As expected, all TAC hearts displayed a significant reduction in SERCA-2A and a significant induction of β-MHC mRNA levels. Interestingly, opposite histone H3 modifications were identified in the promoter regions of these genes after TAC, including H3 dimethylation (me2) at lysine (K) 4 (H3K4me2) and K9 (H3K9me2), H3 trimethylation (me3) at K27 (H3K27me3) and dimethylation (me2) at K36 (H3K36me2). Consistently, a significant reduction of lysine-specific demethylase KDM2A could be found after eight weeks of TAC at the Atp2a2 promoter. Moreover, opposite changes in the recruitment of DNA methylation machinery components  were found at the Atp2a2 or Myh7 promoters after TAC. Taken together, these results suggest that epigenetic modifications may underlie gene expression reprogramming in the adult murine heart under conditions of pressure overload, and might be involved in the progression of the normal heart towards HF.Re-induction of fetal genes and/or re-expression of postnatal genes represent hallmarks of pathological cardiac remodeling, and are considered important in the progression of the normal heart towards heart failure (HF). Whether epigenetic modifications are involved in these processes is currently under investigation. Here we hypothesized that histone chromatin modifications may underlie changes in the gene expression program during pressure overload-induced HF. We evaluated chromatin marks at the promoter regions of the sarcoplasmic reticulum Ca Heart failure (HF) represents an end-stage phenotype of a number of cardiovascular diseases, frequently anticipated by cardiac hypertrophy, the earliest and general response of the heart to injury or increased workload 2+ ATPase (SERCA-2A) In response to cardiac overload, changes in the gene transcription program are required to obtain de novo synthesis of contractile, structural or regulatory proteins The impact of epigenetic mechanisms, i.e. heritable changes in gene expression without alterations in the gene sequence, is now emerging as a major player in cardiovascular pathophysiology The presence and extent of DNA or histone methylation have been also recently addressed in murine cardiac hypertrophy In the present study, we hypothesized that epigenetic modifications may underlie transient and/or stable changes of the gene expression program occurring in HF induced by pressure overload. To test this possibility, we used a well-established mouse model of cardiac hypertrophy and HF induced by transverse aortic constriction, and analyzed chromatin marks at the Atp2a2 and Myh7 promoters after one or eight weeks of pressure overload.All experiments involving animals were conform to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health , to the guidelines from Directive 2010/63/EU of the European Parliament and were approved by the animal welfare regulation of University Federico II of Naples, Italy. Wild-type C57/BL6 mice were purchased from the Jackson Laboratory. Male adult mice  were included in the study and maintained under identical conditions of temperature (21±1°C), humidity (60±5%) and light/dark cycle, and had free access to normal mouse chow.Pressure overload was induced in wild-type C57BL/6 mice  by transverse aortic constriction (TAC) as previously described Cardiac function was non-invasively monitored by transthoracic echocardiography using the Vevo 770 high resolution imaging system  before the surgery and right before termination, 1w and 8w after surgery. Briefly, the mice were anesthetized with an intraperitoneal injection of 0.1 ml/kg of mixture of 50% Tiletamine and 50% Zolazepam (Zoletil 100) and Xylazine 5 mg/Kg (Sigma-Aldrich), and echocardiograms were performed with a 30-MHz RMV-707B scanning head .5′-CGGAAACTGAAAACGGAAAG-3′, reverse 5′-TCCTCGATCTTGTCGAACTTG-3′; SERCA-2A: forward 5′-TCGACCAGTCAATTCTTACAGG-3′, reverse 5′-CAGGGACAGGGTCAGTATGC-3′; GAPDH: forward 5′-TGCAGTGGCAAAGTGGAGATT-3′, reverse 5′- TCGCTCCTGGAAGATGGTGAT-3′. The initial denaturation phase was 5 min at 95°C followed by an amplification phase as detailed below: denaturation at 95°C for 10 s; annealing at 60°C for 30 s; elongation at 72°C for 30 s; detection at 72°C for 40 cycles.Total RNA was prepared using TRIzol , according to the manufacturer's instruction. Oligo-dT first strand cDNA were synthesized using the SuperScript VILO cDNA Synthesis Kit  according to the manufacturer's instructions. mRNA expression was determined in cardiac samples from different experimental groups by real-time quantitative PCR (RT-PCR) using a IQ-5 Multicolor Real-Time PCR Detection System (BIORAD). The primers used were: β-MHC: forward 5′-AGCCAAGGACACCAGTGC-3′ (position from −547 nucleotides to −530) and reverse 5′-GGGATAGAGCGCGGAGTT-3′ (position from −422 nucleotides to −405) amplifying a 143 bp fragment. For amplification of the Myh7 promoter fragment the following primers were used: forward 5′-ACGACCTCCGGATCTGAGT-3′ (position from −75 nucleotides to −57) and reverse 5′-GCGCGCGCTCTTATATAGTT-3′ (position from −34 nucleotides to −15) amplifying a 61 bp fragment. The quantitative PCR conditions were: 95°C for 10 min followed by 40 cycles of 95°C for 15 s, 62°C for 1 min. All PCR signals from immunoprecipitated DNA were normalized to PCR signals from non-immunoprecipitated input DNA. Non-immune Serum or IgG (Mock) was the negative control for the non-specific pull-down of DNA during immunoprecipitation, and provided a measurement of the background or noise for the ChIP system. Results are expressed as percentage (%) of the input with the method described previously Protein bound to DNA was cross-linked by treating tissues of mouse heart with 1% formaldehyde at room temperature, stopping the reaction 10 min later by the addition of 2.5M glycine to a final concentration of 125 mM, followed by 5 min incubation at room temperature. The ChIP assays were performed using the EpiQuik Tissue Chromatin Immunoprecipitation Kit from Epigentek Group Inc. . Antibodies