D) moves freely around the chromosome in 10 kb domains, and constrained supercoil density (σC) results from binding abundant proteins that bend, loop, or unwind DNA at many sites. Diffusible and constrained supercoils contribute roughly equally to the total in vivo negative supercoil density of WT cells, so σ = σC+σD. Unexpectedly, Escherichia coli chromosomes have a 15% higher level of σ compared to Salmonella enterica. To decipher critical mechanisms that can change diffusible supercoil density of chromosomes, we analyzed strains of Salmonella using a 9 kb “supercoil sensor” inserted at ten positions around the genome. The sensor contains a complete Lac operon flanked by directly repeated resolvase binding sites, and the sensor can monitor both supercoil density and transcription elongation rates in WT and mutant strains. RNA transcription caused (−) supercoiling to increase upstream and decrease downstream of highly expressed genes. Excess upstream supercoiling was relaxed by Topo I, and gyrase replenished downstream supercoil losses to maintain an equilibrium state. Strains with TS gyrase mutations growing at permissive temperature exhibited significant supercoil losses varying from 30% of WT levels to a total loss of σD at most chromosome locations. Supercoil losses were influenced by transcription because addition of rifampicin (Rif) caused supercoil density to rebound throughout the chromosome. Gyrase mutants that caused dramatic supercoil losses also reduced the transcription elongation rates throughout the genome. The observed link between RNA polymerase elongation speed and gyrase turnover suggests that bacteria with fast growth rates may generate higher supercoil densities than slow growing species.Gyrase catalyzes negative supercoiling of DNA in an ATP-dependent reaction that helps condense bacterial chromosomes into a compact interwound “nucleoid.” The supercoil density (σ) of prokaryotic DNA occurs in two forms. Diffusible supercoil density (σ Salmonella Typhimurium chromosome. The sensor includes a Lac operon flanked by a pair of directly repeated DNA–binding sites for the γδ recombinase. Measurements of chromosomal supercoil levels and the RNA polymerase elongation rates were made at various positions within the 6 potential macrodomains of the chromosome. Transcription and gyrase catalytic rates were mechanistically linked. Gyrase mutants with impaired activity caused the loss of from 30% to >95% of the diffusible supercoiling throughout most of the chromosome, while treatment with rifampicin that temporarily blocked transcription restored most of the lost supercoils in gyrase mutants. A gyrase defect also caused transcription elongation rates to decrease across the chromosome, and a mutation that reduced RNA polymerase efficiency increased average chromosome supercoiling levels. A model in which topoisomerases act close to highly transcribed operons to equilibrate the supercoil flux generated by transcription suggests that matched rates of gyrase turnover and transcription elongation speed determine the average supercoil density in bacterial chromosomes.A 9-kb module called the “supercoil sensor” was used to measure supercoil density at 10 positions in the 4.8-Mb  In vivo, about half of chromosomal σ is constrained (σC) and half is diffusible (σD) so that when σ = −0.060, σD≥0.030. The scale on the right Y axis shows the resolution response to σDin vivo. Calculations of the apparent supercoil densities use the bottom half of the curve because after X-ray-induced relaxation of diffusible supercoiling, the E. coli chromosome retained a constrained supercoiling value of σD = 0.030 D = −0.030 is about 50% (blue arrow). When resolution efficiency approaches 100%, the σD≥−0.040 (red arrow). We assume that in vivo reactions fall to 0 at σD≤0.004.The graph in Salmonella ribosomal rrnG operon have different supercoil properties during exponential growth in rich medium rrnG operon was less than 1% because the presence of 60–80 RNA polymerases in the transcribed track blocked supercoil branching and slithering required for synapse. RNA polymerase unwinds a segment of the template strand at the active site, which represents −1.7 constrained supercoils per enzyme rrnG operon approaches σC = −0.290. When σC increased, σD decreased, but temporary interruption of RNA transcription by addition of rifampicin increased resolution efficiency 60 to 100-fold Three regions near the rrnG promoter and downstream of the transcription terminator. The upstream sensor had a 75% resolution efficiency compared to 28% for the downstream sensor D = +0.014 In 1987, Liu and Wang proposed that RNA polymerase generates two supercoiling domains during transcription topA217) and TS gyrA205 and gyrB1820 mutants rrnG operon. In cells with the topA217 mutation, upstream resolution efficiencies rose to 97% compared to 38% downstream. Conversely, a gyrA205 mutant caused downstream resolution to fall to 9% compared to 60% resolution upstream. Most strikingly, a gyrB1820 mutation caused downstream resolution to fall to 1% while recombination efficiency was 11% in the upstream domain. Local supercoiling levels were able to rise and fall dramatically at opposite ends of a highly transcribed operon in cells growing at permissive temperatures.Previously, we measured what happens to twin domain supercoiling in strains with a mutant of Topo I . Transcription played a causal role in the supercoil losses because supercoiling rebounded after addition of rifampicin (Rif), which blocked transcription initiation. Our model is that transcription kinetics determine the optimal catalytic speed for gyrase, and the average chromosome supercoil density is an integral function of topoisomerases and RNA polymerase working in tempo together.Here, we measured rrnG operon gidB and atpI  supercoils generated by fork movement and atpI . The AtpNH6016 carries the gyrA213TS allele (R358-H), which has a mutation located in the DNA-binding and cleavage domain All 4 subunits of gyrase and Topo IV were tested. GyrA contains the catalytic tyrosine residue that carries out DNA cleavage and re-ligation during the supercoiling reaction  A. NH601NH6016 had a significant MIF of 1.4. A gyrA209TS allele (G597-D) in NH6019 alters the second ß-propeller of GyrA, which contributes to DNA-looping that forms a chiral (+) node gyrA209 doubling time increased from 39±1 min to 45±3 min, which is a 15% decrease in growth rate. The resolution efficiency in this strain fell to 30±12%, resulting in a MIF of 2.7. Thus, two gyrATS mutants had reduced resolution efficiency, which indicates a loss of (−) supercoiling even at the permissive temperature of 30°.Strain NH6016 had the same doubling time as WT (39±1 min) but the resolution efficiency fell from 81±3% for WT to 58±1%, representing a 28% loss in recombination efficiency. To compare alleles, we define a term Mutant Impact Factor (MIF) to be the resolution efficiency of the WT strain divided by the resolution efficiency of an isogenic mutant. A large MIF indicates a dramatic change in supercoiling. NH6028 contains the gyr652 mutation (R436-S), which alters a Mg++ binding domain that coordinates structural conformation changes during DNA cleavage cat relative to WT gyrase NH6028 was 7±3%  alters the ATP binding domain that dimerizes and then hydrolyzes two ATPs during the catalytic cycle Salmonella gyrase collection. The doubling time at 30° increased by almost 50% to 58±4 min. NH6037 had a resolution efficiency of 8±6%, resulting in a MIF of 10. Thus, the supercoil sensor shows that two TS gyrA and two TS gyrB alleles cause significant losses in (−) chromosome supercoiling at the ATP operon location.The GyrB subunit encodes the ATP-binding domain of gyrase, which couples ATP binding to a large conformation shift that drives negative supercoiling reactions  B. Two Gwas 7±3% , and thein vitro and contributes to the dissipation of (+) supercoils during DNA replication in vivoNH6040 has the parC281TS (P556-L) mutation, which resides in a region with no known function. This mutant showed no difference in growth rate from the WT (39±1 min) and resolution efficiency was 76±1%, which is close to the WT (81±4%) with a MIF of 1.1. ParE functions like GyrB, binding and hydrolyzing ATP to fuel cycles of strand transfer. The parE206 (V67-M) mutation in strain NH6043 is in the ATP binding domain of Topo IV, and the doubling time at 30° increased by 33% to 52±2 min. The resolution efficiency was 59±4% . The Salmonella chromosome has a gene order that is highly congruent with E. coli, but with numerous inserted gene islands, the genome size is 5% larger. To compensate, map coordinates in Salmonella are described in units of 100 centisomes (Cs) with the same starting position as in E. coli. The largest E. coli macrodomain is Ori, which spans 930 kb of DNA. The corresponding segment in Salmonella extends from Cs 81 to Cs 1 in NH6008, resolution efficiency was tested in another segment of the Ori domain in replichore 1. The sensor disrupts the Salmonella gene STM4442, which encodes a small putative “cytoplasmic protein” at Cs 96. NH6008 matched NH6000 with a resolution efficiency of 85±3% , it extends from Cs 1 to Cs 13 in Salmonella. A sensor was inserted at Cs 9 in NH6007 between ampH, which encodes a beta-lactam binding protein, and sbmA, a gene encoding an inner membrane ABC transporter. NH6007 had a resolution efficiency of 73±12%. The Right macrodomain of E. coli spans 600 kb from Cs 13 to Cs 26. In NH6006, a sensor was inserted at Cs 21. This is the only position in which a reporter lies between two divergently transcribed genes. These genes are STM0951, which encodes a “cytoplasmic protein” transcribed in the counterclockwise direction, and STM0952, which is a transcription regulatory protein transcribed in the clockwise direction. The recombination efficiency in NH6006 was the highest measured at 92±2%.Two domains reside exclusively in replichore 1. The Right Unstructured region is shown in black  clockwisE. coli macrodomains reside entirely in replichore 2. The Left Unstructured region is a 550 kb sector. The comparable region of Salmonella is shown in oriC running from Cs 81 to Cs 62. A sensor inserted at Cs 71 lies between STM3261, which encodes a galacticol-1-phosphate dehydrogenase, and STM3262, a putative repressor in strain NH6001. The resolution efficiency was 82±2%. , which is a 780 kb region of E. coli. Ter has 24 copies of a unique 14 bp site called matS that is found uniquely in this segment. The matS sites bind MatP, which may organize them into a single focus in cells with a chromosomal MatP-GFP fusion. One model is that 23 Ter domain loops are formed with a central hub of MatP protein Salmonella, the Ter domain may be a smaller 560 kb region with only 14 predicted matS sites NH6005, a sensor was inserted at Cs 33 between the pseudogene STM1553 and STM1554, which encodes a putative “coiled coil protein.” The resolution efficiency here was lower than any other site tested in the survey, 45±6%.The macrodomain that lies across from Ori in D = −0.038±.002. There was no statistically significant variation in