Polycomb bodies are foci of Polycomb proteins in which different Polycomb target genes are thought to co-localize in the nucleus, looping out from their chromosomal context. We have shown previously that insulators, not Polycomb response elements (PREs), mediate associations among Polycomb Group (PcG) targets to form Polycomb bodies. Here we use live imaging and 3C interactions to show that transgenes containing PREs and endogenous PcG-regulated genes are targeted by insulator proteins to different nuclear structures depending on their state of activity. When two genes are repressed, they co-localize in Polycomb bodies. When both are active, they are targeted to transcription factories in a fashion dependent on Trithorax and enhancer specificity as well as the insulator protein CTCF. In the absence of CTCF, assembly of Polycomb bodies is essentially reduced to those representing genomic clusters of Polycomb target genes. The critical role of Trithorax suggests that stable association with a specialized transcription factory underlies the cellular memory of the active state. We have studied the nuclear localization of genes that are regulated by Polycomb mechanisms. The genomes of higher eukaryotes contain hundreds of genes that are regulated by Polycomb mechanisms. Once repressed by Polycomb complexes, they tend to stay repressed; but, when activated, they bind Trithorax protein and tend to maintain the active state epigenetically. Polycomb repression has been reported to make these genes associate in the nucleus to form “Polycomb bodies.” We find that this association is not caused by Polycomb complexes but by insulator elements accompanying the genes. We show that, when these genes are in the active state, the binding of Trithorax targets them to other nuclear regions where transcription occurs, so-called transcription factories. In these nuclear re-positionings the insulator provides the associative power while the state of activity determines whether a gene goes to a Polycomb body or to a transcription factory. The strong effect of Trithorax suggests the possibility that the stable association with a transcription factory it produces may account for the epigenetic memory of the active state. The default state for the organization of genomic material is the chromosomal territory occupied by the folding of the continuous chromatin fiber constituting a chromosome. From this territory, individual regions may loop out to partake in molecular activities such as transcription, heterochromatic silencing, Polycomb repression, etc. A question debated in the past few years is whether these different chromatin states are physically partitioned in the nuclear volume by targeting transcriptional activity to a transcription factory Drosophila, have been mapped at thousands of sites throughout the genome that differ little from one cell type to another Insulator-binding proteins organize the genomic material by forming networks of chromatin loops that govern both local higher-order folding and distant interactions between remote genomic sites Drosophila nuclei by immuno-staining with antibodies against PcG proteins. Interactions between PcG target genes have been detected by 4C or Hi-C approaches Hox gene clusters in Drosophila co-localize at significant frequencies within the same Polycomb body, resulting in enhanced silencing of both genes Fab-7 and Mcp are so-called boundary elements that separate cis-regulatory regions of the Bithorax Complex in Drosophila. Each has been shown to contain two separable functional parts: a core Polycomb Response Element (PRE) and an insulator Fab-7 boundary region can interact with the endogenous Fab-7 and co-localize at relatively high frequencies inside Polycomb bodies when both are repressed by PcG proteins Polycomb bodies have been observed in mammalian and bxd PRE, Fab-7 and Mcp elements, we have shown Fab-7 or Mcp transgenes. A comparison of our results with previous work Using constructs containing the Mcp transgene inserted at remote sites can physically co-localize in the nucleus. Constructs containing a minimal Mcp element associated in ∼7% of the nuclei in both eye and wing imaginal disc cells, dropping to less than 0.5% when the insulator part of Mcp was deleted white gene, active in the photoreceptor and pigment cells of the eye imaginal disc but not in the wing disc. The Mcp-Eye-B and Mcp-Eye-A constructs contain the 820-bp Mcp and the eye enhancer, flanked by FRT and Lox respectively, but with different position and orientation relative to the white reporter gene  in the eye imaginal disc cells, but low co-localization (5–10%) in wing disc or in the cells of the membrane surrounding the eye disc (Mcp and enhancer. This and the eye colors of the flies (Mcp-Eye-B lines show pairing-dependence while the Mcp-Eye-A lines (in which the Mcp insulator is interposed between the PRE and the promoter) are not pairing-sensitive , using the Cre recombinase. This dramatically decreases the frequency of co-localization in the eye disc cells in all five combinations   or when both are Polycomb-repressed (Polycomb body).These results suggest that, surprisingly, transcriptional activation greatly increases the association of two <0.0001) . Similar<0.0001) . WithoutMcp part of the transgene (ΔM) to see if it is necessary to mediate the long-range interaction. The results (2 test p<0.0001). Double deletion of both Mcp and Eye enhancer gives similar results  on the same chromosome arm. Therefore enhancer-dependent transcriptional activity is not sufficient to promote long-range interactions in the absence of the Mcp insulator+PRE.Next, we deleted the  results  show tha results , ΔMΔE exMcp-Eye-B4–Mcp-Eye-B19) to show that the B4 and B19 transgenes, inserted on two different arms of chromosome 2, interact physically with one another in the eye disc but not in the wing disc. This interaction is still strong after enhancer deletion, but disappears after Mcp is deleted from one of the two transgenes  fragment , a histone methyltranferase homologous to mammalian MLL1, known to methylate H3K4 and to antagonize PcG repression sc cells . TogetheMcp-Ubx-B and Mcp-Ubx-A  after deletion of one or both Mcps . The 3C assay was also used to confirm that the interaction between the two transgenes decreases after enhancer deletion and is not detected after Mcp deletion from one or both transgenes  and activated by the arm-GAL4 driver  and Mcp-Ubx , both inserted in chromosome 3. As shown in Mcp-Eye and Mcp-Ubx transgenes are in different transcription states, they are directed into different nuclear domains. When both transgenes are active, the fact that they interact only at the basal level suggests that, while they may chance to land in the same active compartment, they lack high frequency co-localization, which probably requires sharing transcriptional activators.To look at interactions between transgenes containing different enhancers we used combinations of Mcp-containing transgenes co-localize with one another when they are both repressed and, much more frequently, when they are both activated by the same enhancer. In an earlier paper we showed that they can also interact with the endogenous Mcp element Antp gene of ANT-C has been reported to co-localize in a Polycomb body with the Abd-B gene of the Bithorax Complex (BX-C) when both genes are PcG-silenced, but not when one is active and the other silenced Mcp transgene and other endogenous Hox genes. Since the eye enhancer is active only in a subset of cells of the eye-antenna disc, we turned to the Mcp-Ubx constructs containing the Ubx imaginal enhancer, active in all wing and eye disc cells, as are also ANT-C genes, unless repressed.So far, our results have shown that two Mcp-Ubx line containing an intact transgene Mcp-Ubx B4  is greatly decreased relative to that in BG3 cells. It is equally low upon CTCF knockdown but increases when TRX or ASH1 are knocked down: in their absence the pnt gene is again repressed by Polycomb and is able to interact with the repressed C15 gene. Interactions between Abd-B and lbe follow a similar pattern: in BG3 cells where both are PcG-repressed they show CTCF-dependent interaction not affected by trx or ash1 knockdown. They do not interact in Sg4 cells where one is active and the other repressed but, in the absence of TRX or ASH1, we presume that the active gene becomes at least partially repressed and interacts with the repressed gene. These interactions