used for Protein-DNA immunoprecipitation were: anti-FBXL11 , anti-dimethyl- H3K36 , anti di-methyl-H3K9 , anti tri-methyl-H3K27 , anti-MeCp2 , anti-DNMT1 and anti-DNMT3b , and normal mouse IgG as a negative control antibody. DNA from these samples was subjected to quantitative PCR analyses, using Power SYBR Green PCR Master Mix  in a Chromo4 Real Time thermocycler . Amplification of the Atp2a2 promoter fragment was performed using the primers: forward Data are expressed as mean ± standard deviation (SD). Comparisons between 2 groups were performed using the unpaired Student t test. Multiple comparisons were made by 2-way analysis of variance (ANOVA) or by 1-way ANOVA with Bonferroni correction and Dunnett's post-test. For all analyses, a minimum value of p<0.05 was considered significant; when present, a p value<0.01 was specified. All the analyses were performed with GraphPad Prism version 5.01 .To investigate the mechanism(s) involved in left ventricular (LV) gene expression re-programming during cardiac hypertrophy and failure, we analyzed a well-known murine model of cardiac pressure overload induced by transverse aortic constriction (TAC) To further investigate whether epigenetic mechanisms may mark gene expression reprogramming occurring in the progression of the normal heart towards HF, we analyzed several different chromatin marks associated with transcriptional activation or repression at the Atp2a2 and Myh7 gene promoters. First, we analyzed the well-established mark of active state, H3 di-methylation (me2) at lysine (K) 4 (H3K4me2) at Atp2a2 and Myh7 gene promoters in SHAM or TAC mice. Chromatin immunoprecipitation analysis (ChIP) demonstrated in both 1w and 8w TAC hearts a remarkable decrease of this mark at the Atp2a2 gene promoter, while a significant increase of H3K4me2 at the Myh7 promoter was observed in 8w TAC . ConversThe epigenetic dynamics at the Atp2a2 and Myh7 gene promoters were analyzed in further detail, by testing a potential recruitment of epigenetic modifying factors. The first analysis was performed on the histone lysine-specific demethylase 2A (KDM2A), previously shown to be responsible for the maintenance of the unmethylated state of dense CpG islands In the present study we demonstrate that transcriptional reprogramming of Atp2a2 and Myh7 genes in pressure overload-induced cardiac hypertrophy and failure is associated with significant epigenetic changes, modifying chromatin dynamics at the promoter regions of these crucial genes. These results suggest that specific chromatin conformational changes might be crucially involved in the progression of the normal heart towards heart failure.Cardiac hypertrophy requires gene expression reprogramming to promote de novo synthesis of contractile and structural proteins, and usually promotes a “fetal” gene expression program. Since the fetal gene program has been consistently linked to adverse cardiac remodeling, both in humans and in mouse models Several epigenetic modifications, including acetylation, methylation, phosphorylation, ubiquitination, and sumoylation of histone proteins have been shown to modify chromosomal structure and DNA accessibility In recent years, histone acetylation has been largely investigated in different experimental models of cardiac hypertrophy and failure Interestingly, distinct genome-wide patterns of H3K36me3 enrichment have been identified in end-stage human HF or normal hearts Here we show that expression reprogramming of the Atp2a2 and Myh7 genes in adult murine failing hearts is associated with remarkable, opposite epigenetic modifications at their promoter regions. Significant changes of specific chromatin marks, including H3K4me2, H3K9me2, and H3K27me3 were identified at both genes promoter regions. All the observed chromatin changes were consistent with Atp2a2 gene repression and Myh7 reactivation. In particular, H3K27me3 may also favor the recruitment of DNA methyltranferases and may associate with more stable changes in the expression program Atp2a2 promoter is embedded in a very dense CpG island, and silencing of SERCA-2A in murine HF is associated with significant loss of KDM2A and increase of K36me2 levels. We hypothesize that this chromatin switch at a CpG rich promoter, together with the increase of K27me3 levels and the recruitment of DNA methylation machinery components  may represent a “high risk” epigenetic state predisposing to detrimental DNA hypermethylation of Atp2a2 gene and consequent stable repression. In fact, hypermethylation of several genes has been observed in end-stage human HF per se, and change the epigenetic status in comparison to the living heart. Indeed, it is now widely recognized the epigenetic reprogramming could be even initiated and perturbed by luminous stimuli While other groups performed their analysis in cardiomyocytes isolated from TAC hearts In conclusion, this study demonstrates that specific epigenetic modifications underlie gene expression reprogramming of Atp2a2 and Myh7 gene promoters in the adult murine left ventricles under conditions of chronic pressure overload, and might be involved in the progression of the normal heart towards HF. Further investigation will be necessary to establish whether the observed chromatin switch in HF, loss of KDM2A and enrichment of H3K36me2 at CpG islands may represent a general mechanism operating in the transition of gene promoters from an unmethylated to a methylated state. In this case, the proposed mechanism would be critical to identify novel potential targets and new emerging epigenetic drugs in the therapy of HF.File S1Table S1 & Figure S1. Table S1. Morphometric and echocardiographic evaluation of mouse hearts from different experimental groups. Figure S1. β-MHC and SERCA-2A mRNA levels in failing murine hearts β-MHC (A) and SERCA-2A (B) mRNA levels in murine hearts after 1 week or 8 weeks of transverse aortic constriction (*p<0.05 vs. SHAM).(PDF)Click here for additional data file.