supercoil levels from the Ori to the terminus. At Cs 33, the resolution efficiency of 45% is roughly half that measured at the other 7 sites. At this location, a Res site is only 470 bp from dif. We believe that resolvase binding to the site nearest dif may be occluded by DNA-binding proteins unique to the region. These proteins include matS-MatP complexes dif to catalyze complex topological reactions that untangle and separate sister chromosomes The cumulative average resolution efficiency of sensors located at 7 regions (excluding the Ter domain) was 81±7% and the apparent σNH6019-NH6027, each strain has the TSgyrA209 allele, which showed a MIF of 2.7 at the Cs 85 position  and at Cs 96 – NH6036 (3±1%). Averaging across 7 points on the chromosome, the mean recombination efficiency was 2±2% and the MIF was 40. Surprisingly, this strain with a greatly relaxed chromosome has a doubling time only 36% longer than WT (53±3 vs. 39±1).Supercoil losses at 7 locations in at Cs 85 . The resparE206TS allele of Topo IV was tested . Similar to cultures with the gyrB652 mutation, the resolution efficiency was at the detection limit (≥1%) at all locations other than Cs 85 , which included sensors upstream and downstream of rrnG, increasing the number of test locations to 10  makes a form of RNA polymerase with a constitutive low transcription rate for stable RNA, including the 7 ribosomal RNA operons ns to 10 . The douns to 10 . The WT E. colilacZ transcription/translation kinetics at 8 locations in WT and gyrB1820 mutants. The method is outlined in In 1973 Pato, Bennett, and von Meyenberg discovered that the rates of transcription elongation and translation were closely matched for most genes in Salmonella genome. The fastest transcription speeds were 69±9 nt/sec at Cs 85 and 62±10 nt/sec at Cs 58. These sites were 45% faster than the 38±1 nt/sec rate measured at Cs 9. The average elongation rate in WT cells across all positions was 52±10 nt/sec. The impact of a TSgyrB1820 mutation was tested in strain set NH6222-NH6229. Elongation at 7 positions fell to a uniform mean of 32±6 nt/sec, which is 40% slower than the average of these positions in WT. Again, the Ter domain at Cs 33 was different. Transcription/translation rates at dif fell from 56±6 nt/sec to 16±2 nt/sec in gyrB1820. These results together with the experiments using Rif suggest to us that unique factors influence resolution efficiency and transcription near dif. Nonetheless, throughout most of the genome, and in at least 5 macrodomains, transcription/translation rates and gyrase supercoiling efficiency were covariant.Unexpectedly, coupled transcription/translation rates varied at different positions in the Salmonella DNA is determined by a mechanism that links the catalytic efficiency of gyrase to the elongation rate of transcription. First, TS alleles of GyrB caused a broad and dramatic depletion of (−) supercoiling throughout the Salmonella genome . These observations, along with older experiments showing that mutations in RNA polymerase influence cellular resistance to the gyrase inhibitors novobiocin and nalidixic acid Many components are now known to contribute to the transcription/translation enterprise. The list of factors includes DksA, NusA, NusG, MFD, Rho, RfhA, GreA, GreB, RNAP, ppGpp, tmRNA, Topo I, cAMP, cyclic GMP, and ribosomes. Interestingly, when the Cozzarelli lab set up a genetic screen to identify genes that might encode “domainins,” i.e. proteins controlling supercoil density, they uncovered a surprise gene, In vivo, 300 E. coli genes are reported to change expression within 5 min after DNA relaxation by drug treatment E. coli genes are induced by increased (−) supercoiling, a different mechanism would be needed to turn them off.DNA supercoiling has generally been studied as a mechanism to control gene expression by modulating promoter activity around the chromosome E. coli supercoil responsive genes actually improve fitness and how many represent regulatory noise that is insignificant from a physiological perspective.Second, most of the chromosomal changes in transcription detected after DNA relaxation are 2-fold differences. The transcription rate of the Lac operon, which varies several hundred fold from the uninduced state to maximum expression, also changes elongation rate by 2-fold, according to its chromosome position . This isSalmonella gyrB652 mutant chromosome gyrB652 strains were compared, they differed by only 1 topoisomer. One cause for these chromosome/plasmid differences is that plasmids are single domain elements, except when they have an anchoring element or two active transcription units moving in opposing polarities Third, many investigators rely on plasmids to estimate chromosomal supercoiling and to gauge the effects of mutations on chromosomal structure. But plasmids can be misleading. By assuming that pUC19 was a good reporter of chromosome supercoil density, we completely missed the impact of transcription and the large supercoiling change in a E. coli and Salmonella have a 15% supercoil difference that changes the phenotype of multiple proteins contributing to chromosome dynamics E. coli and Salmonella GyrA proteins is the amino acid sequence and length of the acidic amino acid-rich C-terminal tail (E. coli gyrase E. coli GyrA ortholog was compared to M. tuberculosis (M. tb.) GyrA, the latter protein lacked C-terminal features present in E. coliIn vitro supercoiling tests confirmed that both the speed and endpoint of M. Tb gyrase supercoiling are lower than those measured for the E. coli enzyme. Secondly, the GyrB subunit has the ATP binding site that fuels the supercoiling reaction. Whereas Salmonella GyrB protein is toxic in E. coli, the reverse is not true. Salmonella tolerates the E. coli GyrB chromosomal substitution, and the average supercoiling level of this strain increased at multiple chromosomal locations, including the region immediately downstream of rrnG  Therefore, both gyrase subunits contribute to the enzyme vmax and supercoil endpoint.nal tail . This C-Salmonella/E. coli comparison for other bacterial species, and the relevance of this work to gene expression in eukaryotes. First, our data represent the mean values of an ensemble of cells in different states of the cell cycle during rapid division in rich medium and during slower growth in medium containing a defined carbon sources. Many investigators assume that supercoil density in a bacterium is maintained at a static modulus so that small changes in supercoil density can be used to modulate gene expression. Our view is more dynamic. The constant thrust from highly transcribed genes causes local gradients of supercoil density to arise throughout the genome. If topoisomerase efficiency is changed by metabolism or mutation, supercoil change spreads across the genome. However, little is currently known about single cell metabolism. For example, yeast cells go through an ultradian cycle that oscillates between periods of reductive reactions of the TCA cycle followed by an oxidative phosphorylation phase that increases ATP concentration Three untested issues related to these finding are worth mentioning. They involve current limitations on the fluxuation of supercoil density that we can monitor, the implications of the E. coli and SalmonellaE. coli cells grow faster than Salmonella and they double every 25 min at 37° in rich medium where transcription elongation rates top out at 90 nt/sec Caulobacter crescentus is 2 h M. tb. has a doubling time of 14 h, contains only 1 ribosomal RNA operon, and encodes a gyrase with significantly slower vmax and a lower supercoiling endpoint than E. coliE. coli and Salmonella, methods will be needed to measure in vivo transcription/translation rates and to define supercoil density at multiple chromosome locations.The second interesting issue is the relationship between optimum growth rates and supercoil levels in different bacterial species. A significant supercoiling difference exists between in vivo transcribed DNA was analyzed, camptothecin treated yeast cells produced highly (+) supercoiled DNA, and further transcription was impeded Third, might a pattern of covariant tempos of transcription elongation and topoisomerase turnover apply to eukaryotes? The short answer seems to be yes. In yeast, a type 1B topoisomerase (Topo I) relaxes both positive and negative supercoils and is active during transcription. Single molecule studies All strains in this work are derivatives of S. Typhimurium LT2, and their genotypes are listed in + colonies on minimal lactose medium. In each case the expected recombinant genotype was verified by PCR analysis using flanking PCR primers. Each recombinant was tested and shown to contain a cassette-modified allele with no WT allele present. Transduction crosses were performed as described previously using P22 HT105/1 int-201, a high-efficiency transducing variant of bacteriophage P22 Chromosomal recombinants were selected as antibiotic-resistant colonies on LB medium or as Lac650 between 0.01 and 0.4. Each strain was tested, starting from three independent colonies grown overnight and diluted 100 fold in fresh LB at 30°. The growth rate of individual strains was measured in early-mid log phase and calculated from the log slope of change over time of the ODPL promoter using the TS cI857 repressor E. coli and S. Typhimurium cells Plasmid pJB γδ 30′ was used to induce the expression of a modified form of γδ resolvase. In this plasmid, resolvase is controlled from the λ−6 dilutions of each culture were plated on LB medium or on NCE glucose minimal medium containing chloramphenicol and 5-bromo-4-chloro-3-indolyl β-D-galactoside  plus 200 µM IPTG lacZLog-phase cultures growing in LB at 30° were sampled at a density of 50 Klett units. A 0.1 ml aliquot of each culture was placed in a 42°C shaking water bath for 10 min to induce Resolvase expression. The induced cells were immediately diluted with 2 ml of LB+Cm and incubated overnight at 30°C. On the following day, 100 µl aliquots of 10Salmonella, the β-Gal method described by Vogel was used 600 between 0.2–0.4 were added to 500 ul ZS buffer (chilled at 4°C) containing 200 ug/ml chloramphenicol. Three samples were taken at 10 s intervals for the background measurement, and IPTG at a final concentration 1.5 mM was added to each culture. Aliquots of 500 ul were withdrawn every 10 sec and mixed with 500 µl ZS buffer for about 4 min. 100 µl chloroform was added to each sample followed by 200 ul ONPG which initiated a timed enzyme reaction. Reactions incubated at 30° for 1.5–4 hrs to allow development of appropriate levels of color were stopped by the addition of 500 ul Na2CO3. The OD420 and OD550 values were taken, and standard Miller Units were calculated as described To measure elongation rates in rpoC gene of Salmonella, we used the method described by Sharan et al. CGCGAAGATGGGGGCGGAAG) and RpoC rev del (aaggcttccagcagtttgat acgcttggtttcggagttgg) and RpoC frd del2 (ccaactccgaaaccaagcgt atcaaactgctggaagcctt) and Rpoc rev (CCATCCAGCGGAACCAGCGG) both make 130 bp PCR products carrying the upstream and downstream region of RpoC with a