [−95/−50 bp] in the EMSA experiments  found these CTCF binding sites in all the species studied except for rat and vole. In addition, we were not able to confirm CTCF binding with the regions V-1 [−61/−17 bp] and V-II [−49/−5 bp] by EMSA in vole. The presence of CTCF binding site in the minimal promoter is likely to be a species-specific peculiarity of Xist transcription regulation in rodents.A CTCF binding site was experimentally found in the mouse and human region homologous to the onserved , Fig. 2BXist allele in vole female hybrids and males using ChIP. A multiple alignment of Xist 5′ region of different species revealed a 25 bp region (CNS3) which is conserved and comprises a potential CTCF binding site. ChIP-seq data published on UCSC Genome Browser have demonstrated that a strong peak of CTCF binding is observed at positions −1020 bp and −3000 bp relatively Xist transcription start site in mouse and human, respectively  of cases for the promoter region . It can also recruit co-repressors (HDAC), co-activators (HAT), and components of chromatin remodeling complexes Xist 5′ region of all the mammalian species analyzed, although they are located at different positions relative to the Xist transcription start site. Its expression is detected in embryogenesis and embryonic stem cells. During X-inactivation, SATB1 is involved in formation of loop domains and compartmentalization of the inactive X chromosome in the nucleus. Xist is located in such a domain SATB1 is expressed only in the T- and B-lymphocyte precursors Xist RNA–dependent silencing in tumor T cells The level of pCx10 [−450/+67 bp] activity is comparable to that of pCx9. The region [−450/−400 bp] comprises the protected motifs 3, 4, II, III, and IV. The first three motifs overlap with the potential SRY binding site. SRY may regulate C-terminal Binding Protein) and inhibits expression of target genes Xist. In addition, E3 ubiquitin ligase Rnf12 being an activator of Xist expression is an ERα cofactor and apparently elevates the transcription level of estrogen-sensitive genes Xist transcription during X-inactivation at the implantation stage may be activated by Rnf12 and ERα.Repression of pCx11 luciferase activity is connected with addition of the region [−500/−450 bp] containing the protected motifs V, VI, and 2. The vole region [−480/−450 bp] is quite conserved in the species analyzed . The traXist 5′ region on the future inactive X chromosome, leading to “open” chromatin structure and activation of Xist expression. No potential transcription factor binding sites were observed in the protected motifs VIII and IX. Some other yet uncharacterized regulatory proteins may be discovered in this region.pCx12 [−550/−500 bp] comprises CNS2 [−540/−498 bp] and its luciferase activity is 2.5-fold higher than that of pCx11. This appears to be related to interaction of this region with the transcription factors STAT3 and HMGIY (HMGA1). Potential binding sites for STAT3 and HMGA1 overlap with motifs 1 and VII . The potXist upstream of position −550 bp. pCx14 has a lower level of luciferase activity than pCx13 and contains a CTCF binding site. However, a 600 bp difference in size between constructions does not allow us to make any conclusions about repressive role of CTCF based on reporter construct assay.The constructs pCx13 and pCx14 containing the regions [−843/+67 bp] and [−1453/+67 bp], respectively, demonstrated the same luciferase activity as pCx12 [−550/+67 bp] , implyinXist expression in vole is regulated by numerous regulatory elements. The core promoter bears weak TATA box and initiator element and is involved in recruiting, correct orientation, and assembly of the preinitiation complex. These elements are necessary but not sufficient to initiate transcription at a high level. Therefore, additional elements of promoter  may be involved in activation and fine-tuning of Xist transcription. Since the status of Xist allele and whole X chromosome is established at the implantation stage, we paid close attention to potential binding sites for factors acting in early embryonic development. For example, a potential binding site for Jarid2 which recruits PRC2 in embryonic stem cells and participates in gene silencing was observed in the Xist promoter. The regulatory factors RAR_RXR, AR, GR, RORA, and ERα/Rnf12 can take part in Xist activation during initiation of X-inactivation in undifferentiated cells. HMGA1 and SATB1 belonging to architectural proteins appear to form specific chromatin structures on the future active and inactive X chromosomes, providing a platform for binding the corresponding transcription factors.Thus, Xist expression can be regulated not only by transcription factors but also by CpG methylation of the promoter region. The function of such CpG methylation is to provide active state of one X chromosome by repressing Xist expression. Indeed, vole Xist minimal promoter is completely methylated on the active X chromosome and not methylated on the inactive one .Xist 5′ region of different mammalian lineages were extracted from the GenBank. They were analyzed using the mVista (http://genome.lbl.gov/vista/mvista/submit.shtml) software http://www.ebi.ac.uk/Tools/clustalw2/index.html) http://www.genomatix.de) http://www.gene-regulation.com) The nucleotide sequences of M. rossiaemeridionalis male (MsEf3♂) and female (Sd10♀), M. arvalis male (Maf♂) and female (Maf2♀) and primary female mouse fibroblasts (EFM2♀) were derived as previously described M. rossiaemeridionalis ♀×M. arvalis ♂. In all Sa006 cells, the M. rossiaemeridionalis X chromosome is inactive while that of M. arvalis is inactive in Sad4 line Primary lung and embryonic fibroblasts of M. rossiaemeridionalis were isolated as described Nuclear extracts from liver cells and fibroblasts (line sd10) of 5′-tatgtggcctttcctataagc-3′/Supr7 5′-tagatggacagagaccacagagg-3′ and ERR 5′-atagcccctcgtcttggtgac-3′/ERF 5′-cgctgagctgtttctctaccg-3′, respectively. Before PCR one primer was labeled at the 5′ end with [γ_32P]ATP and T4 polynucleotide kinase (Promega). The binding reactions were carried out according to the protocol recommended for Core Footprinting System (Promega). Reaction mixtures were supplemented with 5–50 µg of the nuclear protein extract or 2 µg of the recombinant transcription factor SP1. Nonspecific protein binding was inhibited by adding 0.1–1.0 µg of Poly(dI-dC) • Poly(dI-dC) (Sigma). To determine the nucleotide sequences of the bound regions Maxam–Gilbert sequencing reactions were performed concurrently with the footprinting. The samples were analyzed in a 4–6% denaturing polyacrylamide gel.Two DNA fragments corresponding to the regions [−267/+54 bp] (Pmin) and [−551/−372 bp] were used as probes. These probes were amplified by PCR with the primer pairs Msx7 32P]ATP and T4 polynucleotide kinase (Promega). The single-stranded oligonucleotides are given in Double-stranded oligonucleotides  and PCR product (probe Pmin) were radioactively labeled and used in EMSA. The double-stranded oligonucleotides were obtained by annealing complementary single-stranded oligonucleotides and were labeled with  (Promega). The nucleotide sequence of the insert was verified by sequencing. The cells were transfected using Lipofectamine™2000 (Invitrogen). The fibroblast cell line of female M. rossiaemeridionalis was concurrently transfected with the reporter and control  plasmids at a ratio of 10∶1. Luciferase activity was determined 48 h after transfection by a Dual-Luciferase® Reporter Assay System (Promega) according to the manufacturer's protocol. The luciferase activity was recorded in a Wallac 1420 multilabel counter using the following parameters: measurement time, 1 s; and delay duration, 0.1 s. The ratio of Firefly to Renilla luciferase activities was taken as the activity of reporter construct. The activity of each reporter construct was measured in three independent experiments. The significance of differences in the reporter gene expression was estimated using Fisher's test.Fragments of vole The cytosine residues in reporter constructs were methylated with M.SssI CpG methyltransferase (NEB). The components of reaction mixture - 12 µl S-adenosylmethionine , 12 µl of 10× NEB buffer 2, 24 U of M.SssI methyltransferase (NEB), and 6 µg of plasmid DNA - were mixed and the volume was adjusted to 120 µl with bidistilled water. The reaction components were mixed and incubated for 1 h at 37°C. The reaction was stopped by heating for 20 min at 65°C. DNA was extracted with phenol–chloroform and precipitated with ethanol.5′-tttgttatgagttttggtataatta-3′ and reverse primer 5′-aaaaactaaaaatattcccaaaaac-3′) were used in the first PCR round. The following PCR conditions were used: 95°C for 5 min; 30 cycles of 95°C for 15 s, 54°C for 15 s, and 72°C for 20 s, and final extension at 72°C for 5 min. In the second round, the PCR product from the first round was used as a template and the primers confined a shorter fragment located inside the PCR product (forward primer 5′-tttaattattttttttagaaaatagtttgt-3′ and reverse primer 5′-aaaaaccacaaaaaaaactcacatc-3′). The reaction was carried out under the same conditions. The bisulfite PCR products were gel purified and cloned into pGEM-T Easy (Promega), and independent clones were sequenced. Sequence analysis was visualized using MethTools Genomic DNA (2 µg) was treated with bisulfite according to the manufacturer's protocol  and eluted in 20 µl of elution buffer. The PCR product was amplified by nested PCR using the bisulfite-converted DNA as a template. The primers specific for the modified DNA  Bos taurus; (Cf) Canis familiaris; (Dn) Dasypus novemcinctus (armadillo); (Ec) Equus caballus (horse); (Hs) Homo sapiens; (La) Loxodonta africana (elephant); (Oc) Oryctolagus cuniculus (rabbit); (Sa) Sorex araneus (shrew); (Ss) Sus scrofa (pig), (Cp) Cavia porcellus (guinea pig), (Mm) Mus musculus; (Rn) Rattus norvegicus; (Mr) M. rossiaemeridionalis (vole). Conserved regions are shown with red frames; arrow indicates Xist transcription start site.The alignment of 5′ regulatory regions and parts of the first exon of (PDF)Click here for additional data file.Figure S2M. rossiaemeridionalis Xist gene. Consensuses of several identified potential transcription factor