Class II gene transcription commences with the assembly of the Preinitiation Complex (PIC) from a plethora of proteins and protein assemblies in the nucleus, including the General Transcription Factors (GTFs), RNA polymerase II (RNA pol II), co-activators, co-repressors, and more. TFIID, a megadalton-sized multiprotein complex comprising 20 subunits, is among the first GTFs to bind the core promoter. TFIID assists in nucleating PIC formation, completed by binding of further factors in a highly regulated stepwise fashion. Recent results indicate that TFIID itself is built from distinct preformed submodules, which reside in the nucleus but also in the cytosol of cells. Here, we highlight recent insights in transcription factor assembly and the regulation of transcription preinitiation. The Preinitiation Complex PIC •Architectural models of human and yeast PIC were proposed.•Mediator core–ITC complex structure reveals novel interactions.•TFIID submodule residing in the cytoplasm has been discovered.•Complex assembly emerges as key concept in transcription regulation. Class II gene transcription is a tightly regulated, essential process controlled by a highly complex multicomponent machinery. A plethora of proteins, more than a hundred in humans, are organized in often very large multiprotein assemblies including General Transcription Factors , RNA polymerase (RNA pol II), and a large number of diverse complexes that act as co-activators, co-repressors, chromatin modifiers and remodelers . Class ITranscription of RNA pol II-dependent genes is triggered by the regulated assembly of the Preinitiation Complex (PIC). PIC formation commences with the binding of TFIID to the core promoter. TFIID contains the TATA-box binding protein (TBP). Binding of TFIID to the core-promoter is followed by the recruitment of further GTFs and RNA pol II. Several lines of evidence suggest that this process occurs in a defined, stepwise order and undergoes significant restructuring Recent landmark studies on human and yeast PIC formation provided more differentiated views of the first steps in the transcription initiation process, corroborating the concept of stepwise assembly while also hinting at significant differences that may be present between the species Notably, the described studies above used TBP instead of holo–TFIID. TBP has been shown to suffice for basal transcription, whereas holo–TFIID is required for activated transcription In vivo experiments showed reduced mRNA synthesis when the observed Mediator–TFIIB–RNA pol II interactions were perturbed, confirming the importance of the identified interfaces Recently, single particle cryo-electron microscopy (cryo-EM) and crosslinking mass spectrometry (CLMS) studies of a yeast ITC bound to a Mediator core complex revealed important first insights into transcription initiation and PIC assembly, suggesting that Mediator is involved in stabilizing PIC and in activating RNA pol II A key component in transcription initiation is GTF TFIID, a large megadalton-sized multiprotein complex with around 20 subunits made up of 14 different polypeptides: TBP and the TBP-associated factors (TAFs) (numbered 1–13)  [25]. AnTFIID was shown to adopt an asymmetric, horse-shoe shape with three almost equal-sized lobes , exhibiting a considerable degree of conformational flexibility with at least two distinct conformations (open and closed) Drosophila nuclei Further support for the remarkable structural flexibility of TFIID comes from recent studies of a physiological TFIID core complex. Core-TFIID consists of a subset of TAFs, TAF4, TAF5, TAF6, TAF9, and TAF12, and was first identified in in vivo. Evidence suggests that TFIID formation is a unique and stepwise process, not just a random accretion of different subunits in vivo, and their unique roles in transcription regulation have been postulated Not much is known to date about the mechanisms of TFIID assembly in vitro and in vivoA recent study provided a further piece to this puzzle by reporting the discovery of a novel TFIID submodule formed by TAF2, TAF8, and TAF10 in the cytoplasm of human cells  [45]. ThThe existence of stable TFIID subcomplexes in the nucleus  and in the cytosol (e.g. TAF2–8–10) supports the concept that preformed TFIID submodules exist, which then combine into the holo-complex, regulated by cellular processes such as nuclear import. It is tempting to speculate that these subassemblies may have holo–TFIID-independent physiological functions, in transcription regulation but possibly also in other unrelated cellular processes. Further study is required to scrutinize and elucidate these exciting possibilities.Substantial research effort has been dedicated to uncover the molecular structure of TFIID, and much has been learned from the outcomes to date. X-ray crystallography has provided atomic structures of TFIID components, mostly conserved domain(s) in isolation or small complexes with other TAF domains, notably HFDs . A consiIt is noteworthy that the TAF1 fragment used in the TAF1–TAF7 studies encompasses a region that was previously proposed to confer histone acetyltransferase (HAT) activity in vivo.Recently, the structure of TFIIB-related factor 2 (Brf2) in complex with TBP and different natural promoters was determined, providing a detailed view of key interactions in RNA pol III transcription initiation  [67]. ThUnraveling the molecular mechanisms of transcription regulation has fascinated generations of researchers and this trend is unbroken. Advances notably in single particle cryo-EM technology are unlocking molecular structures of multiprotein transcription factors that are forming ever larger and more complex architectures. Crucial new insight about the supramolecular organization of key players, their structural plasticity and unexpected and pronounced dynamic rearrangements are emerging. Classical views of complexes existing as clearly defined, discrete, and uniform entities are challenged by the discovery of multiple isoforms and partial but physiological complexes. Complex assembly from preformed submodules is emerging as a key concept in transcription regulation. We have discussed just a few recent highlights in the present contribution. We anticipate many more thereof in the near future.EMBL, the European Commission Framework Programme (FP) 7 ComplexINC project (contract no. 279039), the Agence National de Recherche (ANR) (contract number ANR-13-BSV8-0021) Projet Blanc DiscoverIID, and from the Wellcome Trust (WT106115AIA) through a Senior Investigator Award. M.H. was a Kekulé fellow of the Fonds der Chemischen Industrie . S.T. was supported by the European Commission through the Marie-Curie post-doctoral fellowship progamme.We thank all members of the Berger laboratory for helpful discussions. I.B. acknowledges support from the