are all between different genes with different enhancers and are therefore analogous to those represented in trx or ash1 affects interactions between active genes but not those between repressed genes.The associations detected with the various trol see . The 3C Hox clusters: the Antennapedia Complex (ANT-C) and the Bithorax Complex (BX-C). In the absence of CTCF the ANT-C genes would continue to be clustered and so would the BX-C genes but the two clusters would no longer associate . Egregious examples are the two Drosophila ssociate . To see ssociate  or transssociate . Compareensities . The resensities .Some basic rules can be deduced from our results. A background level of interactions is proximity-dependent and insulator-independent. When two transgenes both contain an insulator, interaction frequency rises to a level of 5–20%. Interactions are more frequent between sites on the same chromosome arm, as has also been reported by others The results of a large number of experiments with different transgenes and different fly lines Mcp insulator. Loss of CTCF function has the same effect as deletion of the Mcp insulator, reducing co-localization to generally less than 1%, which we take to be the level due to chance encounters. It is highly unlikely that this effect is due to misregulation of some unknown gene that affects our transgenes because a) it mimics the effect of deleting the insulator, b) it affects both localization to Polycomb bodies and to transcription factories, c) similar effects are observed for several endogenous genes, d) genome-wide studies in cultured cells show that the loss of CTCF does not have major global effects on gene expression gypsy insulator, which binds SU(HW), CP190 and MOD(MDG4) proteins, has an effect similar to that of the Mcp insulator, which binds CTCF and CP190, on the interaction between remote transgenes Both CTCF and CP190 insulator proteins bind to the Drosophila, however, there is no apparent relationship between cohesin and CTCF in chromatin binding or in insulator function. Consistently, co-localization is not affected by mutations in the major cohesin components Smc1 or Rad21 although these mutations abolish the pairing-dependent silencing effect (results not shown) seen typically with transgenes containing Mcp and other PREs CTCF function in mammalian genomes is tightly linked to cohesion Lei and Corces The powerful effect of TRX mutations, perhaps the most intriguing feature of the high-level co-localization, suggests that TRX provides an additional level of stability to the association with a common transcription factory. Loss of co-localization when TRX levels are reduced is not because the transgene is no longer active. Transcription is only slightly reduced, as shown by the eye color of the transgenic flies and by the expression of endogenous genes Together with recent work in flies and in mammals, our results indicate that insulator binding proteins have much broader functions than blocking inappropriate action of enhancers and silencers. Our results assign to them a key role in the association of genes repressed by the PcG machinery and in the congress of these genes when they are in the active state. We have not determined directly whether the genes tested are in Polycomb bodies or in transcription factories when they co-localize and, in fact, in any one nucleus a gene may be located in one compartment at one time and another compartment at a later time. However, we have shown that all co-localization of our transgenes is dependent on CTCF, which must therefore be responsible for targeting in both the repressed and active state. In the model shown in How could the same insulator-binding proteins direct a gene to PcG bodies when it is PcG-repressed but to transcription factories when it is in the transcriptionally active state? Although our experiments give no answers to this question, we have proposed that insulator complexes might be post-translationally modified depending on adjacent silencing activity or transcriptional activity and that such modifications might select the appropriate nuclear compartment Ubx is more strongly expressed when the two homologous copies are paired than when pairing is prevented by chromosome rearrangements Is there a functional advantage to co-localization in either case? This is difficult to evaluate but the following arguments suggest that there is. PcG repression in Drosophila displays well-known pairing-dependent effects. A transgene containing a PRE that is partially repressed when present in one copy, usually becomes much more strongly repressed when the transgene insertion is homozygous LacO-Mcp construct was described in Mcp fragments were PCR amplified using primers with appropriate restriction sites. The eye enhancer used is a 1110 bp HincII-BanHI fragment described in Drosophila larva genomic DNA. The 820 bp Mcp was the widely used SalI-XbaI fragment, amplified from BX-C clone BAC R24L18 . The amplified fragments were ligated into plasmids containing LoxP and FRT cassettes and the resulting plasmids were sequenced to verify the inserted sequence. The resulting plasmids were cut with KpnI, releasing the Lox-Eye En-Lox-FRT-Mcp-FRT fragment, which was inserted in either orientation into the acceptor plasmid containing the mini-white gene, yielding products with different arrangements of Mcp and eye enhancer relative to mini-white. The tandem array of 128 copies of LacO was isolated from pAFS150 (a gift from J. Vazquez) and inserted into the pC4Yellow vector LoxP-flanked Mcp insulator part, the FRT-flanked Mcp PRE part and mini-white gene.The McpΔPRE-Eye, Mcp-UAS and Mcp-Ubx were similarly constructed, except using different PCR-amplified fragments. The insulator part of Mcp used in McpΔPRE-Eye was amplified from BX-C clone BAC R24L18, the 5×UAS fragment was amplified from pUASTattB (GenBank: EF362409.1), and the 2250 bp Ubx imaginal enhancer fragment was previously described The plasmids Transgenic fly lines were made according to standard procedures LacI from the ubiquitous Ubiquitin promoter was described in LacI fly line was kindly provided by Dr. A. Csink Pc3 and trxE2 are loss of function mutations . Mutants CTCFy+2, CP190p11, CP1904-1, CP190H31-2, AGO2v966m, AGO251B, piwi1, piwi2, aubQC42, aubp-3a, Rm62sh(3)029, Rm6201086 were generously provided by Drs. E.P. Lei and V.G. Corces. Mutants Smc17-13a, Smc1ex46, Rad21ex15, and Rad2136RipP were kindly provided by Dr. D. Dorsett. Experiments with mutations other than trx and Pc were done using trans-heterozygous allele combinations.The fly line expressing EGFP-2HPO4, 2 mM KH2PO4, pH 7.4) containing 10% fetal calf serum. The tissue was fixed 10 min in 2% paraformaldehyde/PBS at room temperature. The cells were lysed in lysis buffer  on ice for 10 min, followed by 20 strokes of a Dounce homogenizer. The nuclei were recovered, washed and resuspended in 400 µl 1.2×NEB3 buffer  with 0.3% Sodium dodecyl sulfate (SDS). After shaking for 1 hr at 37°C, Triton X-100 was added to 1.8%, and shaking was continued for another 1 hr at 37°C. After digestion with EcoRI (200 units) overnight, the enzyme was inactivated with 1.5% SDS at 65°C for 25 min. 1% Triton X-100 was used to neutralize the SDS at 37°C for 1 hr. The DNA was ligated in 2.4 ml with 20 µl ligase  at 16°C for 4.5 hrs, then 1 hr at room temperature. The 3C template DNA was then un-crosslinked overnight at 65°C, extracted with phenol-chloroform, and dissolved in 100 µl Tris buffer (10 mM Tris·Cl).3C experiments were done as previously described EcoRI fragments in the Brk gene (X chromosome) and pointing in the same direction. All 3C primers are listed in 3C primers were designed for the regions flanking the religated restriction sites, close to the insertion sites of transgenes. As a control for the crosslinking and ligation procedure we used Primers K1 and K2, close to adjacent Drosophila medium (Invitrogen Co.). The dissected eye and wing imaginal discs were aligned on a coverslip bottom dish (MatTek Co.) with a drop of Drosophila medium and then covered with a coverslip. In similar imaging conditions, tissue cells have been found to stay alive for up to 12 hours. Usually, the dissected tissues were immediately subjected to direct microscopy, which finished within 1 hour. Z-stack images across at least one layer of cells were taken with a DeltaVision Image Restoration Microscope system  using a 100×/1.35 UplanApo objective, deconvoluted and processed with