deletion in both fragments. These products were combined and amplified with the RpoC fwd and RpoC rev primers to generate a 220 bp PCR with the 6 amino acid deletion at the center. The PCR DNA was introduced by electroporation of WT LT2, which had been pre-induced for recombineering function encoded on the pSIM6 plasmid. After incubation for 2 hrs in LB to allow recovery, 200–400 cells were plated onto 5 LB plates and incubated at 30° for 2 days. Small colonies were observed in both WT and fis mutant plates at a frequency of 1/500 to 1/1000. Three colonies were picked and subjected to PCR sequencing using the outside primers and DNA template from a negative control. Every small colony we tested carried the deletion called RpoC (ß′ Δ115–220) by Bartlett et al. To make a 6 amino acid deletion (ΔKKLTKR) in the Figure S1Salmonella chromosome. In each strain, a single 34 bp Frt site was introduced into the chromosome using the λ red recombination methods atpI gene and the gidB gene. The circular form of the Lac-Gn Res module isolated from a donor strain with a chromosomal copy by thermo-induction of the Flp recombinase was re-inserted in new locations by transformation of cells induced for the Flp expression (center). Each module was transferred by P22 transduction to strains with the desired test gene plus the pJB-γδ-Res-Ssra-L9D plasmid, which encodes a 30 min half-life resolvase.Strain construction for supercoil analysis in the (TIF)Click here for additional data file.Figure S2S. typhimurium gyrase. In A the GyrA protein is shown with the catalytic tyrosine-122 (Y122*). The gyrA213TS mutation is caused by a change of Arg 358 to His in the DNA binding/cleavage domain (aqua), and the gyrA209TS allele changes Gly 597 to Asp in the second ß-propeller domain (Blue). There are 72 codon differences between WT E. coli and Salmonella GyrA (black hatches); most changes are in the carboxyl-terminal segment of the protein that involve DNA looping (blue) and regulation of looping by the acidic tail (Red) gyrB used in this study are the gyrB1820TS mutation of Cys 56 to Tyr in the ATPase domain (red) and the gyrB652TS substitution of Arg 436 to Ser in the magnesium binding/DNA cleavage activation domain (green). Only 28 amino acids have diverged between E. coli and Salmonella GyrB (black hatches.)Map of GyrA and GyrB subunits of (TIF)Click here for additional data file.Figure S3Salmonella enterica. Bacteria were grown to an optical density of 50 Klett units. Aliquots (4 ml) of each culture were harvested before and after temperature induction. Cells concentrated by centrifugation at 4°C were suspended in sterile 100 µl TGED buffer . 8 µl aliquots were mixed with 2 µl 5× SDS PAGE loading buffer , boiled 5 minutes and spun down. 5 µl of each supernatant was loaded onto an SDS 15% polyacrylamide gel. Membranes washed twice in TBST and once in TBS  were developed using PerkinElmer Western Lightning Plus-ECL kit according to manufacturer recommendations. Two cell proteins run near the resolvase protein react with the rabbit antiserum; one lies above and one much lighter band runs at the same position as Resolvase (21 kDa) in the control lane. Lane 1) NH2002 (WT LT2) without a plasmid after 10 min at 42°. In all other lanes each strain has the pJBRES 30′; 2) NH6000 (LT2 WT) uninduced; 3) NH6000 10 min at 42°; 4) NH6000 10 min at 42° followed by 30 min incubation in Rif at 30°; 5) NH6018 (gyrA213) uninduced; 6) NH6018 10 min at 42°; 7) NH6019 (gyrA209) uninduced; 8) NH6019 after 10 min at 42°; 9) NH6037 (gyrB1820) uninduced; 10) NH6037 10 min at 42°; 11) NH6206 (rpoC) uninduced; 12) NH6206 10 min at 42°.Western Blot analyses of Resolvase expression in WT and mutant strains of (TIF)Click here for additional data file.Table S1List and genetic structure of all strains used in this study. All strains were created for this or previous studies related to this work.(DOC)Click here for additional data file.


There is considerable evidence that transcription does not occur homogeneously or diffusely throughout the nucleus, but rather at a number of specialized, discrete sites termed transcription factories. The factories are composed of ~4–30 RNA polymerase molecules, and are associated with many other molecules involved in transcriptional activation and mRNA processing. Some data suggest that the polymerase molecules within a factory remain stationary relative to the transcribed DNA, which is thought to be reeled through the factory site. There is also some evidence that transcription factories could help organize chromatin and nuclear structure, contributing to both the formation of chromatin loops and the clustering of active and co-regulated genes. Cellular processes like differentiation or the response to physiological stimuli all require coordinated and efficient regulation of many genes. This regulation can now be monitored at thousands of genes simultaneously using high throughput microarrays and next-generation sequencing. These technologies have revealed temporally coordinated changes in the transcription levels of many genes in response to developmental or environmental changes. However, in addition to this temporal coordination of gene transcription, there is strong experimental evidence that transcription is also spatially coordinated within each cell nucleus. Specifically, it is now recognized that transcription occurs at discrete foci located throughout the nucleus. Here we review first the data supporting this observation, and then discuss its potential consequence for polymerase function, gene regulation, and nuclear organization.Figure 1), which are now commonly referred to as transcription factories.The term transcription “factory” was first used in 1993 by Jackson and colleagues. They exploited Br-UTP labeling to visualize mRNA synthesis in permeabilized HeLa cells that were encapsulated in agarose microbeads. Using confocal microscopy to visualize the transcripts, they found that transcription occurred at 300–500 discrete sites in the nucleus , rather To further investigate transcription factories, It is interesting to note that these analyses of transcription factories were mirrored by parallel studies of DNA replication using biotin-11 UTP or Br-UTP. Like RNA synthesis, these studies demonstrated that DNA synthesis occurred at discrete sites throughout the nucleus  leading in vivo in much thinner bacterial cells  combined with counting ~2,400 transcription foci (which were detected by electron microscopy) yielding 75,000/2,400 ≈ 30 polymerase molecules per factory . The spectroscopic images detect very few phosphorus atoms (marking the backbone of nucleic acids) in the proteinaceous core, suggesting that the DNA template and the nascent transcript must lie outside of the core, presumably attached to the surface as well.High-resolution microscopy has also started to provide information about the ultrastructure of transcription factories. Electron spectroscopic imaging permits the detection of nitrogen and phosphorus distributions at electron microscopy resolution, which can be used to infer the distribution of protein and nucleic acid in a structure . When apIn addition to RNA polymerase and mRNA, transcription factories appear to contain virtually all of the other molecules that one might expect to find at a transcription site. The molecular composition of factories was determined by isolating factory components and then performing mass spectrometry. Factory components were isolated by subjecting cells to permeabilization and DNaseI digestion, which had previously been shown to leave the polymerase factories in an insoluble fraction , 1993. FThe number of transcription factories in a nucleus appears to depend on the cell type and species, and also in part on the measurement procedure. For example, in cultured mouse embryonic fibroblasts ~1500 transcription factories have been detected by immunofluorescence of active RNA polymerase II , whereasde novo in response to transcriptional demands, or whether they are relatively stable structures whose number in a cell nucleus remains relatively constant.There is conflicting evidence about whether transcription factories can assemble uPA gene prior to its activation (D-ribofuranoside (DRB) to inhibit elongation , also addresses the question of whether factories are stable or dynamic structures . ConsistIn vitro experiments have provided support for this view by visualizing single prokaryotic RNA polymerase molecules sliding along immobilized stretches of DNA  to ~2,070 bp (at 30 min), they still occupied the same small volume .Additional evidence in support of a stationary polymerase comes from Another piece of indirect evidence supporting the stationary polymerase model comes from an immunofluorescence analysis of polymerase factories, nascent transcripts and histone modifications at a tandem gene array . Here itin vivo evidence will be required to demonstrate convincingly that the DNA of transcribed genes is actually moving relative to the factories. It also remains to be investigated how a stationary polymerase can transcribe genes on the sense and antisense strand of the same region at the same time. In such a “tug-of-war” scenario the DNA would be pulled in opposite directions simultaneously, thereby potentially abrogating any movement.These preceding studies are all consistent with a stationary polymerase but more direct Another missing piece of evidence in the stationary polymerase model is how the polymerase is tethered and what it is tethered to in order to remain stationary. Several lines of evidence suggest that the polymerase could be associated with a nuclear matrix . JacksonFigures 3B). In terms of chromatin structure, there is also some evidence that factories can lead to the formation of chromatin loops .Factories have been suggested as a key organizing principle for both nuclear and chromatin structure . In termFigure 3C). This could be a chance occurrence, or if specialized factories for certain transcription factors exist, then it could arise because the two genes are both regulated by the same transcription factor(s). There is evidence that such specialized factories do exist (see below), giving rise to speculations that interaction among the shared transcription factors could be involved in recruiting the genes to the same factory .Two mechanisms have been suggested by which factories could drive chromatin loop formation . First,  factory . In any A second reason that transcription factories could induce loop formation is the consequence of a physical phenomenon known as the “depletion attraction”. This occurs when larger objects (such as large transcription complexes) are in a crowded environment containing many smaller, soluble objects (such as proteins). The larger objects will tend to aggregate because their clustering excludes the smaller objects from the region of overlap, and this reduces the entropy of the system as a whole . Thus twThere is now experimental evidence from various sources that chromatin loops exist and in fact are commonly found within nuclei. Such loops were initially visualized by light microscopy of lampbrush