Deinococcus radiodurans exhibits an extraordinary resistance to ionizing radiation. Previous studies established that a protein named PprI, which exists only in the Deinococcus-Thermus family, acts as a general switch to orchestrate the expression of a number of DNA damage response (DDR) proteins involved in cellular radio-resistance. Here we show that the regulatory mechanism of PprI depends on its Mn(2+)-dependent protease activity toward DdrO, a transcription factor that suppresses DDR genes’ expression. Recognition sequence-specificity around the PprI cleavage site is essential for DNA damage repair in vivo. PprI and DdrO mediate a novel DNA damage response pathway differing from the classic LexA-mediated SOS response system found in radiation-sensitive bacterium Escherichia coli. This PprI-mediated pathway in D. radiodurans is indispensable for its extreme radio-resistance and therefore its elucidation significantly advances our understanding of the DNA damage repair mechanism in this amazing organism.The extremophilic bacterium  Deinococcus radiodurans is one of the commonly used model organism for studying DNA repair due to its exceptional tolerance to extensive DNA damage inflicted by various DNA-damaging agents such as ionizing radiation (IR), ultraviolet light (UV), chemical mutagens and desiccation [DNA repair is an essential process for cells to maintain their genomic stability. Cells have evolved elaborate systems to recover its DNA from damage caused by hazardous environmental stresses. The Gram-positive bacterium iccation –4.Deinococcus-Thermus family, has been identified as one of the essential proteins for the DNA damage response and repair processes [pprI gene renders the bacteria sensitive to various DNA damage. It has been observed that PprI can bind to the promoter regions of recA and pprA, two genes important for DNA repair [pprI-knockout mutant confirms that PprI regulates the expression of a series of genes, including those involved in the DNA repair process [PprI , a protein that is unique to the rocesses , 6. DisrA repair –10. Comp process , 11. Cry process . Mutants process , indicatet al [Deinococcus deserti. They first detected the interaction between PprI and DdrO using a bacterial two hybrid method and far western method. Then they observed that PprI can cleave DdrO in vitro and when co-expressed in Escherichia coli. The cleavage of DdrO in D. deserti was found to occur after irradiation. While unable to detect the binding of DdrO to the promoter region of DNA damage response (DDR) genes directly, Ludanyi et al used a reporter gene approach to show that DdrO can regulate these genes.DdrO, a putative XRE (xenobiotic response element) family transcription regulator, has been predicted to play a role in DNA damage response as it is one of the few transcription regulators which are up-regulated following radiation damage , 14. Recet al  reportedD. deserti at the time, we came to essentially the same conclusion independently regarding PprI’s cleavage of DdrO in D. radiodurans. In addition to what have been reported by Ludanyi et al, we discovered that this protease activity is Mn(2+) dependent. Furthermore, in this report, the direct binding of DdrO to DDR gene promoter regions were confirmed with gel mobility shift assay. The sequence specificity of the proteolytic reaction was further explored and mutations that can render DdrO uncleavable were identified. Replacing the native DdrO with the uncleavable mutant dramatically sensitized D. radiodurans toward various DNA damage. These results further clarify the mechanism by which DNA damage response were regulated in this amazing organism.Unaware of the results in Unless otherwise stated, standard chemicals were of high purity available from Sigma-Aldrich. Tryptone, yeast extract were purchased from Oxford. Enzymes for molecular biology were from Takara. HiTrap Heparin HP column and Superdex 75 were from GE healthcare.D. radiodurans strains were grown at 30°C in TGY broth  or on TGY plates with 1.5%(w/v) agar powder. E. coli strains, containing DH5α and BL21(DE3), were cultivated in LB broth  or on LB medium with 1.5%(w/v) agar at 37°C. Antibiotics used to select E. coli were kanamycin (50 μg/ml) and ampicillin (50 μg/ml). For D. radiodurans, concentrations of kanamycin, chloramphenicol and streptomycin were 10 μg/ml, 4 μg/ml, 10 μg/ml, respectively.All plasmids and strains were listed in E. coli BL21(DE3) for overexpression of 6xHis-MBP tagged proteins, respectively. The expression strains were grown in LB broth containing 30 μg/ml kanamycin at 37°C to exponential phase. 0.5 mM isopropyl-β-D-thiogalactoside (IPTG) was added when OD600 reached 0.6. Induced expression lasted for 4 hours before the cells were harvested by centrifugation. For purification, cells were washed twice with phosphate saline and re-suspended in lysis buffer , then disrupted using a High Pressure Cell Cracker (JNBio). Cell debris was removed by centrifugation . An AKTA Purifier system was used for purification. The supernatant was loaded onto a Ni-NTA superflow column. The unbound impurity was removed using 3 column volumes of buffer A . The protein was then eluted with buffer B . The collected sample was flown through an amylose column (NEB), eluted by 500 mM NaCl, 20 mM Tris–HCl pH 8.0 and 10 mM maltose. Cleavage of the 6x His-MBP tag with a His-tagged TEV protease was carried out at 15°C overnight. After short centrifugation, the supernatant was purified by Ni-NTA column and MBP column again to discard the 6xHis-MBP tag. NaCl concentration of the sample was lowered to 100 mM by dialysis. Further purification was carried out using a HiTrap Heparin HP column . A gradient elution from 100 mM to 600 mM NaCl was applied. Final samples were analyzed using gel filtration chromatography (Superdex 75) and SDS-PAGE.All the interested gene fragments obtained by PCR amplification (primers listed in pprI or ddrO gene as template (primers listed in Site directed mutagenesis was carried out using the pET28-HMT expression plasmid containing ddrO-R109E in vivo was constructed as previously described [ddrO-R109E gene in pET28-HMT vector and downstream fragment of dr2574 in strain R1 were amplified by appropriate