the SoftWoRx software (Applied Precision Instruments). Each tissue in the dish was imaged no more than twice to avoid photo-bleaching. We mainly imaged eye imaginal discs and wing imaginal discs. For eye discs, only the region posterior to the morphogenetic furrow was examined. The dots in each nucleus were manually scored by moving the 3D images up and down along z-axis, one dot as co-localization and two non-overlapping dots (center-to-center distance greater than 0.3 µm) as no co-localization.The live-imaging was done as described previously Mcp, more than 99% of the nuclei have two dots, arguing that we are not likely to overlook one of the two transgenes Since the expression of LacI-EGFP was driven by the Ubiquitin promoter, all the cells in all tissues under investigation showed one bright GFP dot per cell containing a single inserted transgene, showing that transgene detection is 100%. Since the LacI-EGFP contains a nuclear localization signal, a weak diffuse GFP signal demarcates each nucleus. In lines lacking 2 tests were used for comparison of each fly line and its derivative lines or mutant backgrounds to calculate the probability that the differences might be due to chance. All statistical analysis was done with the software JMP (SAS Institute Inc.) and the results are tabulated in The percentage of one-dot cells over total cells was used to measure the frequency of co-localization for each individual fly line. More than a dozen eye or wing discs were counted for each specific fly line. Variation in the percentages among eye discs or among wing discs in a given line was less than 2%. We therefore added the counts from a given tissue to represent each fly line by more than 500 cells for the eye discs or wing discs. χ2, the number of bodies and the mean intensities of the bodies were computed by the software. The maximum intensities in each nucleus were also measured. The Pooled t-test , and Satterthwaite t-test  were used to compare the difference in the number of bodies, the mean intensities, and the maximum intensities of the two sets of data (LacZ control and CTCF RNAi cells) using SAS software (SAS Institute Inc.). The two t-tests gave similar results and box plots were used to present the data, with the median shown as a line across the box and the mean indicated by “+”.Eye and wing imaginal discs were dissected from third instar larvae and fixed with 2% para-formaldehyde for 20 minutes at room temperature. The fixed tissues were subjected to extensive washing with PBTr , then incubated with blocking buffer and mouse monoclonal antibody against RNA Pol II large subunit, clone 3 (generously provided by H. Saumweber) or with mouse monoclonal anti-PSC (Santa Cruz). After extensive wash, the tissue was stained with anti-mouse Cy3 secondary antibody. The slides were sealed with VECTASHIELD mounting medium (Vector Laboratories) and image acquisition was done at 100× magnification using the DeltaVision Image Restoration Microscope system. NIH ImageJ software was used to analyse the images. The 3D stack was first projected, and the background fluorescence was subtracted by adjusting the threshold. The particle size was set at >2 µm7 cells, or used for the immunofluorescence experiments as described above.Drosophila ML-DmBG3-c2 cells (abbreviated: BG3) cells and Sg4 cells were cultured in Schneider's Drosophila Medium (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum. Double-stranded RNAs for CTCF and for lacZ as a negative control were prepared according to the user's manual using the RiboMAX Large Scale RNA Production System—T7 (Promega). Genomic DNA was used to amplify the template for dsRNA synthesis. The primers are listed in Figure S1Mcp-Eye fly lines. Three Mcp-Eye-B lines all show pairing-sensitive silencing of the mini-white gene, while Mcp-Eye-A22 flies do not. In each image, the head on the left is heterozygous for the transgene insertion while the head on the right is homozygous but has lower expression of the white gene and lower eye pigmentation. (B) Single enhancer deletion has similar effects on co-localization of two transgenes as double enhancer deletions. Three Mcp-Eye transgene pairs were tested by live-imaging either intact, or with the enhancer deleted in one transgene, or with the enhancers deleted in both. As a negative control, deletion of both Mcps was also tested.Eye color phenotypes and co-localization effects of the Eye enhancer. (A) Eye colors of the (TIF)Click here for additional data file.Figure S2Mcp-Eye insertions (intact) after deletion of the eye enhancer (ΔE), of Mcp (ΔM) or with loss of function mutations of Rm62, piwi or aub. AGO2V966M, a mutation in the AGO2 catalytic site, has no effect but the loss of function allele AGO251B reduces high level co-localization to the basal level. (B) Effects of cohesin mutations. Co-localization is not affected by heteroallelic loss of function mutations Smc17-13a/Smc1ex46 or Rad21ex15/Rad2136RipP.Effects of RNAi and cohesin mutations on co-localization. (A) The effects of co-localization of a pair of (TIF)Click here for additional data file.Figure S3Mcp-Eye with Polycomb bodies or transcription factories. (A) The transgene labeled with LacI-RFP (red) was visualized in eye or wing discs of flies expressing PC-GFP (green) and in (B) the transgene was labeled with LacI-EGFP (green) and the imaginal discs were stained with anti-RNA pol II (red). The transgene  associates with PC in ∼20% of wing nuclei but in <1% of eye nuclei . It associates with RNA pol II in ∼10% of wing nuclei  but in 80% of eye nuclei .Co-localization of (TIF)Click here for additional data file.Figure S4Mcp-UAS containing 5 GAL4 consensus sequences, and lower panel shows the co-localization of pairs of Mcp-UAS insertions in the presence of the LacI-EGFP alone or of LacI-GFP plus the Arm-GAL4 driver to activate transgene expression. A weak but significant increase in co-localization is observed in both eye and wing discs, consistent with the ubiquitous expression of Arm-GAL4.Arm-Gal4/UAS enhancer can also promote co-localization both in the eye and wing disc cells. The upper panel shows the map of (TIF)Click here for additional data file.Figure S5LacZ dsRNA (control) or CTCF dsRNA, or TRX dsRNA, or ASH1 dsRNA for three rounds, then lysed and extracts were subjected to western blot with α-CTCF antibody (A), or α-TRX antibody (B), or α-ASH1 antibody (C), β-Tubulin was used as loading control. (D) CTCF knockdown does not affect the expression level of PSC. BG3 cells were treated with either LacZ dsRNA (as control) or CTCF dsRNA for three rounds, then lysed and extracts were subjected to western blot with α-PSC antibody, β-Tubulin was used as loading control.CTCF, TRX and ASH1 RNAi treatments knock down the target proteins in both Bg3 and Sg4 cell cultures. The cells were treated either with (JPG)Click here for additional data file.Figure S6Abd-B and Antp genes, which are both PcG-repressed in BG3 cells. The CTCF dependence of this emblematic Polycomb body interaction implies that those Polycomb bodies that are formed by the association of remote PcG targets would fall apart in the absence of CTCF.CTCF knockdown abrogates the long-range interaction detected by 3C between (TIF)Click here for additional data file.Figure S7Effect of CTCF RNAi on transcription factories. A monoclonal antibody against RNA polymerase II large subunit was used to illuminate the transcription factories in both LacZ and CTCF RNAi treated Bg3 cells (red). DNA was stained with DAPI (blue). CTCF knockdown does not have major effects on the number or intensity of the transcription factories.(TIF)Click here for additional data file.Table S1List of PCR primers.(DOC)Click here for additional data file.Table S2Statistical analysis.(DOC)Click here for additional data file.Video S1rd instar larva eye imaginal disc eye columnar cells were imaged under wide-field fluorescence microscope. The images were taken along the z-axis of the tissue every 0.3 µm, then reconstituted into a 3D movie, and the dots in each nucleus were counted. The movie shown here shows typical Mcp-Eye-B4–Mcp-Eye-B15 fly line eye columnar cell clusters. Notice that the eye membrane cells could be seen at the beginning of the movie and appear always with two dots in each nucleus, while the ommatidial columnar (photoreceptor) cells mostly (∼86%) have only one dot.Z-stack scan of co-localization in the eye imaginal disc. The 3(WMV)Click here for additional data file.