chromosomes isolated from oocytes of many animals  since there are not enough factories per cell to provide every active gene with its own factory. Specifically, factory numbers per nucleus range from a few hundred to a few thousand at most, whereas in the human genome, there are currently ~21,000 protein-coding genes and about 12,000 RNA genes known  were frequently spatially associated. They found that virtually all (~90%) active alleles of these genes were associated with distinct RNA polymerase II foci, as demonstrated by RNA FISH signals that co-localized with an antibody against initiating and elongating RNA polymerase II phosphorylated at Ser5 on the C terminal domain (CTD). In contrast to active alleles, they found that otherwise identical but inactive alleles in the same cells were repositioned away from transcription factories. Finally, they showed in a triple-labeling experiment that when transcripts from two different genes were co-localized they were often associated with the same RNA polymerase II factory.Direct experimental proof for this possibility has come from a study in mouse erythroid cells. Myc and Fos rapidly relocated to pre-assembled transcription factories and preferentially co-localized with the highly active immunoglobulin heavy chain (Igh) locus (In a follow-up study h) locus . Before Hbb-b1 (β-globin) gene, which – when activated – contacts the LCR and forms an active chromatin hub (ACH) in a tissue-specific manner , which account for about 10–20% of all factories in that cell type. Consistent with this, Hbb, Hba, Hmbs, and Epb4.9) were transcribed at KLF1-enriched factories. These association frequencies far exceeded the 10–20% association rate that would be expected if these genes were randomly associating with factories. The authors also found that active pairs of KLF1-regulated genes were co-associated with KLF1 transcription factories at unexpectedly high frequencies (63–79%), although this high degree of co-localization might be largely explained by the tendency of individual KLF1-regulated genes to associate with KLF1 factories, thereby increasing the odds that pairs of co-regulated genes could be at the same factory.Further evidence for this possibility comes from Finally, this clustering of KLF1-regulated genes was itself dependent on KLF1, since KLF1 knockdown disrupted the co-association. Nevertheless, it was also shown that the KLF1 transcription factories are not absolutely required for transcribing KLF1-regulated genes, since transcription of these genes can also occur at other factories lacking an accumulation of KLF1. The authors propose that specialized factories may enhance the expression of clustered and co-regulated genes by placing them in an environment where all the required factors for increased transcription are concentrated.Hbb, but in contrast to Hbb. Although Hbb alleles are active . However, other studies suggest this is not always the case, indicating that “specialized” transcription factories may be cell type- and transcription factor-dependent.In summary the results of some studies, suggest that “specialized” transcription factories may exist , data from other groups suggest several alternative mechanisms for such co-localization. For example, by combining immunofluorescence with DNA FISH, a number of studies have suggested that SC35 splicing speckles could be responsible for gene clustering . Splicing speckles are nuclear structures of ~0.5–2 μm in size characterized by an enrichment in the SR splicing factor, which has a critical role in transcription and elongation . Rad23 gene, suggesting that ongoing transcription or polymerase binding is not essential for maintaining some of the global genome organization in a cell. Note, however, that these results do not exclude the possibility that RNA polymerase II had a role in establishing this organization before transcription was blocked.Indeed, other data suggest that some genes can cluster without sharing either speckles or factories. Specifically, some studies suggest that certain sets of genes can be co-localized even in the absence of transcription . It remains uncertain what might specify this global organization, but several possibilities have been suggested, namely nucleosomal positioning, histone modifications, or transcription factor binding .After two decades of work there is now considerable evidence in many different cell types confirming the existence of transcription factories. However, several corollaries of the basic transcription factory model remain uncertain. While there is accumulating evidence that the DNA template is drawn through a stationary factory, more direct demonstrations of this process will be required before student text books are re-written. Furthermore, the importance of transcription factories as organizers of nuclear architecture remains to be fully understood. Specifically, it remains uncertain whether factories are the central organizing principle or instead one of many factors that contribute to gene clustering. In addition, more data and experiments are needed to assess a possible role of transcription factories in cancer facilitating chromosome translocation or other types of disease. Progress in deciphering the role of factories in nuclear organization will certainly come from continued application of next-generation sequencing methods to specialized derivatives of 3C. High throughput microscopy should also enable analysis of a much larger number of genomic loci leading to more general insights into the role of transcription in the spatial organization of the genome. Finally, further elucidation of transcription factory structure, including analysis within live cells, will benefit considerably from the emergence of super-resolution microscopy.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Drosophila melanogaster and ~85% of them encode for proteins. We recently characterized the D. melanogastertimeless2 (tim2) gene, showing its active involvement in chromosomal stability and light synchronization of the adult circadian clock. The protein coding gene named 2mit maps on the 11thtim2 intron in the opposite transcriptional orientation.Intronic genes represent ~6% of the total gene complement in 2mit. The 2mit gene is expressed throughout Drosophila development, localizing mainly in the nervous system during embryogenesis and mostly in the mushroom bodies and ellipsoid body of the central complex in the adult brain. Insilico analyses revealed that 2mit encodes a putative leucine-Rich Repeat transmembrane receptor with intrinsically disordered regions, harboring several fully conserved functional interaction motifs in the cytosolic side. Using insertional mutations, tissue-specific over-expression, and down-regulation approaches, it was found that 2mit is implicated in adult short-term memory, assessed by a courtship conditioning assay. In D. melanogaster, tim2 and 2mit do not seem to be functionally related. Bioinformatic analyses identified 2MIT orthologs in 21 Drosophilidae, 4 Lepidoptera and in Apis mellifera. In addition, the tim2-2mit host-nested gene organization was shown to be present in A. mellifera and maintained among Drosophila species. Within the Drosophilidae 2mit-hosting tim2 intron, insilico approaches detected a neuronal specific transcriptional binding site which might have contributed to preserve the specific host-nested gene association across Drosophila species.Here we report the molecular and functional characterization of 2mit, a gene mainly expressed in the nervous system, has a role in the behavioral plasticity of the adult Drosophila. The presence of a putative 2mit regulatory enhancer within the 2mit-hosting tim2 intron could be considered an evolutionary constraint potentially involved in maintaining the tim2-2mit host-nested chromosomal architecture during the evolution of Drosophila species.Taken together, these results indicate that  Drosophila melanogaster, while ~2.1 and 0.5% in Caenorhabditis elegans and Homo sapiens genomes, respectively  and ran the Augustus gene prediction tool for each contig identified by tBlastN. 2MIT orthologs were detected in D. eugracilis, D. rhopaloa, D. biarmipes, D. bipectinata, Delegans. , D. ficusphila, D. kikkawai, Dtakahashii. , and Dmiranda.   database; thus they were excluded from subsequent analyses.Searching for 2MIT orthologs with tBlastN  showed that the 2mit honey bee (Am 2mit) ortholog maps internally to the Am tim2 intron 14 on opposite strands. However, the tim2-2mit host-nested gene structure was not maintained in the 4 lepidopterous species .Comparative analyses of the D. melanogaster genomic region around the c03963 transposon, using a sliding window of approximately 400 nucleotides both upstream and downstream of the insertion site, searching for potential transcription factors (TFs). In particular, we identified a 6 bp TF binding site specific for the zinc-finger neuronal SNAIL repressor within the 11th intron of the D. melanogaster tim2 gene . Comparative analyses performed on the 21 Drosophilidae identified SNAIL binding site conservation in the tim2 11th intron in all species with the exception of D. virilis, D. ananassae, Dgrimshawi. , and Drhopaloa. , which are characterized by incomplete assembly coverage in the region upstream of 2mit.Finally, we analyzed the 2mit was originally identified as an intronic protein-coding gene located in an opposite transcriptional orientation within the 11th intron of the Drosophila melanogaster tim2 locus. Its transcription at all developmental stages gives rise to two 2mit mRNA variants with different 3’ UTR lengths . A rece1 intron .In silico analysis has indicated that D. melanogaster 2mit encodes a transmembrane protein carrying a LRR domain of 16 repeats in the extracellular portion and a Ser-rich region in the cytoplasmic portion, which might represent a putative binding site for different molecular factors. LRR domains are widespread and highly conserved structural motifs with a primary function in protein-protein interactions , 2mitOM4 (insert on 2nd Chr), 2mitOM14 (insert on 2nd Chr). Three independent 2mit KD transgenic lines were obtained in our laboratory: 2mit KD61. (insert on 3rd Chr), 2mit KD16.2 (insert on 2nd Chr) and 2mit KD61.1 (insert on 2nd Chr). Insert localization along polytene chromosomes was determined by in situ hybridization . Data are expressed as mean ± SEM with the number of tested flies indicated above each bar. The CIs of the sham flies were significantly different from those of the conditioned males in all c232Gal4- and 52YGal4>2mit KD lines and relative controls. The number of asterisks indicates the significance level: *: p < 0.05; **: p < 0.005; ***: p < 0.0001.(TIF)Click here for additional data file.File S1Drosophilidae, 4 Lepidoptera and Apis mellifera 2MIT sequences performed by MAFTT software and manually refined.Multiple alignment of 21 (FASTA)Click here for additional data file.Table S12mitc03963 flies.Phototaxis behavior in (DOCX)Click here for additional data file.Table S2Primers used in amplification and cloning experiments.(XLS)Click here for additional data file.