binding sites are shown above and below the nucleotide sequence. The sequence corresponding to the first conserved region, CNS1, is framed with a rectangle. Footprints are shown with red lines and numerals. Roman numerals denote the protected DNA motifs identified in the (+)-strand and Arabic numerals, in the (−)-strand; arrow shows the transcription start site. The nucleotides conserved for vole, human, cow, dog, horse, and rabbit are shown in yellow.Computational analysis of the promoter region of the (TIF)Click here for additional data file.Figure S3M. rossiaemeridionalis Xist gene. Consensuses of several identified potential transcription factor binding sites are shown above and below the nucleotide sequence. The sequence corresponding to CNS2 is framed with a rectangle. Footprints are shown with red lines and numerals. Roman numerals denote the protected DNA motifs identified in the (+)-strand and Arabic numerals, in the (−)-strand. In CNS2, the nucleotides conserved for vole, human, cow, dog, horse, and rabbit are shown in yellow.Computational analysis of the second conserved region (CNS2) and the adjacent sequences in the 5′ region of the (TIF)Click here for additional data file.Figure S4Xist minimal promoter with the liver nuclear extract. (−) [γ-32P]ATP labeled DNA fragments (0.05 pmol) without incubation with the nuclear extract (NE). (+) [γ-32P]ATP labeled DNA fragments (0.05 pmol) incubated with NE (5–10 µg) . The regions [−31/+19 bp] and [−70/−25 bp] of the vole Xist promoter were obtained by hydrolysis of a PCR product with Sau3AI. The primers used were 5′-cacgggaaactggcaaacat-3′ and 5′-cactcctcttctggtctct-3′. The [−95/−50 bp] fragment was amplificated with 5′-gaagtcgggacttttccgc-3′ and 5′-agagaccagaagaggagtg-3′. The promoter region [−110/−78 bp] was amplificated with 5′-taaaacgccaataagaag-3′ and 5′-gcggaaaagtcccgacttc-3′ primers. The fragments [−155/−90 bp] and [−212/−155 bp] were generated by hydrolysis of a PCR product with VspI. The primers used were 5′-cccgacttcttattggcgtttta-3′ and 5′-atatacaaatttggtggttctc-3′. (B) Competitive inhibition of EMSA. (NE) [γ-32P]ATP labeled DNA fragments (0.05 pmol) with nuclear extract only. In the other reactions, unlabeled double-stranded oligonucleotides  containing binding sites for a number of transcription factors were added. (TBP) 5′-gcagagcatataaggtgaggtagga-3′ oligonucleotides with the TBP binding site («Promega»); (SRE) 5′-cagtacaggatgtccatattaggacacatctgcgt-3′ oligonucleotides with the YY1 binding SRE-element 5′-attcgatcggggcggggcgagc-3′ oligonucleotides with the Sp1 binding site («Promega»); (CBF) 5′-cgtctccaccaatgggagggctggc-3′ oligonucleotides with the CBF binding site Xist promoter gave two retardation bands in EMSA. An addition of unlabeled the “TBP” oligonucleotides resulted in a loss of the band corresponding to a more electrophoretic mobile complex. When added the “SRE” oligonucleotides a less mobile complex disappeared. The fragment [−95/−50 bp] comprises a potential Sp1 binding site. In EMSA, it yielded three retardation bands. An addition of the “SP1” oligonucleotides led to a loss of high-molecular retardation bands, which is typical of the Sp1 factor. The region [−110/−78 bp] gave two retardation bands. Formation of a less mobile complex was inhibited with “CBF” oligonucleotides but not with those comprising the Sp1 binding site, suggesting this region binds CBF or another CAAT-binding factor, for example NFY detected by MatInspector.(A) Interaction of the vole (TIF)Click here for additional data file.Figure S5Xist promoter in vitro. C, DNA probe containing consensus binding site for AP2 (positive control); AP2–43G, DNA probe containing the [−54/−36 bp] region with guanine at position −43 bp; (+) denotes the lanes with the DNA probes incubated with AP2 extract and (−) without AP2 extract. Arrow indicates the specific complex formed by AP2 binding to DNA probe.AP2 binding to the vole (TIF)Click here for additional data file.Figure S6Western blot analysis of extracts from vole and human fibroblasts using CTCF antibody - Cell Signaling #2899 (A), Upstate #07-729 (B).(TIF)Click here for additional data file.Figure S7Xist minimal promoter. (+) indicates the lanes with DNA probes incubated with CTCF and (−) without CTCF. Arrow denotes the specific complex formed by CTCF binding to F-II probe.EMSA analysis of the CTCF transcription factor binding to the (TIF)Click here for additional data file.Figure S8Xist minimal promoter is indicated in yellow.Chip-seq data on CTCF binding in mouse (UCSC genome browser). Significant sites are shown with rectangles. The (TIFF)Click here for additional data file.Figure S9Xist minimal promoter is indicated in yellow.Chip-seq data on CTCF binding in human (UCSC genome browser). Significant sites are shown with rectangles. The (PDF)Click here for additional data file.Table S1M. rossiaemeridionalis Xist.Description of the transcription factors with potential binding sites identified in the 5′ region of the (DOC)Click here for additional data file.Table S2Nucleotide sequences used for obtaining DNA probes for EMSA.(DOC)Click here for additional data file.