Remodeling of the local chromatin structure is essential for the regulation of gene expression. While a number of biochemical and bioimaging experiments suggest decondensed chromatin structures are associated with transcription, a direct visualization of DNA and transcriptionally active RNA polymerase II (RNA pol II) at super-resolution is still lacking. Here we investigate the structure of chromatin isolated from HeLa cells using binding activatable localization microscopy (BALM). The sample preparation method preserved the structural integrity of chromatin. Interestingly, BALM imaging of the chromatin spreads revealed the presence of decondensed chromatin as gap structures along the spreads. These gaps were enriched with phosphorylated S5 RNA pol II, and were sensitive to the cellular transcriptional state. Taken together, we could visualize the decondensed chromatin regions together with active RNA pol II for the first time using super-resolution microscopy. Eukaryotic DNA is packaged, together with histones and non-histone proteins, into chromatin fibers148Modulation of the chromatin structure at promoter sites is required for eukaryotic transcription, and this occurs in a highly regulated manner1012141516171911212223252627et al.th serine in the heptad YSPTSPS of the C-terminal domain. This active RNA pol II is recruited to gene promoters during transcription initiation. Colocalization of RNA pol II with gap structures implies that these gap structures may be transcriptionally active. Consistent with this, when cell quiescence was induced via serum starvation, the number of gap structures, as well as RNA pol II signals in those regions, decreased. In conclusion, we show that super-resolution microscopy revealed decondensed chromatin regions with transcriptionally active RNA pol II.In this paper, we visualized the chromatin structure on open chromatin spreads prepared from interphase cells, using both total internal reflection fluorescence microscopy (TIRFM) and binding activatable localization microscopy (BALM), which was recently developed by Schoen, I., Isolated nuclei from HeLa cells were seeded on polylysine-coated glass microscopy slides and swollen using deionized (DI) water for 10–30 minutes. Force, exerted by moderate tapping through a coverslip, resulted in chromatin being spread into strands, which were subsequently visualized by total internal reflection fluorescence microscopy (TIRFM) . This saHistone protein H2B, and the highly dynamic H1Various durations of incubation with DI water resulted in different degrees of nuclear expansion. A short incubation time (<1 min) resulted in poor spreading whilst a long incubation time (>1 hour) produced well-expanded spreads . Statistet al.Materials and Methods). YOYO-1, a DNA intercalating dye that fluoresces around 800–1000 times more upon binding to DNA, has been reported to be a good marker for STORM imaging of DNA in a reducing bufferet al., and YOYO-1's labeling density improved, when its property of enhanced fluorescence following DNA binding was exploited by providing dynamic binding conditions. This gave rise to binding activatable localization microscopy (BALM) and using this imaging method, we were able to obtain more detailed images of chromatin fiber structures  was used to yield further insights into the organization of chromatin fibers. This super-resolution technique was developed by Schoen, I., ructures .Materials Methods, Materials Methods) and images were captured at a rate of 20 Hz. In solution, the YOYO-1 molecules remained dark until binding to DNA, at which point they became bright. This resulted in low background signals. Accumulated images of individual fluorophores localizing to the DNA allowed for the optical reconstruction of stretched double-stranded DNA (dsDNA) molecules  triethoxysilane (APTES) coated coverslip  showed dramatic enhancement in resolution when compared with diffraction-limited TIRFM image of chromatin fibers that have a width of 450 ± 30 nm (mean ± SD) . Gap strMethod and Materials) showed smaller intervals (164 ± 37 nm (mean ± SD)) between two punctate structures in serum (−) chromatin fibers. This was compared to serum (+) chromatin fibers of the same length where the distance between two punctate structures was 673 ± 187 nm (mean ± SD)  .The structural changes of chromatin at actively transcribing and quiescent states are important for the function and localization of transcriptional machinery. Because of this we next investigated regions of chromatin that are enriched with transcriptional machinery. The colocalization of chromatin and RNA pol II in chromatin spreads prepared with and without serum conditions were also compared.Materials and Methods) to each selected point in the images and merging them together. Line intensity profiles were plotted along fibers in the final image, and regions with a mean intensity at least two times lower than that of the neighboring region was defined as a gap structure  was immunolabeled in isolated nuclei and this was followed by chromatin spreading. Chromatin fibers were labeled with YOYO-1. RNA pol II was labeled with a primary antibody (anti-RNA polymerase II CTD repeat YSPTSPS (phosphor S5), ab5131) and a secondary antibody conjugated with Alexa 647. Super-resolution images of RNA pol II were taken via direct STORM (dSTORM) by first increasing laser power to 100% and then decreasing to 2% for imaging. RNA pol II signals were found to be enriched in gap structures. These were characterized by regions of low YOYO-1 fluorescence intensity . To quantructure . Reconsttructure . Gap strtructure  and weretructure .p2c) was within 20 nm  conditions, the normalized density of gap structures along 10-μm chromatin fibers was less than half of that measured under serum (+) conditions . ConcomiThe eukaryotic nucleus is an organelle that is densely packed with DNA and proteins. This density makes visualizing the local chromatin structure, as well as its interactions with functionally relevant proteins such as RNA pol II, particularly difficult. In this study we overcame these difficulties by swelling nuclei and preparing chromatin spreads via a technique that does not disrupt chromatin architecture, as evidenced by the retention of highly dynamic linker histones, as well as the core histones on the chromatin.By combining open chromatin spreads with BALM, we have generated a robust yet simple strategy for visualizing the structure of active chromatin with a spatial resolution of 20 nm. This approach allows for the detection of structural changes in chromatin, specifically in fiber width, or in the characteristic distances between two punctate structures. This detection ultimately arises from the spatial analysis of chromatin fibers and can identify changes when the chromatin is in either an actively transcribing, or quiescent, state. By applying this technique, we noted that fiber width dropped from 150 ± 45 nm (mean ± SD) in the actively transcribing state to 80 ± 40 nm (mean ± SD) in the quiescent state. The reduced distance between two punctate structures reflected a higher density of punctate structures in the quiescent state than in the actively transcribing state.These data implied that in a serum (−) condition, or quiescent state, chromatin fibers were more tightly packed together. This was contrasted by chromatin fibers in a serum (+) condition, or actively transcribing state, which tend to be loosely entangled with each other and form a relatively thick structure . Our resImportantly, the compact organization of serum-starved chromatin fibers may be disrupted if expansion is permitted over an extended preparation time (>1 hrs). When this was explored in our study, a significant increase in chromatin width, and a significant decrease in H1 density became apparent . HoweverThe super-resolution microscopy technique, BALM, provided a direct snapshot of previously unobserved gap structures on the chromatin fiber. These images, together with images captured on a dSTORM system, further revealed a correlation between gap structures and RNA pol II. ~75% of transcriptionally active RNA pol II was co-localized with gap structures, while only ~10% of the heterochromatin protein 1α (HP1α) was found in gap structures. A decrease in the transcriptional activity of cells, which was induced by withdrawing serum from their growth media, leads to a decrease in the number of gap structures, as well as RNA pol II and SRF punctae. We propose that gap structures are likely associated with transcription sites, and although we have not directly visualized transcription occurring at these sites in this work, this will be the subject of future studies. At present however, and despite the BALM technique providing a relatively higher resolution than other imaging techniques, limitations remain that prevent the resolution of single gene promoters. Our super-resolution microscopy based observations, do however show decondensed chromatin regions that are possibly composed of several genes, and are associated with active RNA pol II in mammalian cells.2. To subject cells to serum starvation, cells were cultured in DMEM without FBS at 37°C in 5% CO2 for 36 hours.Wild type HeLa cells and HeLa cells stably transfected with fusion plasmid for core histone H2B tagged with EGFP2, 10 mM Tris-HCl (pH 7.4), 5 mM PMSF  with 1% Triton X-100 for 4–5 minutes at 4°C. The nuclei were separated as pellet from the ruptured cytoplasm by centrifugation for 2 minutes at 400×g. The pelleted nuclei were separated from each other by rigorous tapping and stored in 1× PBS containing 1× protease inhibitor cocktail HeLa cells were suspended in Phosphate Buffered Saline (PBS) after a brief treatment with Trypsin (Gibco). Cells were collected by centrifugation at 200×g and re-suspended in cell rupturing buffer TM2 containing 2 mM MgClAfter rupturing the nucleus of wild type HeLa cells, the DNA, which existed as chromatin fibers prepared as described above, was labeled with either 1 μg/ml Hoechst 33258  or 100 ng/ml of YOYO-1  diluted in freshly made ROXS buffer , 10 mM L-Ascorbic acid , pH7.5) for 5 min and mounted in ROXS bufferDNA-associated proteins were labeled with antibodies in isolated nuclei before the swelling process. For immunolabeling, isolated nuclei were incubated in blocking reagent (1% BSA in PBS), followed by primary antibody and secondary antibodies