In strains of the Anabaena/Nostoc genera NtcA is pivotal for the differentiation of heterocysts in response to N stress.The CRP-family transcription factor NtcA, universally found in cyanobacteria, was initially discovered as a regulator operating N control. It responds to the N regime signaled by the internal 2-oxoglutarate levels, an indicator of the C to N balance of the cells. Canonical NtcA-activated promoters bear an NtcA-consensus binding site . Some filamentous cyanobacteria, such as the model strain Anabaena sp. PCC 7120, are also able to fix N2 in specialized cells called heterocysts , where the input sample was used as the control. The efficiency of the PCR was calculated using the program LinRegPCR  dATP. Additional file 2, 2 mM DTT, 5% glycerol). 0.04 mg/ml poly (dI-dC) and 0.04 mg/ml bobine serum albumine were used as non-specific competitor DNA and protein, respectively. The reaction mixtures with the corresponding DNA fragment were incubated with purified NtcA (100 nM and 400 nM) for 30 min at 30°C. The protein-DNA complexes were separated on native 8% polyacrylamide gels. Radioactive areas of the gels were visualized with a Cyclone storage phosphor systems (Packard).The DNA fragments assayed were obtained by PCR using one of the primers labeled with T4 polynucleotide kinase (Roche) and γ-2P dATP. The data sets supporting the results of this article, which can visualized with the Integrative Genome Viewer (Broad Institute), are available at GEO accession # GSE51865.The authors declare that they have no competing interests.SP participated in the design of the experiments, performed the experiments, analyzed data, and drafted the manuscript. EF analyzed data and helped draft the manuscript. AH participated in the design of the experiments, analyzed data, and helped draft the manuscript. All authors read and approved the final manuscript.Q-PCR verification of the immunoprecipitated material.Click here for fileCompendium of the binding regions found in the ChIP-Seq analysis.Click here for fileScheme of the ChIP-Seq results.Click here for fileAnabaena sp. PCC 7120 in ChIP-Seq target regions.Identification of previously described NtcA binding sites in Click here for fileFunctional category of genes ascribed to target regions with NLQ>300.Click here for fileet al. and Mitschke et al.Comparison of the results of the ChIP-Seq analysis with the proposed NtcA-dependent genes by Su Click here for filePrimers used in this work.Click here for file


Herpes simplex virus (HSV) establishes a latent infection within sensory neurons of humans. Latency is characterized by the transcriptional repression of lytic genes by the condensation of lytic gene regions into heterochromatin. Recent data suggest that facultative heterochromatin predominates, and that cellular Polycomb proteins are involved in the establishment and maintenance of transcriptional repression during latency. This review summarizes these data and discusses the implication of viral and cellular factors in regulating heterochromatin composition. Persistent viral infections, especially those that establish latency, utilize epigenetic modifications to regulate transcription from their latent genomes as well as to regulate their entry and exit from latency. The goals of this review are to: (1) provide an overview of epigenetic regulation of Herpes Simplex Virus type 1 (HSV-1) gene expression during latency; (2) review the evidence for polycomb proteins playing a role in regulating heterochromatin deposition on the latent genomes; and (3) discuss potential mechanisms for the remodeling of heterochromatin to facilitate selective reactivation of HSV-1 from latency.Upon transport into the cell body of neurons, there is an ever-growing list of candidate viral and cellular factors that determine HSV’s propensity towards either productive replication or descent into latency. During the latter, the non-random distribution of histones and functionally distinct PTMs partition the roughly 152 kb HSV-1 episome into regions with different chromatin profiles. As discussed later, regions associated with the three temporal classes of lytic genes display very dense deposition of histone marks typical of facultative or constitutive heterochromatin, conveying reversible or irreversible transcriptional repression. During this time, in an estimated third of latently-infected neurons, only non-coding transcripts are transcribed from the HSV-1 genome ,4,5. BasThe chromatin profile of HSV-1 at any time during infection is commonly probed using chromatin immunoprecipitation (ChIP) of desired nucleosome components followed by DNA analysis. Early applications utilized semi-quantitative methods to quantify enrichment, but qPCR using primers specific to members of different HSV-1 gene classes has become the norm. The use of high throughput sequencing (ChIP-seq) holds promise for generating HSV-1 chromatin profiles with greater resolution and coverage. Though methods of analysis for ChIP data have been converging over the last decade, there remains some controversy as to what are the most meaningful and statistically valid means of quantifying the enrichment of any one chromatin mark on the viral genome. This shortcoming complicates the assembly of data provided by independent groups into a holistic map of heterochromatin on the HSV-1 genome.ex vivo; latently infected ganglia are explanted into culture medium, where isolation from their axonal processes and natural physiological environment serves as a stressor that efficiently and synchronously induces reactivation [et al. [Despite its apparent tight co-evolution with humans, HSV-1 can produce infection in a variety of non-human animals, often with characteristic neuronal invasion and establishment of latency. For the purposes of studying HSV-1 epigenetics, mouse models are frequently utilized with the majority of data regarding latent HSV-1 chromatin dynamics generated using the footpad or corneal routes of infection. Following infection, the virus migrates along the axons of the sensory neurons afferent to these sites, and, for mice surviving the initial infection, latency is reliably and stably established in the dorsal root ganglia (DRG) or trigeminal ganglia (TG), respectively. Though the model’s consistency engenders its utility, the difficulty in detecting any clinical shedding of virus during reactivation at the primary site of infection is one of the more important dissimilarities to human pathology. Some experimentalists have developed protocols to induce reactivation via thermal stress , immunostivation ,10. Altetivation . For a m [et al. .in vitro as a means of emulating latency. For such applications, several human fibroblast cell lines have been employed, and HSV-1 can be compelled to quiescence through the use of mutant strains which lack the ability to drive expression of initial lytic genes [in trans, or removal of inhibition can, in some ways, emulate reactivation. These in vitro systems allow for more precise genetic assays and more defined control over the cellular and viral life cycle progression, but have acknowledged limitations on physiological relevance. More recently, the development of dissociated cultures of primary neurons either from the adult mouse TG [in vivo, including the stable transcriptional repression of lytic genes and the ability to reactivate following stimulation, in a manipulable in vitro setting  ,14,15,16ic genes ,20,21 ormouse TG  or embrymouse TG  show greview see ).i.e., a pathogen evolved to carefully exploit the nuances of a phylogenetically distinct host). As a notable example, an HSV-1 KOS-derived LAT mutant exhibits a 5–10 fold increase in expression of lytic genes in latently infected mouse TGs, whereas a similar mutation in a 17syn+-based mutant results in a 3–154 fold decrease in expression of the same genes for latently infected rabbits [Special effort is required to integrate data obtained in distinct experimental systems; cell cultures lack extrinsic factors that may influence chromatin regulation such as exogenous immune effectors and signaling molecules. The animal systems, though more holistic as models, may have developed significant differences in epigenetic regulation strategies through evolutionary divergence, and the relevance of these differences is compounded in the context of a non-equilibrium host-pathogen interaction  of infection. Therefore, in the familiar orolabial route of primary infection, the latent reservoir is comprised of neurons located in the mandibular/maxillary tract of the trigeminal ganglion with axons afferent to the mouth . In contvs. latent infection are not known, but point out the roles that specific cell populations play in the biology of HSV-1 latency. In addition, it is important to note that while A5+ cells represent a defined population of HSV-1 latent neurons, they make up