primers (2 treatment. Mutant (named MR109E) was selected on TGY plates with streptomycin (10 μg/ml) and validated by PCR and sequencing  were transformed into the mutant strains MR109E and YR1, respectively. Clones were screened on TGY plates with different antibiotics.The complementary plasmids and strains were shown in 600 = 1.0). A fraction of each culture was saved as control at room temperature while the rest was exposed to 2 kGy radiation at the same temperature. Then all samples were incubated at 30°C. Cells were harvested from equal volume of culture at different time points, and re-suspend and washed twice by PBS prior to lysis by sonication on ice [Wild type R1, YR1, MR109E and CMR109E strains were cultivated in TGY culture to late exponential phase (ODn on ice .Western blot assay was performed as previously described . The pro600 = 0.5). For time course of gamma radiation treatment, sample preparation was carried out as described above. RNA extraction and QRT-PCR were performed as previously described [dr0089, a house-keeping gene, was used as an internal control, because its expression in vivo was not influenced by normal treatments. Seven genes were quantified by QRT-PCR  in 200 mM NaCl, 20 mM Tris-HCl pH 8.0, 0.5 mM EDTA at 4°C for 2 hours. Then the EDTA in protein samples was removed by dialysis in 200 mM NaCl, 20 mM Tris-HCl pH 8.0 for 2 hours.in vitro by incubating 450 μM DdrO with 8 µM PprI at 37°C for 30 minutes unless otherwise specified. The final reaction buffer was 200 mM NaCl, 20 mM Tris-HCl pH 8.0, 15% glycerol, 1 mM DTT, 1 mM MnCl2 or other metal ion at the same concentration. SDS–PAGE was applied to detect the cleavage result.The protease activity of PprI was performed D. radiodurans cells were collected in TGY culture at late exponential phase, and then washed twice by PBS. 1% of formaldehyde was added to crosslink DNA-protein in vivo. After 25 minutes, reactions were quenched by the addition of 0.125 M glycine. Cells were then harvested by centrifugation, washed twice with Tris-buffered-saline (pH 7.5), and re-suspended in lysis buffer . Cells were disrupted, and DNA was sheared by a High Pressure Cell Cracker to an average size of 200 to 700-bp at 4°C. After centrifugation, the supernatant was moved to sterile microcentrifuge tubes in 2ml aliquots. The sample was then incubated with 50μl of EZ-CHIPTM protein G beads (Millipore) and 10mg of normal rabbit IgG as the negative control or DdrO rabbit polyclonal antibody for 4 hours at 4°C with agitation. Magnetic field was applied and the beads were washed by re-suspension in 1 ml each of the cold washing buffers (Millipore). 500 μl of elution buffer with RNaseA was added to each tube containing beads followed by incubation at 65°C for 15 minutes. The eluted samples was collected after centrifugation and incubated at 65°C overnight to reverse the cross-linking of protein-DNA complexes in order to free the DNA. Proteinase K was added to degrade the protein. Then DNA was purified by Gel Extraction Kit (Qiagen). QRT-PCR was applied to analyze the relative abundance of seven chosen DDR genes using immunoprecipitated DNA.ChIP assays were performed as described , 19 withD. radiodurans cells in late exponential phase (OD600 = 1.0) were suspended in 10 mM MgSO4 and irradiated by 137CS-γ ray (Dose rate: 1 kGy/h) at several different dosages . For UV radiation, cells were diluted, plated on TGY plates, and exposed to UV-254nm at different dosages . For H2O2 effect, the diluted cells were treated by H2O2 at different concentration . Cells plated on the TGY plates were incubated at 30°C for 3 days before colony counting.The methods for the survival measurements have been reported previously . Briefly2, 1.6 μM DNA and protein at 0, 6, 8, 12, 16 μM. The mixture was incubated at 30°C before loading onto 12% TB-PAGE and run for 2 hours. The bands were visualized on a Typhoon scanner .Promoter regions of DDR genes were amplified by PCR , in which a high performance liquid chromatography (ABI) with a 75 micrometer inner diameter reverse phase C18 column was on-line coupled with an ion trap mass spectrometer. The mass spectrometric data acquired were used to search the most recent non-redundant protein database with ProtTech’s proprietary software suite.D. deserti [D. deserti PprI structure adopts a zinc metallopeptidase fold [D. radiodurans PprI, residual divalent metal cation was first removed from the purified PprI by EDTA. After a second dialysis to get rid of EDTA, various metal ions were added to the PprI solution 1 hours before the reaction was initiated by the addition of purified DdrO. As shown in 2 alone does not cause any visible degradation of DdrO (D. radiodurans was found to accumulate high concentration of Mn(2+) in the cell [Running the reaction mixture of PprI and DdrO on SDS-PAGE gel shows that two lower molecular weight bands emerge while the DdrO band becomes weaker . Mass sp deserti . The N-tase fold . A zinc  of DdrO .. This ithe cell , which wD. deserti. To test the sequence specificity of PprI cleavage, a series of DdrO mutants were constructed. Mutation of residues Glu-107, Leu-108, Gly-110 or Arg-112 to Ala separately makes these mutants uncleavable to PprI, while the R109A and K111A mutants can still be cut (According to the molecular mass detecting . and C-tl be cut . To testl be cut .. These recA (dr2340) and pprA (dra0346), which are also the binding targets of PprI [in vitro, including ddrB (dr0070), ddrA (dr0423), ssB (dr0099), recQ (dr1289), ddrO (2574) itself and so on  (Using gel mobility shift assay (GMSA), we observed that purified DdrO has the capability to bind to the promoter regions of  of PprI . Furthernd so on . ChIP-PC(in vivo . All the), ddrO 274 itselfhermalis . Our suberlined) . Thus, RddrO knockout is lethal in both D. radiodurans and D. deserti, as efforts to construct such knockout strain has failed in several labs including ours [in vivo function of DdrO, we designed another approach. We constructed a ddrO-site disruptant and designated MR109E in which the entire coding region of the ddrO gene has been replaced by the whole coding mutation of R109E with a streptomycin resistance cassette. Western blotting was then utilized to assay whether the cleavage of DdrO happens in vivo. In the wild type R1, the band corresponding to DdrO disappeared immediately after radiation and recovers after one hour , the levels of DdrO remain unchanged before and after radiation  after incubation for recovery, and purified the RNA to analyze transcription