diluted in blocking reagent, each for ~30 min at room temperature. Linker histone H1 , transcriptionally active CTD phosphorylated RNA pol II  and Serum Response Factor, SRF  were immunolabeled on chromatin fibers.Pre cleaned 22 × 22 mm coverslips were rendered positively charged by coating with (3-Aminopropyl) triethoxysilane . Then, 1 μg/ml of λDNA  stained with YOYO-1 (at a dye/bp ratio of 1/150) was added onto the coverslips and incubated for ~30 min, allowing attachment of λDNA via one or more sites on the coverslip. The stretching of λDNA was achieved through capillary effect created by the rapid absorption of the buffer by tissue paper followed by the force caused by the surface tension of the receding liquid surfaceSuper-resolution imaging was performed on a Zeiss Elyra P.1 microscope, equipped with an oil-immersion objective  and Total internal fluorescence (TIRFM) illumination. TIRFM illumination was achieved by using lasers with motorized TIRFM angle adjustment. The resulting illuminated area was 51.1 × 51.1 μm . Excitation was provided by a 488 nm laser line (100 mW) with AOTF-based intensity control. Emitted fluorescence was collected by the same objective and captured by an Andor iXon 897 back-thinned EMCCD camera. Integration time per frame was 50 ms at full laser power. Typically 10,000 frames were collected, which corresponded to measurement duration of ~10 min. XY drift and alignment differences between different channels were corrected by localizing 0.2-μm Tetraspeck beads  immobilized on the sample coverslip.Raw data was processed using Zeiss Zen software to detect single-molecule events above background noise. A Gaussian filter and a Laplace filter were applied to every event of single molecule fluorescence in each frame of the raw image to reduce noise and enhance the detection of events. The image mean (M) and standard deviation (S) were then computed. Single-molecule events were defined when the peak intensity (I) satisfies: I(r) is the mean intensity value at position r, and r0 is the step moving along the fiber. The averaged autocorrelation g(r) was obtained from fibers with the length of 2 μm (n ≥ 15). All data analysis was carried out using LabVIEW 6.1  and graphs were plotted in Origin 8.0 (OriginLab).A high-pass filter was applied in the Fourier domain of the reconstructed super-resolution image of chromatin fibers. This resulted in an image with only periodic node structures along a fiber, which originally existed together with other random structures in chromatin. A pixel-wise autocorrelation analysis was then carried out to determine the compaction of chromatin structures. To obtain characteristic length scales, the starting point of the fiber was set zero, each mean intensity value along the fiber was used as a signal to calculate the autocorrelation function Y.W., S.M., M.W. and G.V.S. designed experiments. Y.W. and S.M. performed experiments. Y.W., S.M., M.W. and G.V.S. wrote the main manuscript text. All authors reviewed the manuscript.Super-resolution microscopy reveals decondensed chromatin structure at transcription sites


Bacillus subtilis, controlling the formation of spores, biofilms, and cells competent for transformation. We have monitored the rate of transcription of the spo0A gene during growth in sporulation medium using promoter fusions to firefly luciferase. This rate increases sharply during transient diauxie-like pauses in growth rate and then declines as growth resumes. In contrast, the rate of transcription of an rRNA gene decreases and increases in parallel with the growth rate, as expected for stable RNA synthesis. The growth pause-dependent bursts of spo0A transcription, which reflect the activity of the spo0A vegetative promoter, are largely independent of all known regulators of spo0A transcription. Evidence is offered in support of a “passive regulation” model in which RNA polymerase stops transcribing rRNA genes during growth pauses, thus becoming available for the transcription of spo0A. We show that the bursts are followed by the production of phosphorylated Spo0A, and we propose that they represent initial responses to stress that bring the average cell closer to the thresholds for transition to bimodally expressed developmental responses. Measurement of the numbers of cells expressing a competence marker before and after the bursts supports this hypothesis. In the absence of ppGpp, the increase in spo0A transcription that accompanies the entrance to stationary phase is delayed and sporulation is markedly diminished. In spite of this, our data contradicts the hypothesis that sporulation is initiated when a ppGpp-induced depression of the GTP pool relieves repression by CodY. We suggest that, while the programmed induction of sporulation that occurs in stationary phase is apparently provoked by increased flux through the phosphorelay, bet-hedging stochastic transitions to at least competence are induced by bursts in transcription.Phosphorylated Spo0A is a master regulator of stationary phase development in the model bacterium  Bacillus subtilis is its ability to enter developmental pathways: forming spores, acquiring the ability to take up environmental DNA, and the formation of biofilms. These pathways are dependent on the transcription factor Spo0A. All are expressed heterogeneously across populations of cells and exhibit characteristic rates of transition to the developmental pathways depending on environmental signals. We have monitored the rate of transcription of spo0A during growth and have detected unexpected fluctuations that correlate with pauses in the growth rate. We present support for a model in which the release of RNA polymerase from transcription of ribosomal RNA genes during the growth pauses permits increased transcription of spo0A. We show that these bursts in transcription increase the still-rare probability of transition to the transformable state, suggesting that this transition is limited by the transcription rate of spo0A. In contrast, it has been shown that the programmed development of spores is determined by the rate of phosphorylation of Spo0A. Thus there are two modes of developmental transition. We also show that a popular hypothesis for the initiation of spore formation by release of repression by the protein CodY is incorrect.A hallmark of the intensively studied model organism  Bacillus subtilis can embark on several developmental programs leading to sporulation, cannibalism, biofilm formation and genetic competence In response to nutritional deprivation and high population density, the model bacterium B. subtilis require the participation of specific signal transduction genes, many regulators are shared, presumably detecting common signals and coordinating the responses. Most notable among the shared regulators is Spo0A, which plays an essential role in each of the developmental pathways listed above. The concentration of Spo0A∼P increases gradually in a controlled manner as cells enter stationary phase, binding to target promoters as an activator or repressor of transcription, with a hierarchy governed by DNA-binding affinities Although the various developmental programs of spo0A is complex. During growth, spo0A is transcribed from the Pv promoter, which is dependent on the major housekeeping sigma factor, SigA. As cultures enter stationary phase in media that support sporulation, transcription from the downstream Ps promoter takes over, dependent on the minor sigma factor, SigH sigH is repressed by AbrB Apart from posttranscriptional regulation involving phosphorylation and dephosphorylation, the transcriptional control of comK gene determines which cells transition to the competent state Competence, sporulation and the early steps in biofilm formation are bimodally expressed in clonal populations B. subtilis and Escherichia coli, nutrient deprivation elicits the synthesis of the alarmones pppGpp and ppGpp. In B. subtilis, ppGpp  inhibits IMP dehydrogenase and hence the synthesis of GTP spo0A repression by CodY, which requires GTP as a co-repressor Classical work by E. Freese and colleagues demonstrated that sporulation could be initiated by the experimental manipulation of the GTP pool and suggested that a drop in this pool is normally responsible for triggering sporulation spo0A during growth and stationary phase and its relationship to the stringent response. To do this we have utilized firefly luciferase as a real-time reporter for the rate of transcription. With this tool, which is new to the Bacillus community, we show that the transcription of spo0A varies dramatically, exhibiting bursts during the growth phase in commonly used media. The onset of each burst in transcription corresponds to a transient decrease in the growth rate of the culture and each decline in the transcription rate from Pspo0A corresponds to an increase in growth rate. As expected, transcription from an rRNA promoter exhibits the opposite behavior, slowing and speeding up in concert with the growth rate. Experimental evidence is presented in support of a “passive regulation” mechanism, in which RNA polymerase stops transcribing the ribosomal RNA genes during the growth arrests, thus becoming available for the increased transcription of spo0A as well as of certain other genes. We have studied the major fluctuations in spo0A promoter activity that occur during the growth phase and show that these are not influenced in a major way by factors known or proposed to act at Pspo0A and are not eliminated in a strain that no longer synthesizes ppGpp. We propose that the increased spo0A transcription that accompanies a decreased growth rate is a stress response that serves to increase the probabilities that cells will enter developmental pathways. We confirm this model for competence. Our data imply that the synthesis of Spo0A∼P is limited by transcription from the vegetative promoter of spo0A during exponential growth. In contrast to these transcription rate bursts, the programmed increase in spo0A transcription that takes place as a culture approaches and enters stationary phase is delayed by mutational inactivation of ppGpp synthesis. However, we show that a popular model for the initiation of sporulation by CodY derepression caused by ppGpp accumulation is unlikely to be correct.We have examined the transcription of Photinus pyralis) luciferase with cultures growing in the