only about 25% of the latent reservoir, and further studies are needed to define the phenotypes of the other cells which support latency, and their contribution to the pool of reactivating virus.It is important to note that sensory neurons in the peripheral nervous system represent a very diverse and highly specialized population of cells that serve to detect a wide range of different types of sensation including hot and cold, pain, vibration, moisture, and touch. Therefore it is not surprising that there are almost 2 dozen types of sensory neurons characterized to date whose representation differs in the various sensory ganglia and individual specialized ganglia, like the TG and DRG  during latency and then turned back on in response to appropriate stressors, resulting in reactivation. The strength of lytic gene repression must be carefully modulated. If the repression is too strict, lytic genes may be silenced permanently, possibly resulting in inefficient or nonexistent reactivation and a subsequent failure to spread to further hosts. If the repression is too weak, then lytic gene activity may invite a vigorous immune response to sensory neurons harboring viral genomes (reviewed in ). HSV-1 Early studies of epigenetic regulation of HSV-1 gene expression focused primarily on DNA methylation. A broad study using methylation-sensitive restriction endonucleases found no extensive methylation of HSV-1 genomes within the central nervous systems of latently-infected mice . A laterDrosophila melanogaster, are a set of proteins that interact with and modify chromatin to effect epigenetic changes and gene silencing  and Polycomb repressive complex 2 (PRC2)  [Drosophila these consist of 5–7 bp binding motifs for different components of the PRC2 complex including Pho/Pho1, as well as transcription factors Sp1 and KLF . PcG pr2 (PRC2) . The moss (PREs) . In Drosview see ). Mammalview see ,53. PRC1view see  and alsoview see . These hview see ,41,42, aview see  and PRC2Drosophila embryos demonstrated that PRC2 blocks recruitment of RNA Pol II to promoters [The mechanisms by which histone modifications promote gene silencing are varied and still under investigation. PRC2 negatively impacts the recruitment and binding of positive chromatin regulators and the deposition of activating modifications, such as histone acetylation. A ChIP-seq analysis of PRC2-negative romoters . The lacromoters . Subsequromoters , or evenromoters ,59.Drosophila, this is achieved primarily by proteins that bind specific PREs and then recruit PcG proteins. However, in mammals, only a few PRE-like elements have been identified [In order to silence the correct genes, PcG proteins must first be recruited to the appropriate loci. In earlier models, PRC2 was designated the “establishment” complex where it was recruited to select loci and deposited the H3K27me3 mark. This mark then served as a binding site for PRC1, the “maintenance” complex which compacted nucleosomes and preserved silencing. While not inaccurate, later evidence shows that this model is incomplete and that PRC2 and PRC1 may be targeted by independent mechanisms . In Drosentified ,62. It ientified  and alsoentified ,48, perhin trans in transgenic mice, reduces H3K27me3 enrichment on latent viral genomes. There is also evidence for an interaction between the stable 2.0 kb LAT intron and PRC1 component PHC1 (unpublished data). The nature of this interaction and whether it directly impacts lytic gene silencing is still under investigation.PcG proteins may be targeted to select regions by multiple mechanisms, including interactions with transcription factors and non-coding RNAs (ncRNA). Several ncRNAs have been demonstrated to interact with EZH2, a member of PRC2. One example of these is RepA, a repeat region of the X-inactive specific transcript (XIST) . A deletsyn+ in a mouse footpad/DRG model of infection, published seemingly contradictory results [syn+ reactivates efficiently but 17ΔPst, the LAT deletion mutant, does not [Several mechanisms have been proposed for the observed effects of LAT transcription on PcG-based silencing of lytic genes. One study, using HSV-1 strain KOS in a mouse ocular/TG model of infection, found that a deletion of the LAT promoter resulted in decreased H3K27me3 at lytic genes . It was  results . Deletiodoes not . All of As previously noted, the above studies used strains of virus that are already known to differ greatly in virulence and reactivation potential. The fact that they behave differently in regard to PcG recruitment and heterochromatin deposition is not surprising. The differing routes of infection and sites of latency must also be taken into account. The subunit composition of Polycomb complexes is likely to differ between TG and DRG, and perhaps even between subtypes of neurons within these tissues. These cell specific differences may explain why the PRC1 component Bmi1 was found to associate with latent genomes in one study , but notH. sapiens, M. musculus, D. melanogaster, C. elegans, S. pombe, S. cervisiae, as well as some prokaryotes. Their presence in the latter suggests an ancestral role divergent from histone modification. The following section will review the relevant demethylases with potential roles in regulation of alpha-herpesvirus infections; LSD1 , JMJD3 , and UTX  histone demethylases. The known histone demethylases that would be predicted to play a role in remodeling HSV chromatin to facilitate reactivation are described in There are currently several histone demethylases with a large assortment of names but relatively focused functions. Homologs have been identified in several organisms including in vivo and requires the neuronal silencer co-repressor of RE1-silencing transcription (CoREST) factor to demethylate nucleosome-associated histones [Lysine-specific demethylase 1 (LSD1) specifically demethylates H3K4me2/me1 as well as H3K9me2/me1 and is unable to demethylate tri-methylated lysine due to biochemical and biophysical constraints. In addition, LSD1 is dependent on protein partners within the Set1/MLL methyltransferase complex such as the transcriptional coactivator host cell factor-1 for its activity histones . The usehistones . LSD1 alhistones . Displachistones . RNAi dehistones ,73. The vast majority of lysine demethylases contain a conserved Jumonji-C domain motif (JmjC). The existence of a large family of Jumonji proteins that can demethylate mono-, di-, and tri-methylated lysine in a reaction mediated by Fe(II) and α-ketoglutarate catalysis provides an additional demethylation mechanism fundamentally different from LSD1. in vitro [in vivo. The association of UTX and JMJD3 with the H3K4 methyltransferase MLL family of proteins and components of the MLL complex  suggests a physical role in balancing activation and repression [UTX and JMJD3 belong to a subfamily of proteins that require a catalytically active JmjC domain to maintain demethylase activity. As well as possessing 84% sequence similarity, their Jumonji-domains share high structural conservation. Temporal expression of Hox genes—which are silenced in pluripotent cells—is mediated through the demethylation of H3K27me3 by UTX and is critical in mammalian embryogenesis. In addition to having a role in activated macrophages, JMJD3 has been identified as a protein specifically upregulated at the outset of neural commitment ,75. Previn vitro . DecreasThe analysis of HSV-1 epigenomes during latency reveals a general consensus that: (1) the HSV-1 lytic genes are associated with heterochromatic histone modifications; (2) while both constitutive (H3K9me3) and facultative (H3K27me3) marks are present, the H3K27me3 marks predominate; and (3) the LAT-encoding regions of the genome display bivalent modifications of both repressive (H3K27me3) and active  marks. A number of studies suggest that a transient and rather global re-modeling of both the lytic and latent gene regions occur rapidly following stressors that induce reactivation, though productive reactivation occurs in only a fraction (<5%) of latently infected neurons. These observations suggest that while there are initial stress-induced changes in chromatin to a large proportion of the genomes, there are downstream effectors of productive phase transcription that operate only in a sub-set of cells. Whether this reflects different neuronal population that regulate transcription differently, differences in established epigenome profiles that vary from cell to cell, or the magnitude of stress-induced signaling that reaches a given cell remains to be determined, and ultimately may require single-cell analyses to sort out. None-the-less, the identification of the spectrum of epigenetic marks that are present on the latent genomes sets the stage for identifying the remodeling proteins that ultimately play an essential role in the initial stages of HSV reactivation.