of the chosen DDR genes. QRT-PCR analysis show that the transcription of DDR genes including adiation , while tadiation . DDR genDeinococcus radiodurans is an important model organism valuable for the study of DNA repair. PprI protein has been implicated as a switch for DNA damage response. In this study, besides confirming the recent report that PprI can cleave a putative transcription factor DdrO [ddrO in D. radiodurans by using a mutant strain where the native ddrO is replaced with an uncleavable version. Furthermore, the in vivo cleaved N-terminal of DdrO (N-DdrO) was not observed after radiation as well as the in vitro interaction between N-DdrO and PprI  as its metal co-factor. The unanswered question here is whether Mn(2+) plays any role in the activation of PprI. One can speculate that PprI binds metal ion other than Mn(2+) before stress and thus protease activity was inhibited. When DNA damage occurs, the relative affinity of metal ions change and Mn(2+) displaces other ions at the binding site, thus activating PprI. The change in metal affinity can be caused by protein conformational change brought on by factors such as post translational modification or ligand binding in the GAF-like domain. This is a hypothesis that needs to be further investigated.Given the abundance of Mn(2+) in the Deinococcus radiodurans and the closely related Deinococcus geothermalis, Marakova et. al. identified a common palindromic DNA motif, designated as the Radiation /Desiccation Response Motif [et al confirmed that DdrO represses the RDRM-containing promoter of ddrD in E. coli through promoterless lacZ reporter gene [DdrO is annotated as XRE family transcription regulator protein with a predicted HTH (helix-turn-helix) domain for binding to the target. It was speculated to be a general regulator for DDR, because microarray assays show that it is the only up-regulated putative transcription factor at low gamma dose (3 kGy) . The resse Motif , in a coter gene . In thister gene  are bindEscherichia coli, the SOS response is activated after exposure to ultraviolet, ionizing radiation or chemical mutagens [Deinococcus radiodurans [in vitro. The truncated DdrO then dissociates from its DNA target. This proteolytic reaction also happens in vivo after ionic radiation. In the mutant where PprI was absent, this cleavage does not take place, further confirms that PprI is the enzyme that cuts DdrO. Mutation that renders DdrO uncleavable to PprI sensitizes the bacterium towards ionic radiation, UV and peroxide. Microarray and proteomics data reported by several groups including us all show that the seven genes under DdrO control are up-regulated after irradiation [Deinococcus-Thermus phylum. Based on these findings, we propose that the PprI and DdrO are the key players in this novel DNA damage response pathway.In bacteria such as mutagens –26. Howeiodurans , 28. Altiodurans , 28. In adiation , 11, 14.To summarize the proposed model : Throughin vitro. The mechanism by which PprI is activated is still not clear. The reason that ssDNA-RecA filament is not used for signaling may be related to the observation that in D. radiodurans RecA binds preferentially to dsDNA over ssDNA therefore the ssDNA-RecA filament is not readily formed after DNA damage [While the pathway we described here shares some similarities to the classic SOS response, there are some important differences. The protease activity is shifted to a new player, PprI, from the transcription repressor itself. This affords more control points to the process. In the SOS pathway, the signal that triggers the response is the single strand DNA , 30. It A damage –33. So tS1 Table(DOC)Click here for additional data file.S2 Table(DOC)Click here for additional data file.S1 Protocol(DOC)Click here for additional data file.S1 FigddrO gene in mutant MR109E was checked by sequencing and sequence alignment.(A) Schematic representation of the uncleavable mutant MR109E. AF1, AR1, AF2 and AR2 refers to primers, respectively. (B) The uncleavable mutant MR109E was confirmed by genomic PCR using AF1 and AR1. (C) The direct site-mutation of (TIF)Click here for additional data file.S2 FigMolecular weight of the cleavage product was checked using SELDI-TOF-MS. The peak (15194.3+H) is the entire DdrO protein left after the cleavage, and the other peaks  are the two cleaved fragments of DdrO, respectively.(TIF)Click here for additional data file.S3 Fig(A) PprI protease activity does not change after incubating with metal ions for longer time. After incubation with 5 mM of metal ions for 2 hours, 8 μM of PprI was added and mixed with 450 μM of DdrO at 37°C for 1hour. (B) More DdrO was cleaved by increasing amount of PprI .(TIF)Click here for additional data file.S4 FigConsensus amino acids and similar amino acids are shown in black and gray backgrounds, respectively. And the protease cleavage sequence is underlined.(TIF)Click here for additional data file.S5 FigrecA and ddrO. The concentrations of promoters and DdrO are 1.6 μM, and 10 μM, respectively. With the increasing PprI concentration , the bands of DNA-protein complex were gradually weakened.GMSA was applied to detect PprI effect on the binding of DdrO to the promoter regions of (TIF)Click here for additional data file.S6 FigrecA promoter region was analyzed by GMSA. The concentration of DNA is 1.6 μM and the DdrO concentrations are 0, 6, 8, 12μM, respectively. (B) GMSA for uncleavable mutant performed in different binding buffers(Mg(2+) or Mn(2+)) show no noticeable difference. The concentrations of DNA and DdrO are 0.8 and 5μM, respectively.(A) The binding of the uncleavable mutant (DdrO-R109E) to the (TIF)Click here for additional data file.S7 Fig1, control blank of PprI; 2, control blank of N-DdrO; 3, the supernatant of the incubated mixture after short centrifugation; 4, the washing buffer of the agarose beads; 5, the eluted sample.(TIF)Click here for additional data file.S8 FigDeinococcus radiodurans RecA (Dr-RecA) and Escherichia coli RecA (Ec-RecA), even with the addition of ATP, single strand DNA (ssDNA), and double strand DNA (dsDNA). (A) Effect of EC-RecA on DdrO. (B) Effect of Dr-RecA on DdrO. CK1, a blank control of the boiled proteins before reaction; CK2, the reaction of RecA and DdrO; dsDNA, the reaction of RecA and DdrO with dsDNA and ATP; ssDNA, the reaction of RecA and DdrO with ssDNA and ATP. The reactions were incubated at 37°C for 1hour, then analyzed by SDS-PAGE.DdrO cleavage could not be activated by (TIF)Click here for additional data file.