presence of luciferin in 96-well plates. Luciferase catalyzes the oxidation of luciferin in the presence of cellular ATP to emit light, which can be measured in a plate reader equipped for luminometry. The Claverys lab has previously used firefly luciferase to study gene expression in Streptococcus pneumoniaeB. subtilis, with a half-life of about 6 minutes  coding sequence are grown in a standard medium supporting sporulation (DSM) luc reporter has been placed under control of all the relevant upstream regulatory sequences and that the wild-type spo0A locus is undisturbed. The transcription rate from the spoIIG-luc promoter increased just before the transition from exponential growth to the stationary phase (T0), followed by further step-wise increases after T0. Restriction of spoIIG transcription to these late growth stages is expected for this sporulation-specific gene that is only transcribed in the presence of a relatively high concentration of Spo0A∼P luc fused to Pspo0A exhibited 5 prominent bursts, two of which occurred during the growth phase, followed by a third at T0, a fourth about 20 minutes later and a fifth more prominent increase in transcription rate beginning at about T1 and followed by a sustained rise after T2. These stationary phase increases in spo0A transcription presumably reflect the early stages of global commitment to spore formation. It is noteworthy that the peak transcription rates achieved before T0 are appreciable, about half to three-fourths the maximum rate achieved by the fifth burst. Thus substantial but transient transcription of spo0A occurs during the growth phase, although the transcription rate from PspoIIG is higher than that achieved at any point by Pspo0A .This experiment was repeated in competence medium spo0A transcription were promoter-specific or whether they reflected global changes in the initiation of transcription or even changes in the rates of mRNA elongation or in the translation of luciferase. Transcription from ribosomal RNA promoters characteristically decreases together with growth rate upon starvation or down-shifts in nutritional sources spo0A. To test this we constructed a fusion of luciferase to the P1 promoter of the rrnB promoter  or sporulation (Ps) promoters sigH markedly decreased the stationary phase expression of spo0A but had only minor effects on the growth stage transcriptional bursts. These results confirm a role for the SigH-dependent Ps promoter after the end of growth, when it is required for sporulation spo0A transcription reflect changes in transcription from Pv.We next studied the pattern of spo0Aspo0A transcription during growth and stationary phase. Only minor differences in the growth curves of a spo0A mutant and the wild-type strain were observed, the pauses in growth were not affected by inactivation of spo0A (not shown) and the growth phase bursts were clearly not eliminated by disruption of spo0A  as demonstrated by the activation of spoIIG transcription  expressed comK-gfp. At 3.4 hours, during the declining portion of the second burst, 0.13%  of the cells expressed this marker of development. In the spo0A null mutant, no fluorescing cells were detected out of 30,000 and 33,000 cells examined at the early and late times respectively. We conclude that rare cells in a growing population undergo transitions to competence dependent on spo0A and that the probability of transition is greater after the two major bursts in spo0A transcription. We do not know if the transition probabilities decreased following the bursts, but they clearly responded to the increased concentrations of Spo0A∼P that were produced by the spikes in spo0A transcription.Spo0A∼P is required for competence  strains . At 0.65B. subtilis, showing that the identity of the initiating nucleotide for transcription is a factor in the regulation of several genes B subtilis is decreased by the direct inhibition of GTP synthesis due to the addition of decoynine and by starvation for amino acids, which induces ppGpp synthesis and thus depresses the GTP pool rrnB transcription from G to A was shown to relieve this inhibition and similar results have been obtained with other promoters that exhibit a stringent response spo0A, it was first necessary to map the +1 nucleotide for transcription from Pv, which we did using 5′ RACE. The Pv transcriptional start site is at the first A in the sequence AAAAGAAGATTT  molecules is occupied with the transcription of rRNA GAAGATTT . A. ChasGGAAGAA and incorporated these changes into the Pv promoter of a spo0A-luc fusion integrated as before at the spo0A locus. The initiating nucleotide hypothesis predicts that in the mutant strain, the bursts in spo0A transcription will be absent or at least blunted. Because the initiating nucleotide and RNAP-availability hypotheses are not mutually exclusive, we might still expect to detect a component of regulation that retains the behavior of the wild type Pv promoter. To test the roles of the initiating nucleotides, we altered the above sequence to spo0A transcription are dependent only on the increased availability of RNA polymerase, they should be evident when expression of the luc reporter is driven by sequences that contain only the −35, the spacer, the −10 and the +1 sequences of the Pv promoter, presumably lacking binding sites for regulatory proteins. Such a synthetic promoter was placed in front of luc at the ectopic amyE locus  it excludes a role for translational regulation. Note that these translational signals have been used in many vectors designed for use with lacZ as a transcriptional reporter and that spoVG is not known to be regulated translationally. These results provide support for the passive model.The passive regulation model predicts that because the bursts in yE locus . The seqB. subtilis to nutritional status spo0A, we monitored spo0A transcription in a codY mutant  and it is likely that these alterations in the timing of the transcription pulses in the codY mutant are secondary to changes in the timing of growth pauses. The complex changes exhibited by the codY mutant, are perhaps not surprising, because of its global effects on metabolism CodY is known to respond to the cellular pool of GTP and is clearly an important player in the response of Y mutant . Althougspo0A transcription, we determined the effects of ppGpp synthase mutations. B. subtilis encodes three ppGpp synthases; RelA, YwaC and YjbM. The inactivation of relA alone is known to increase the level of ppGpp, because RelA is both a ppGpp synthase and hydrolase, while YwaC and YjbM also synthesize ppGpp but lack hydrolase activity relA alone dramatically altered the pattern of spo0A expression . The quadruple codY relA ywaC yjbM knockout strain was constructed and grew well. However, this strain exhibited precisely the same delayed expression of spo0A in stationary phase as the ppGpp-deficient parent (not shown).As noted, it has been suggested that the accumulation of ppGpp in stationary phase depresses the GTP pool, which relieves repression of codY knockout mutant sporulated with a delay, resembling the delay it exhibits in spo0A transcription and reaches a final frequency of sporulation about ten-fold lower than the isogenic parent (∼2% vs 20%). The relA and triple relA yjbM ywaC mutants also exhibited delayed spore formation, and at 48 hours had about 20 and 50-fold lower sporulation frequencies than the wild-type parent, respectively. Strikingly, the quadruple relA yjbM ywaC codY mutant, like the sigH negative control, failed to form any spores, contrary to the prediction of the model. We further conclude that ppGpp plays an important role in spore formation although an alternative pathway exists and functions independently of this alarmone. Also, CodY does not function in stationary phase as a negative regulator of spore formation.To further explore the roles of ppGpp and CodY in sporulation, we determined the frequency of spore formation in mutant strains after 12, 24 and 48 hours of growth in DSM. spo0A transcription operating on the vegetative promoter during the growth phase and of the sporulation promoter beginning at around T0. We will first consider the biological significance of the regulation of spo0A transcription during the growth phase and then discuss the initiation of sporulation.The data presented here demonstrate the existence of complex regulation of spo0A varies in a pulsatile manner together with the growth rate, with a pattern that is specific for different media. In each case when growth slows, a burst of transcription takes place that terminates as the growth rate increases. These changes in transcription do not reflect global changes in non-specific factors like the availability of energy or nucleotides for mRNA elongation because transcription from the P1 promoter of rrnB exhibits the opposite behavior  but were not observed for spoIIG . Finally, and perhaps most tellingly, because it is known that stable RNA synthesis engages a major fraction of RNAP during rapid growth Several findings are supportive of the passive model for  blunted . When th blunted  or growtckground . Third, ild-type , and mosth PrrnB  and PspoB  is selected sigH mutant decreased the stationary phase transcription of spo0A, reflecting the well-documented phenomenon of promoter switching spo0A transcription after T0, probably due to the inducing effect of Spo0A∼P on sigH as well as the direct positive effect of Spo0A∼P on its own promoter spo0A transcription is secondary to increased flow of phosphoryl groups through the phosphorelay spo0A transcription at T0 and in early stationary phase is characteristic and complex, exhibiting waves and stepwise increases. The third wave, which occurs at T0, is somewhat affected by the sigH mutant  strain shows a delay in the rise in the stationary phase induction of spo0A transcription. The relA mutant, which is expected to accumulate excess ppGpp, shows a more extreme effect, perhaps with a delayed rise beginning at about T2 , and all the strains were therefore isogenic. The details of strain and plasmid constructions are respectively presented in the 600) of 2. Cells were then centrifuged and resuspended in fresh DSM, adjusting all the cultures to an OD600 of 2. These pre-cultures were then diluted 20 fold in fresh DSM and 200 µl was distributed in each of two wells in a 96-well black plate (Corning). 