TMEFF2 is a protein containing a single EGF-like domain and twofollistatin-like modules. The biological function of TMEFF2 remains unclearwith conflicting reports suggesting both a positive and a negativeassociation between TMEFF2 expression and human cancers.Here we report that the extracellular domain of TMEFF2 interacts withPDGF-AA. This interaction requires the amino terminal region of theextracellular domain containing the follistatin modules and cannot bemediated by the EGF-like domain alone. Furthermore, the extracellular domainof TMEFF2 interferes with PDGF-AA–stimulated fibroblast proliferationin a dose–dependent manner. TMEFF2 expression is downregulated inhuman brain cancers and is negatively correlated with PDGF-AA expression.Suppressed expression of TMEFF2 is associated with its hypermethylation inseveral human tumor types, including glioblastoma and cancers of ovarian,rectal, colon and lung origins. Analysis of glioma subtypes indicates thatTMEFF2 hypermethylation and decreased expression are associated with asubset of non-Proneural gliomas that do not display CpG island methylatorphentoype.These data provide the first evidence that TMEFF2 can function to regulatePDGF signaling and that it is hypermethylated and downregulated in gliomaand several other cancers, thereby suggesting an important role for thisprotein in the etiology of human cancers. TMEFF2, also known as tomoregulin Platelet-derived growth factors (PDGFs) not only play important roles indevelopmental and physiological processes, but also are directly implicated in humancancer and other proliferative disorders  TMEFF2 is predicted to contain a transmembrane (TM) domain with an amino terminal(NT) signal peptide sequence (SP) . RecombiSince follistatin (FS) module-containing proteins have been shown to interactwith PDGF ligands To confirm that the binding observed is indeed due to the interaction betweenPDGF-AA and TMEFF2-ECD, we then immobilized the PDGF ligands on the plates, andapplied the TMEFF2-ECD fused to a different tag, TECD-Fc, as an analyte.Consistent with the results obtained with immobilized TECD-FLAG, TECD-Fcexhibited significant dose-dependent binding only to immobilized PDGF-AA, butnot AB, BB, CC or DD . SimilarTo determine if the ECD of TMEFF2 can interact with PDGF-AA when expressed on thesurface of mammalian cells, we transfected 293 cells with constructs containingthe full-length TMEFF2 (TMEFF2-FL), or a truncated TMEFF2 without theintracellular domain (TMEFF2-ΔICD) . PDGF-AATMEFF2 contains 2 FS modules and an EGF-like domain. To dissect which domains ofTMEFF2 are involved in its interaction with PDGF-AA, we made Herpes simplex type1 glycoprotein D (gD)-epitope tagged deletion mutants of TMEFF2 and examinedtheir ability to bind PDGF-AA when expressed on the surface of 293 cells . As expePDGF ligands are potent mitogens of connective tissue cells, includingfibroblasts, smooth muscle cells, chondrocytes, and some endothelial cells We next examined the effect of TMEFF2-ECD on PDGF stimulated proliferation. Themurine fibroblast cell line NR6 expresses both PDGF receptors α and βIn situ hybridization experiments confirmed high levels ofTMEFF2 mRNA expression in normal adult and fetal central nervous systems, aswell as both malignant and non-malignant prostate tissues  have been classified into three molecular subtypesbased on similarity to defined expression signatures: Proneural (PN),Proliferative (Prolif) and Mesenchymal (MES) −14). TMEFF2 expression is barely detectable inalmost all colon adenocarcinoma, rectal adenocarcinoma, lung adenocarcinoma andlung squamous cell carcinoma samples. While the majority of these tumor samplesshow methylation beta values greater than 0.1, there are insufficient dataavailable to determine whether different thresholds of methylation beta valuesexist in different tumor types for suppressed TMEFF2 expression, or othermechanisms exist to suppress its expression. Nevertheless, taken together withother published reports of TMEFF2 methylation in other tumor types, these dataare consistent with the hypothesis that TMEFF2 is silenced through DNAmethylation in a significant proportion of human cancers, including glioma andcancers of ovarian, rectal, colon and lung origins.Hypermethylation of the TMEFF2 gene in human cancers has been reported in severaltissues including colorectal, gastric and esophageal cancers Recently, a subset of gliomas with characteristic promoter DNA methylationalterations, referred to as G-CIMP, have been identified in the context of TCGAGBM samples That TMEFF2 hypermethylation is not found in the G-CIMP and IDH1-mutant GBMsamples is consistent with our observation that higher levels of TMEFF2 areassociated with the Proneural HGGs, the subclass that the G-CIMP tumors belongto, while suppressed expression of TMEFF2 is associated with the Proliferativeand Mesenchymal subclasses of HGGs . TherefoFollistatin domain–containing proteins have been shown to interact with growthfactors or their binding partners and modulate their signaling Pdgfra-activating mutations have alsobeen identified in a subset of human gastrointestinal stromal tumors First identified in a search for serum factors that stimulate the proliferation ofarterial smooth muscle cells The significance of the previously reported hypermethylation of TMEFF2 gene in humancancers including colorectal, gastric and esophageal cancers We have found that TMEFF2 hypermethylation is associated with non-Proneural subtypesof GBMs, in contrast with G-CIMP methylation and IDH1 mutation status, which areassociated with the Proneural subtype and lower-grade gliomas. These associationsare consistent with our finding of higher levels of TMEFF2 expression in theProneural subtype. Moreover, we observe an exclusivity relationship between TMEFF2hypermethylation and G-CIMP methylation, in that none of our samples show both typesof methylation patterns. These data suggest that TMEFF2 is preferentiallyhypermethylated and suppressed in a subset of non-Proneural and non-G-CIMP HGGs, andthat TMEFF2 methylation may be associated with worse prognosis.We also observed an anti-correlation between TMEFF2 expression and PDGF-A expressionin the GBM and HGG samples, with lowest levels of PDGF-A expression observed in theProneural subtype compared to other subtypes. Interestingly, despite the high levelsof TMEFF2 and low levels of PDGF-A expression, PDGFRα amplification appears tobe associated with the Proneural signature of GBM, which may also display elevatedPDGF signaling signature through increased PDGF-B protein levels and elevatedphosphorylation of PDGFRβ Our findings not only suggest a connection between the role of TMEFF2 in PDGFsignaling and the potential tumor suppressor function of TMEFF2, but also providepossible explanations for the seemingly conflicting roles of TMEFF2 in humancancers. It was previously reported that soluble forms of TMEFF2 extracellulardomain could weakly stimulate erbB-4/HER4 tyrosine phosphorylation in MKN 28 gastriccancer cells 2 in DMEM/Ham's F-12 (1∶1) containing10% fetal bovine serum (FBS) and RPMI 1640 containing 10% calfserum, respectively. Recombinant human PDGF-AA, AB, BB, CC and DD, recombinanthuman PDGF receptor α extracellular domain (PDGF sRα), recombinant humanPDGFRβ-Fc, goat anti-human PDGF, and biotinylated goat anti-human PDGF-A andPDGF-B antibodies were obtained from R&D Systems . Rabbitanti-PDGF-A polyclonal antibody was obtained from Santa Cruz Biotechnology. Mouse anti-FLAG antibody was obtained from Sigma . Other recombinant proteins and antibodies were generated at Genentech.The HEK 293  fused in-frame to the CT ofthe end of the EGF domain. TECD-Fc was generated similarly using the sameforward primer and reverse primer 5′-CTGGGCGCGCCACTTTTTTTCACAGTGTT-3′ and clonedinto the same vector containing the human Fcγ sequence which was fusedin-frame 3′ to the end of the EGF domain. The gD-tagged full-length TMEFF2was cloned into the same vector with a 5′ gD tag (amino acid sequenceKYALADASLKMADPNRFRGKDLPVLSGR) attached in-frame to the predicted start of themature protein. gD-TMEFF2-ΔFS I and TMEFF2-ΔFS I/II were PRC amplifiedwith the same reverse primer 5′-CGACTCTAGATTAGATTAACCTCGTGGACGCT-3′ andeither 5′-CTGCTCGAGTGTGATATTTGCCAGTTTGGTG-3′ or5′-CTGCTCGAGACACCACATACCTTGTCCGGAAC-3′ asforward primer, respectively.The full-length TMEFF2 open reading frame (GenBank Accession No. NM_016192) wascloned into a modified pRK vector containing a CMV promoter. The FLAG-taggedextracellular domain of TMEFF2 (TECD) was cloned into the same vector by PCRamplification using forward primer For the TMEFF2 coat format, MaxiSorp 96-well microwell plates  were coated with 1 µg/ml TECD-FLAG (Genentech) in50 mM carbonate buffer, pH 9.6, overnight at 4°C. Plates were washed withPBS, pH 7.4, containing 0.05% polysorbate 20 and blocked with 0.5%bovine serum albumin, 15 parts per million Proclin 300, in phosphate bufferedsaline (PBS), pH 7.4 for 1 hour at room temperature. Serially diluted PDGF-AA,PDGF-AB, PDGF-BB or Fc-fusion proteins in PBS containing 0.5% BSA,0.05% polysorbate 20, and 15 parts per million Proclin 300 were added tothe plates and incubated for 2 hours. Bound PDGF was detected by addingbiotinylated goat anti-human PDGF-AA, PDGF-AB or PDGF-BB to the plates andincubating for one hour, followed by adding horseradish peroxidase (HRP)conjugated streptavidin  and incubating for 30min, with a wash step in between. Bound Fc-fusion protein was detected by addinggoat anti-human Fc-HRP . After a finalwash, the substrate 3,3′,5,5′-tetramethyl benzidine (Kirkegaard& Perry Laboratories) was added. The reaction was stopped by adding 1 Mphosphoric acid and absorbance was read at 450 nm on a Multiskan Ascent reader. The titration curves were fitted using afour-parameter nonlinear regression curve-fitting program .For the PDGF coat format, plates were coated with 1 µg/ml PDGF-AA, PDGF-AB,PDGF-BB, PDGF-CC, or PDGF-DD. Serially diluted TECD-Fc (Genentech) or otherFc-fusion proteins were added to the plates. Bound protein was detected usinggoat anti-human Fc-HRP .To measure binding of soluble PDGF receptor α to PDGF, recombinant human PDGFreceptor α extracellular domain (PDGF sRα) was biotinylated usingbiotin-X-NHS . Serially diluted