Here we review recent findings and offer a perspective on how the major variant RNA polymerase of bacteria, which contains the sigma54 factor, functions for regulated gene expression. We consider what gaps exist in our understanding of its genetic, biochemical and biophysical functioning and how they might be addressed. O). Closed promoter complexes (RPC) rarely spontaneously isomerised to open complexes, and so sigma54 dependent systems showed a typical major dependence on cognate activators (reviewed in [rpoN gene or genes (some organisms have two rpoNs) encoding sigma54, although informatics suggests sigma54 use is rooted in the control of cell envelope functions in response to stress [Along with the recognition that bacterial RNA polymerases were heterogeneous with respect to their sigma factor content, came the finding that two classes of sigma factor existed in many different types of bacteria . Unlike iewed in ,3). No ho stress ,7,8,9, trpoN, also called ntrA) was a dissociable sigma factor which directed RNA polymerase as a holoenzyme to the −12, −24 promoter sequences from which it transcribed [rpoN encoded genes was to provide clear indications that the sigma54 protein was unrelated to the major sigma70 class of sigma factors at the level of primary sequence and most likely its fold and tertiary structure. For the most part this lack of relatedness has been upheld by what limited structural data sets for fragments of sigma54 have been obtained to date. The lack of relatedness suggests that sigma54 may direct a different set of structural transitions in taking RPc to RPo to that orchestrated by sigma70 type factors, rather than just changing the kinetics of the same pathway of conformational change.Work from Sydney Kustu’s lab provided the first biochemical evidence that the sigma54 factor  of sigma54 have not been obtained, in contrast to sigma70, and so quite how sigma54 contributes to opening DNA from −10 to −1 is unknown. However mutants in sigma54 able to allow RPO formation in vitro without activation suggest the barrier to RPO formation is distinct from determinants that allow formation and maintenance of (at least unstable) forms of RPO. Although not widely surveyed across promoters, it seems that the sigma54 holoenzyme forms fewer abortive RNA products before elongation than does the sigma70 containing enzyme [Apparently sigma54 RNAP finds promoters by a direct binding route and not sliding, at least in vitro . At its  DNA RPC  is now ag enzyme , and so C, recognition of the −24 promoter sequence seems relatively well understood, and involves a HTH motif and the RpoN box amino acid sequence, as solved by the Wemmer group by NMR [In contrast to a complex recognition of the promoter −12 intimately linked to maintaining RPp by NMR . In contp by NMR .C to RPO. Cryo-EM reconstitutions carried out by the Zhang lab revealed up to three sigma54 activators within a hexamer could directly contact the RPC structure for isomerisation [I) when the ADP-AlFx hydrolysis analogue was added. The DNA melting site was misaligned with the DNA loading site in the RPI in the proposed model, which could constitute an ancillary inhibitory mechanism. The Stockley and Tuma labs further addressed sigma54 domain movements in relation to promoter DNA and ATP hydrolysis by smFRET analysis  belonging to the AAA+ protein family assemble into hexamers and fuel the rearrangement of RPrisation . These crisation , and werrisation . A bridgO requires a repositioning of the sigma54 Region I, which occurs through the use of at least two GAFTGA loops of the activator ATPases that bind there. Whether the activator binds a rare state of the holoenzyme in RPC, or drives the formation of an entirely new functional state is not yet clear. However, the R336A sigma54 variant phenocopies the action of the binding of the activator to RPC in that the Region I has moved compared to that in RPC, and so the barrier to passing from RPC to at least an RPI if not RPO is not so large. Release of the activators from the sigma54 Region I may be key in passing to RPO from RPI, as may a contact of the activator with upstream promoter DNA. Kinetic studies on the glnAP2 promoter using COSMO methodologies indicates that the transition from RPC to transcript generation takes around ninety seconds, and activators interact with both RPC and RPO [C exists without the DNA base unstacking at −12 [54 within the RPC. A processive functioning of the activators may not be necessary for activation, because the activator bound RPI has been shown to possess RPO like characteristics [O with sigma54 is not known, and could involve other functions of the activators ATPase rather than a direct sigma DNA melting activity. Notably when double stranded DNA is outside of the RNAP in RPI, extensive cross links can be made with the bound activator, which might guide dsDNA into the holoenzyme for making RPO, or indeed act on it directly for making RPO [C  when promoter DNA from −10 to −1 is unpaired allows transcription initiation, suggesting the holoenzyme can accept ssDNA and then open up the start site to allow RNA synthesis, without the need for ATP hydrolysis by the activator protein [Overall it is clear that activation for making RP and RPO . These sg at −12 . Given tg at −12 ,25. Theieristics . Howeverking RPO . An inteking RPO . Further protein .Genes under control of the sigma54 are required under specific stress conditions or when a particular C or N source is available. Classical examples include the NtrC dependent activation of genes in response to nitrogen limitation, the use of XylR in expressing genes for catabolism of aromatic hydrocarbons and PspF in activating the psp response for inner membrane stress. Although all contain the critical AAA+ domain needed for remodelling the closed promoter complex, the activators are all signaled to via a range of N-terminal regulatory domains. These fall into a wide range of different classes ranging from the response regulator type aspartic acid phosphorylation target in two component members, to GAF domains in NifA and CARF domains in RtcRs (reviewed in ). In manC, RPI and RPO are now needed to work out quite what it is in structural terms that