10 µl of luciferin were added to each well to reach a final concentration of 1.5 mg/ml (4.7 mM). The cultures were incubated at 37°C with agitation in a PerkinElmer Envision 2104 Multilabel Reader equipped with an enhanced sensitivity photomultiplier for luminometry. The temperature of the clear plastic lid was maintained at 38°C to avoid condensation. Relative Luminescence Unit (RLU) and OD600 were measured at 1.5 min intervals. Further details are presented in For the detection of luciferase activity, strains were first grown in LB medium to an optical density at 600 nm  listed in Mutagenesis of the B. subtilis chromosome. A single nucleotide was inserted in the primer to restore the correct reading frame. Primers are listed in KpnI/NcoI in sites present at the extremities of the primers used for the amplification. In parallel, the luciferase gene was cut from the pGL3 plasmid (Promega) by NcoI/BamH1 digestion. A three-fragment ligation was then carried out between the promoter of interest, the luciferase gene and plasmid pUC18Cm digested with KpnI and BamH1. The resulting plasmid, pCU18cm-promoter::luc, which cannot replicate autonomously in B. subtilis, was used to transform B. subtilis where it integrated, by single crossover. This event reconstructs the “normal” regulatory region in front of the fusion and a complete copy of the gene of interest, downstream of the fusion.A 1 Kb fragment ending with the initiating codon of the gene of interest, and containing the promoter, was amplified by PCR from the luciferase gene was amplified from the plasmid pGL3 (Promega) using the primers ‘OAcorepromo’ and ‘luc2’  of each sample were fixed by the addition of 20 µl of Na2HPO4, pH 7.4 and 100 µl of formaldehyde. The samples were kept on ice for one hour and then washed twice in PBS. One µl of each treated sample was placed on a 1% agarose pad. All images were acquired using Volocity v 4.1 (Perkin Elmer) and a Nikon 90i fluorescence microscope with filters appropriate for detection of GFP.From cultures of BD5486 and BD5487 growing in the plate reader in DSM, samples were taken after 0.65 and 3.4 hours, before and after the two major bursts in spo0A::luc), overnight cultures grown at 30°C in LB were diluted 50-fold into fresh DSM. At T−1 and T3, 15-ml samples were taken, rapidly chilled, pelleted by centrifugation for 10 min at 4°C, and resuspended in 1 ml RNApro solution (MPbio). RNA was then extracted using FastRNA Pro Blue Kit (MPbio). 5′ RACE-PCR was carried out using the 5′ Race System for Rapid Amplification of cDNA Ends Kit (Invitrogen). Sequences of gene-specific primers (GSP1-0A and GSP2-0A) used for the mapping are shown in BamHI and SalI sites of pUC18 and sequenced, using the primer seq+1-0A  Maximum light output at the fifth burst (solid line) and the first burst (dashed line) plotted as a function of the initial luciferin concentration in the growth medium. (B) Luciferin was added to 1.5 mg/ml at the times indicated by the vertical arrows. (C) Puromycin (200 µg/ml) was added to one of duplicate cultures at the time indicated by the arrow. (D) The data from panel C, plotted with an expanded time scale.Properties of the luciferase assay. All of these curves were obtained with the P(TIF)Click here for additional data file.Figure S2spo0A::luc) from two independent experiments are shown. The duplicate growth curves and light output curves from each experiment are shown in shades of red and blue.Reproducibility of the luciferase assay. The results of duplicate samples of the strain PP530 (P(TIF)Click here for additional data file.Figure S3spo0A transcription and the concentration of Spo0A protein. Strain PP530 was used for this experiment. Panels (A) and (B) show results in S7 medium with the indicated concentration of Glucose and Arabinose (Correlation between rabinose  and in D(TIF)Click here for additional data file.Figure S4spo0A expression. In each panel, light output from a wild-type strain (dark lines) is compared to that from an isogenic mutant (gray lines). (A) ΔkinA (B) ΔkinB (C) ΔkinC (D) ΔkinD (E) ΔkinE (F) Δspo0J (G) Δ(soj spo0J) (H) ΔepsH (as a control for panel I) (I) ΔepsH ΔsinR. The downward-facing arrows show T0 for the wild type (black) and mutant (gray) strains.Effects of gene inactivations known to affect (TIF)Click here for additional data file.Figure S5spo0A. (A) Growth curves of strains with deletions of yjbM (green), ywaC (purple), ywaC and yjbM (blue) and the wild type strain (black). (B) Growth curves of strains with deletions of relA (red), relA and yjbM (green), ywaC and relA (purple), relA, ywaC and yjbM (blue), and the wild type strain (black). (C) Transcription of spo0A in wild type (black) and yjbM (gray) backgrounds. (D) Transcription of spo0A in wild type (black) and ywaC (gray) backgrounds. (E) Transcription of spo0A in wild type (black) and ywaC yjbM (gray) backgrounds. (F) Transcription of spo0A in wild type (black) and relA (gray) backgrounds. (G) Transcription of spo0A in wild type (black) and relA yjbM (gray) backgrounds. (H) Transcription of spo0A in wild type (black) and relA ywaC (gray) backgrounds.Effects of mutations in ppGpp synthases on growth and transcription of (TIF)Click here for additional data file.Figure S6rrnB transcription. In both panels the black lines show rrnB expression in a wild-type background and the gray lines show results from mutant strains, as follows: (A) relA (B) relA yjbM ywaC.Effects of ppGpp synthase mutations on (TIF)Click here for additional data file.Table S1Bacterial strains used for this study.(RTF)Click here for additional data file.Table S2Sequence of oligonucleotide primers used.(RTF)Click here for additional data file.Text S1rate of transcription of a given gene during the growth of B. subtilis using the Firefly luciferase as a reporter gene is described in detail.The method that allows us to measure the (RTF)Click here for additional data file.


Although protein recognition of DNA motifs in promoter regions has been traditionally considered as a critical regulatory element in transcription, the location of promoters, and in particular transcription start sites (TSSs), still remains a challenge. Here we perform a comprehensive analysis of putative core promoter sequences relative to non-annotated predicted TSSs along the human genome, which were defined by distinct DNA physical properties implemented in our ProStar computational algorithm. A representative sampling of predicted regions was subjected to extensive experimental validation and analyses. Interestingly, the vast majority proved to be transcriptionally active despite the lack of specific sequence motifs, indicating that physical signaling is indeed able to detect promoter activity beyond conventional TSS prediction methods. Furthermore, highly active regions displayed typical chromatin features associated to promoters of housekeeping genes. Our results enable to redefine the promoter signatures and analyze the diversity, evolutionary conservation and dynamic regulation of human core promoters at large-scale. Moreover, the present study strongly supports the hypothesis of an ancient regulatory mechanism encoded by the intrinsic physical properties of the DNA that may contribute to the complexity of transcription regulation in the human genome. Gene expression in eukaryotes is a complex process regulated by a myriad of molecular mechanisms. The protein recognition of specific DNA sequence motifs located on promoter regions, upstream of transcription start sites (TSSs), has been traditionally considered as the most important regulatory element in transcription ,2. NeverWe and others  have sugIn this manuscript, we have revisited our presumptions about the existence of a physical code involved in gene activity regulation. To this end, we have evaluated de novo promoter predictions arising from the location of regions with unusual physical properties . A repreOur ProStar promoter prediction program is able to predict TSSs based on the presence of an unusual profile of physical properties  , simplifhttp://www.repeatmasker.org), or that did not allow unique polymerase chain reaction (PCR) primer localization to the human genome assembly by in silico PCR BLAT search (http://genome.ucsc.edu). This process yielded 119 genomic regions (1200 bp long) located around 72 putative TSS .To be coherent with the ProStar training, we applied our predictor using ENSEMBL (v47)  as a refAs a negative prediction set, we randomly selected 100 positions, where ProStar suggested no TSS in a 1200 bp window, and for which unique PCR primers could be located. To make the test unbiased, we did not perform any filtering based on the presence of 2006 known promoters in these ProStar negative predictions. Both ProStar-positive and ProStar-negative predicted promoters were subjected to experimental validation.The positive set was further compared against the latest gene and transcript reference annotations GENCODE (v7)  and ENSEhttp://genome.ucsc.edu). Primers were ordered in 96-well plates to Sigma-Aldrich. PCR was performed in a 96-well format using AccuPrime GC-rich DNA polymerase (Invitrogen) for the amplification of selected regions. PCR products were analyzed in a 1% agarose gel. Successfully amplified regions were inserted into the promoterless pGL4.21 (luc2P/Puro) vector and ligated through Sfi I restriction sites  that enable directional cloning. Escherichia coli competent cells  were transformed with the ligation products. Two independent colonies were selected from each transformant and were verified by sequencing from both the 5′ and 3′ ends. The experimental approach for luciferase activity assays in a high-throughput approach is outlined in Supplementary Figure S1.We designed hybridization primers suitable for high-GC content regions. The presence of a unique hybridization site was subsequently verified by a BLAT genome alignment  vector (Promega) using TransFact reagent (Promega) according to the instructions of the manufacturer. DMEM supplemented with 10% FBS was added to the cells 1 h after co-transfection to allow correct growth and protein expression. Dual Luciferase Reporter Assay (Promega) was performed 36 h after co-transfection using a GloMax Multidetection Luminometer (Promega) with dual injector system allowing rapid reagent addition. Light emission was measured 2 s after addition of each of the substrates and integrated over