biotinylatedhuman recombinant PDGF sRα was added to PDGF-AA, PDGF-AB or PDGF-CC coatedwells. Bound receptor was detected using streptavidin-HRP.To measure blocking of TECD-Fc binding to PDGF-AA by PDGF sRα, seriallydiluted PDGF sRα was pre-mixed with TECD-Fc and added to the PDGF-AA coated plate. Bound TECD-Fc was detected using goatanti-human Fc-HRP.For binding of PDGF ligands to membrane-bound TMEFF2 proteins, 293 cells weretransfected with the various TMEFF2 constructs and changed to fresh growthmedium containing 5 µg/ml PDGF-AA or AB 48 hours after transfection. After30 minutes of incubation unbound PDGF ligands were washed away with ice cold PBSand cells were lysed in lysis buffer containing 50 mM Tris pH 8.0, 150 mM NaCl,1 mM EDTA, 1% NP-40, protease and phosphatase inhibitors, pre-clearedwith protein G sepharose, and immunoprecipitated with anti-TMEFF2, anti-PDGF, oranti-gD antibodies. The immune complexes were dissociated with SDS sample bufferwith β-mercaptoethanol and resolved by 4–20% Tris-Glycine SDSPAGE, transferred to nitrocellulose membranes, and detected with the indicatedantibodies using enhanced chemiluminescence.2, BrdU labeling solution was addedto each well and the subsequent labeling and detection were carried outfollowing the manufacturer's protocols. BrdU incorporation was measured asabsorbance at 405 nm with a reference wavelength at 490 nm.The NR6 proliferation assay was carried out using a5-Bromo-2′-deoxy-uridine (BrdU) labeling and detection kit (Roche). Theindicated concentrations of PDGF-AA or AB were added to quiescent confluentcultures of NR6 cells in RPMI 1640 supplemented with 1× Serum Replacement1 (Sigma) on 96-well microplates, either alone or after pre-mixing withincreasing concentrations of TECD-Fc or sRα for 1 hour at 37°C. After 18hours at 37°C and 5% COGene expression profiling and analysis of microarray data were performed aspreviously reported We obtained data from the Cancer Genome Atlas (TCGA) that was publicly availableas of July 29, 2010, on both the Illumina Infinium methylation microarray andthe Agilent G4502A expression microarray. We correlated samples based on theMAGE tables provided and found dual methylation and expression measurements for86 colon adenocarcinomas, 226 glioblastoma samples, 36 renal papillary cellcarcinomas, 21 lung adenocarcinomas, 69 lung squamous cell carcinomas, 535ovarian carcinomas, and 53 rectal adenocarcinomas.Methylation was measured using the beta value taken from the Level 2 filesprovided by TCGA. From the TCGA array description files, we identified two CpGsite methylation probes for TMEFF2: cg06856528 and cg18221862. These probesequences are located at (−204 to −155) and (−29 to +20)relative to the translation start codon, within a CpG island describedpreviously Expression was measured using the antilog of the log2 lowest normalized valuesfrom the Level 2 files provided by TCGA. The array description files showedthree probes belonging to TMEFF2: A_23_P125382, A_23_P125383, and A_23_P125387.Pairwise correlations among these expression values were 0.94–0.95Click here for additional data file.Figure S1A) PDGF-AA, but notAB, BB, CC or DD, binds to TECD-Fc. TECD-Fc was applied to wells coated withrecombinant human PDGF-AA, AB, BB, CC or DD and detected with HRP-conjugatedanti-human Fcγ. (B) sRα binds to all three recombinanthuman PDGFs: AA, AB and BB. Biotinylated recombinant sRα (sRα-bio)was applied to wells coated with recombinant human PDGF-AA, AB or BB anddetected with streptavidin-HRP. (C) 70 ng/ml TECD-Fc was mixedwith increasing concentrations of sRα and applied to PDGF-AA coatedwells. Binding between TECD-Fc and PDGF-AA was detected using goatanti-human Fc-HRP.TECD-Fc selectively interacts with PDGF-AA. ((TIF)Click here for additional data file.Figure S2gD-tagged TMEFF2 proteins are expressed on the cell surface as detected by ananti-gD antibody. FACS analysis of 293 cells expressing the gD-taggedfull-length TMEFF2 or deletion mutants lacking either FS I or both FSmodules using anti-gD mAb (black) and four mAbs  recognizing the FS I module of TMEFF2. Biotinylated anti-mouse IgG wasused as a secondary reagent followed by streptavidin-PE. Filled purple, noprimary antibody control.(TIF)Click here for additional data file.Figure S3Comparative transcript expression profiles of TMEFF2 in human tissues basedon GeneLogic data. The mRNA expression patterns for TMEFF2 across thousandsof human cancer (red) and normal (green) tissue specimens using probe223557_s_at on chips HG-U133A and B are shown.(TIF)Click here for additional data file.Figure S4A)Bar-graphs of mean TMEFF2 mRNA expression levels in indicated tissues basedon GeneLogic data. Error bars represent standard errors of the mean.(B) Number of tissues analyzed in each category.[N], Normal tissues; [C], Cancer tissues; [M],metastatic tissues; * p<0.05 and **p<0.005 compared to normal.TMEFF2 expression is down-regulated in some cancers. ((TIF)Click here for additional data file.Figure S5In situ hybridization (ISH) analysis of TMEFF2 mRNAexpression in normal adult brain and cerebellum (A), fetalspinal cord and spinal ganglion (B), non-malignant prostate(C) and prostate cancer tissues collected on tissuemicroarrays (TMA) (D). Upper panels, H & E stains; lowerpanels, ISH signals (white).(TIF)Click here for additional data file.Figure S6A) Correlations between the beta values of two TCGA arraymethylation probes for TMEFF2 in the tissues analyzed: colon adenocarcinoma(coad), lung adenocarcinoma (luad), lung squamous cell carcinoma (lusc),glioma (gbm), rectal adenocarcinoma (read), ovarian carcinoma (ov), andrenal papillary cell carcinoma (kirp). (B) Pairwisecorrelations among the three expression probes belonging to TMEFF2.((TIF)Click here for additional data file.Figure S7A) vs. PDGF-A expression (B) inGBM subtypes. Each GBM sample is classified according their classificationby both Verhaak and Phillips schemes (denoted as Verhaak scheme:Phillipsscheme).TMEFF2 methylation ((TIF)Click here for additional data file.Figure S8A) Efficiency of anti-TMEFF2 immunoprecipitation of full-lengthor intracellular domain–truncated TMEFF2 expressed on 293 cellscompared to inputs in the whole cell lysates (WCL). (B)Efficiency of PDGF-A co-immunoprecipitation with full-length TMEFF2 with orwithout a gD tag compared to 5 ng of recombinant PDGF-AB or the amount ofsurface-bound PDGF-A in the whole cell lysates (WCL).((TIF)Click here for additional data file.


Understanding the role of these factors in epidermis is complicated by the fact that each protein is expressed, at different levels, in multiple cells layers in differentiating epidermis, and because AP1 transcription factors regulate competing processes . Various in vivo genetic approaches have been used to study these proteins including targeted and conditional knockdown, overexpression, and expression of dominant-negative inactivating AP1 transcription factors in epidermis. Taken together, these studies suggest that individual AP1 transcription factors have different functions in the epidermis and in cancer development and that altering AP1 transcription factor function in the basal versus suprabasal layers differentially influences the epidermal differentiation response and disease and cancer development.AP1 (jun/fos) transcription factors ( Keratinocytes are the major cell type responsible for the structure of the epidermis. They begin as stem cells in the basal epidermal layer and hair follicles –3. Durin The process of activation and suppression of gene transcription is controlled by a diverse family of regulators called transcription factors. Transcription factors mediate the final steps in the relay of information from the cell surface to the nucleus and the gene. This is accomplished by the interaction of the transcription factor with specific DNA elements that are usually located immediately upstream of the sequence that encodes the gene. DNA elements are generally a short DNA sequence of 8–20 nucleotides that encode a specific consensus sequence. A host of transcription factors has been implicated in control of epidermal differentiation and function, including activator protein 1 (AP1), AP2, Sp1, POU domain proteins, and CCAAT enhancer binding proteins . AP1 traα, β, δ, and γ). δ MAPK pathway which regulates expression of differentiation-associated genes during keratinocyte differentiation anthracene (DMBA) to produce initiated cells, and then inducing TAM67 expression. Surprisingly, TAM67 expression, and the associated increase in cell proliferation, did not drive tumor formation in DMBA treated mice. This is interesting, because cell proliferation is thought to predispose tissue to enhanced tumor formation [α]anthracene, DMBA) followed by tumor promoter  is known to cause tumor formation [ Because of the hyperproliferative phenotype, it was anticipated that mice expressing TAM67 in the suprabasal epidermis would be more susceptible to tumor formation. This was tested by treating control and suprabasal TAM67 mice with a DNA mutagenic agent, 7,12-dimethylbenz[ormation . Treatmeormation . However Thus, although the basal and suprabasal targeted TAM67 mice produce very different epidermal phenotypes, these mice share features in common , 71. Firin vivo role of AP1 transcription factors in epidermis. It is clear from these studies that AP1 transcription factors play a key role in controlling differentiation of epidermal keratinocytes and that perturbing this process results in a variety of disease phenotypes including psoriasis and cancer. It is also clear that some AP1 transcription factors function as procancer proteins , while others inhibit cancer development . Additional studies suggest that a host of cytokines and chemokines is involved in generation of the disease and cancer phenotypes that develop when AP1 transcription factor function is perturbed, and these studies suggest that the epidermis can act as an endocrine organ to influence the function of other organs. It also appears that AP1 transcription factors have differing roles in basal and suprabasal epidermis, as inactivation of AP1 transcription factor function in these compartments produces no change  or hyperproliferation .A variety of genetic approaches have been used to study the