a 10-s interval. The firefly luciferase activity results were normalized with the renilla luciferase activity from the pGL4.74 (hRluc/TK) plasmid to account for differences in transfection efficiency. The previously characterized SPG4 gene promoter  supplemented with 10% of fetal calf serum (FBS). All cultures were grown as a monolayer in a humidified incubator at 37°C in an atmosphere of 5% COpromoter  was usedAfter luciferase assays, 80 regions from both the positive and negative promoter sets were further divided into four subsets for further analysis: subset 1 contains 20 high-confidence ProStar sequences with high luciferase activity (PS+L+); subset 2 contains another 20 high-confidence ProStar sequences with low luciferase activity (PS+L−); subset 3, 20 low-scored ProStar sequences with luciferase activity ; and subset 4, 20 low-scored ProStar sequences with no luciferase activity (PS−L−).To measure transcription initiation in the different promoter subset regions, profiles of cap analysis gene expression (CAGE) 5′-ends were computed. For this purpose, ENCODE stranded CAGE data from polyadenylated cytosolic RNA of seven different cell lines  and generated in two bio-replicates were used . For eacTo measure transcription activity in the different promoter subset regions, profiles of RNA-seq 5′-ends were computed. For this purpose, ENCODE CSHL stranded paired-end RNA-seq data from polyadenylated cytosolic RNA of seven different cell lines  were used . For eacThe chromatin structure was inferred from DNase I hypersensitivity sites as reported in ENCODE through the UCSC Table Browser data retrieval tool . From thhttp://www.biomart.org) (76 905 transcripts) as a background control. To determine the significant enrichment, we used a Fisher’s exact test and represented the magnitude of enrichment as odds ratios, which is the ratio of enrichment for a given TFBS. The corrected significant P-value after applying a Bonferroni’s correction for all tests was 0.05/885 = 5.65 × 10−5. The analyses were performed using the R statistical environment (http://www.r-project.org).We investigated if different subsets, including the PS+ predictions , the experimentally tested PS+ predictions (119 sequences) and PS− predictions (100 sequences) or luciferase positive (49 sequences) and negative (23 sequences) regions, were enriched within the 1200 bp in any of the currently annotated 885 TFBSs. To this end, we systematically compared them with a full list of transcripts described in the BioMart database  Table Browser data retrieval tool . The levIf physical signals were indeed significant in regulatory regions, as we presume, we would expect a high proportion of ProStar predictions to be promoters, despite the lack of experimental annotation. As described elsewhere , promoteSupplementary Table S1). We additionally defined a negative set consisting of 100 sequences corresponding to nonsignaled promoter regions by ProStar  (Supplementary Table S1).Therefore, to validate our hypothesis, we first defined a TSS prediction set for experimental screening from the ProStar genome-wide calculations. To better validate the prediction power, we used the original ProStar outcome based on the 2006 release of the human genome , withoutSupplementary Table S1). Yet, up to 48 ProStar predicted regions are not proximate (<1.2 kb) to any 2012-annotated TSS. Intriguingly, the attempt of validating ProStar predicted regions using methods based on interspecies sequence conservation, such as ReLA . By applying a threshold of at least 3-fold higher activity than the control vehicle, 85 putative promoters regions were scored as functional, with a validation rate of 71.4%, while only 34% of the analyzed regions in the negative set displayed activity (Supplementary Table S1). From those 85 positively active sequences, 8 correspond to sense strand, 5 to antisense and 72 to both directions , accounting for 49 distinct TSSs. Interestingly, a significantly large number of the suggested promoters (37.8%) displayed high activity (10-fold above the vehicle). Furthermore, almost 98% of active promoters in one cell line also displayed activity in three additional cell lines, indicating that the identified regions would mainly generate transcripts involved in housekeeping activities, rather than in tissue-specific processes. Taken together, these findings suggest that physical properties would signal promoters of the loosely regulated ‘housekeeping’ genes, whereas highly specific sequence signals would be required for the activation of development or tissue-specific genes.We evaluated the ability of the selected putative regions to activate transcription in mammalian cells by using luciferase reporter gene expression assays (Supplementary Table S1). Nevertheless, we should also consider that the resulting activity could be an artifact for some regions, as the activity measurements were based on plasmid-inserted regions rather than on their native structure like in bulk chromatin. Alternatively, the activity might result from the absence of methylation or other posttranslational modifications of a true cellular environment, which can modify the DNA physical properties and ultimately lead to a transcription repression in vivo .Consequently, we complemented our first validation with a CAGE ,32,33 toThe results summarized in Interestingly, subset 3 (PS−L+) regions contain few cage tags, although they showed some activity in luciferase expression assays c. We couSupplementary Figure S1).Even more intriguingly, subset 2 regions (PS+L−) did show clear CAGE enrichment although they did not provide a luciferase response b. These Supplementary Figure S2, see ‘Materials and Methods’ section for details)  analysis to survey the transcription profiles of the selected regions and to identify putative exons near the suggested TSSs. We performed the analysis of 2000 bp regions centered on the predicted TSSs, using RNA-seq data of subcellular-fractionated RNAs from the ENCODE Consortium (details) ,25. InteSupplementary Figure S1 for method details). Interestingly, luciferase assays measured a 4-fold higher activity on average than the original sequences .We first analyzed chromatin accessibility to DNase I degradation profiles, as DNase I hypersensitive sites (DHS) are expected to correlate with loosely packed regions in bulk chromatin and hence with gene transcriptional activity . AnalysiWe also evaluated the occurrence of histone modifications correlated with epigenetic modulation of gene transcription, in particular H3K4Me1, H3K27Ac and H3K4Me3, which are specifically prevalent in regulatory regions . The resP < 5.65 × 10−5 (Supplementary Table S3). Again, PS− regions showed little enrichment, whereas 17 human TFBSs were found to be overrepresented in at least one of the PS+ groups, the larger part being annotated as ubiquitous (Supplementary Table S3). Interestingly, TFBSs overrepresented in the regions capable of driving luciferase transcription were also enriched in PS+ predictions , we further investigated the conservation of ProStar regions across species, as biologically relevant sequences should display some level of sequence conservation. As expected, PS+ regions were enriched for conserved DNA elements a, particA comprehensive analysis of ProStar predicted TSSs has enabled us to identify novel functional core promoters in the human genome exclusively detected by their differential physical deformability pattern and not simply by sequence-based signals such as the CG content alone. A large percentage of ProStar seemingly ‘false positives’, i.e. regions with unusual physical properties but not associated to any annotated promoter, are indeed transcriptionally active. In particular, highly active regions containing a differential physical pattern display typical chromatin features of housekeeping gene promoters involved in cell survival and maintenance, as proven by an overwhelming amount of direct  and indirect evidence . Interestingly, physical signaling also appears to be able to detect promoter activity even in cases where the TSS is located 500 bp upstream of the prediction. Whether this displacement is indicative of a particular feature of genes with closely related alternative TSSs, as indicated by massive CAGE and RNA-seq mappings b and 4, We have also observed that most of the active core regions signaled by physical properties do not exhibit directionality in transcript initiation, indicating that physical properties might signal zones where the binding of regulatory proteins and the deformation of DNA are less intricate, as we had previously suggested . Yet, thOn the whole, our study provides insights into the role of DNA physical properties in ascertaining an ancestral coarse regulatory mechanism. Thereby, regions with high chance of undergoing spontaneous transcription would be recognized by protein effectors and favor nucleosome depletion aside from the purely sequence-based signals encoded as H-bond patterns in the DNA major and minor grooves. In fact, recent genome-wide nucleosome mapping analyses from our group have revealed that housekeeping genes display unique nucleosome architectures, with large nucleosome refractory regions upstream the TSS (unpublished data). In general then, the interplay between DNA physical properties and regulatory regions could be rationalized in terms of nucleosome positioning , favorinYet, the physical code type of mechanism could have been evolutionary deactivated in specific genes where fine regulation is required, but seems to be still active in many other cases, where such a stringent regulation is not essential. This convoluted regulatory signaling present in complex organisms could partially explain the failure of traditional promoter location methods to identify a significant number of TSSs, implying the presence of many hidden promoter regions in the human genome.Supplementary Data are available at NAR Online: Supplementary Tables 1–3 and Supplementary Figures 1 and 2.Spanish Ministry of Science and Innovation [BIO2012-32868 and Consolider E-Science Project]; Instituto de Salud Carlos III ; European Research Council (ERC) Advanced Grant; Fundación Marcelino Botín. M.O. is an Institució Catalana de Recerca i Estudis Avançats (ICREA)Academia Researcher. Funding for open access charge: Fundación Marcelino Botín.Conflict of interest statement. None declared.