establishes the barrier to making RPO, and how RPC and RPO interconvert. Comparisons to the use of the sigma70 Region I as a place keeper for DNA and that of sigma54 Region I as a target for activation, maintaining the repressive fork junction structure and escaping the action of certain phage inhibitors of RNAP activity such as T7 phage gp2 will be one valuable outcome from such studies. Similarly, knowing what the functional state the core RNAP is in when bound to sigma54 will provide insights into the importance of the sigma-core interface in gene control and potentially antibiotic action. For example in the presence of sigma54 is the core enzyme in a catalytically competent state, and can smFRET data be reconciled with RNAP clamp opening and clamp closing, and the processive closed state of the core enzyme? Do the sigma54 and its activators take the RNAP down a new set of conformationally distinct changes for making RPO, or is it simply altering the kinetics of a single pathway for making the RPO from the RPC?Detailed structural insights into RPCs seem not to be served by cognate activators, and amongst these a subset seem to repress transcription by other forms of RNAP holoenzyme. In combination with genomic approaches to better understand roles of sigma54 binding sites on bacterial chromosomes, sufficient knowledge of sigma 54 and sigma70 structure-function relationships will allow us to make orthogonal non-native chimeric sigma factors-to tackle issues of promoter use in synthetic biology and an increased insularity from the bacterial chassis used in such work. Since sigma54 is served by cognate activators of the AAA+ protein family, detailed atomic structures of their co-complexes with RPC will help work out how such AAA+ proteins remodel their targets. Lastly, the full role of sigma54 in bacterial physiology is unknown. Many of its activators respond to signals which are poorly characterised, yet underpin important processes such as RNA repair (the rtcBA genes activated by RtcR) and envelope maintenance (the psp operon activated by PspF). As details of how genes turn on and off in individual cells through transient changes in, for example, local DNA superhelicity to yield the so called bursty behaviours of gene transcription revealed by time series studies, one might wonder if the sigma54 factor with its coupled ATPase overlays some special features to such time series.Quite where sigma54 came from remains a mystery-it cannot be rooted and seems to have no obvious structural counterpart . Genomics methods and RNAseq studies will now no doubt offer us perspectives on why have sigma54 at all-is it a relic of a transcription repression mechanism, can it (or has it?) evolve in some cases to activator independence, and does it have repressive functionality alongside its gene activation responsiveness? Currently ChipSeq and RNAseq work with sigma54 and its holoenzyme to define its regulon suggests complexity in the roles of sigma54 ,29. For


Lineage potential is triggered by lineage-specific transcription factors in association with changes in the chromatin structure. Histone H3.3 variant is thought to play an important role in the regulation of lineage-specific genes. To elucidate the function of H3.3 in myogenic differentiation, we forced the expression of GFP-H3.1 to alter the balance between H3.1 and H3.3 in mouse C2C12 cells that could be differentiated into myotubes. GFP-H3.1 replaced H3.3 in the regulatory regions of skeletal muscle (SKM) genes and induced a decrease of H3K4 trimethylation (H3K4me3) and increase of H3K27 trimethylation (H3K27me3). Similar results were obtained by H3.3 knockdown. In contrast, MyoD-dependent H3.3 incorporation into SKM genes in fibroblasts induced an increase of H3K4me3 and H3K27me3. In mouse embryos, a bivalent modification of H3K4me3 and H3K27me3 was formed on H3.3-incorporated SKM genes before embryonic skeletal muscle differentiation. These results suggest that lineage potential is established through a selective incorporation of specific H3 variants that governs the balance of histone modifications. The development of multicellular organisms is accompanied by the acquisition of various differentiated cells. Cells acquire lineage potential toward specific directions during cell fate decision, and the lineage potential can be established by marking genes prior to their expression after differentiation. The expression of selected genes during differentiation is regulated by the structure of chromatin, which includes nucleosomes. Post-translational modifications of histones are regarded as signals for the compaction of chromatin and other protein complexes, acting as ‘on/off’ switches for the gene expression . One exaXenopus  are associated with human pediatric glioblastoma  incorporated into chromatin during replication –10. The blastoma  and are blastoma . In ES cblastoma . These rIn myogenesis, both the selection of the histone variant and the appropriate modification play a critical role in selective gene expression. We previously reported that H3.3 is incorporated into skeletal muscle gene (SKM gene) loci prior to the gene expression . BivalenAll animal procedures were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee (IACUC) at Kyushu University.WT and Chd2KD cells generated previously (C2C12 cells or Chd2eviously  were groeviously .Hist1h3a and H3f3a cDNA (purchased from Operon Biotechnologies) were used for the expression of H3.1 and H3.3. The cDNAs were ligated into the Bidirectional Tet Expression Vector pT2A-TRETIBI (modified Clontech Tet-On system), which contains TolII transposon elements and Enhanced Green Fluorescence Protein (EGFP) cDNA located upstream of the cDNA sequence, which was modified from pT2AL200R150G  was performed as described  using a Cells were plated on cover slips, washed twice with phosphate buffered saline (PBS), fixed with 1% paraformaldehyde in PBS, permeabilized with 0.5% Triton X-100 in PBS and washed twice with PBS. A 15 min incubation with Blocking One  was followed by 2 h incubation with mouse anti-myogenin , centrifuged, and then resuspended in 2x sodium dodecylsulphate (SDS) sample buffer. The samples were separated by SDS-PAGE (10–20% gel for histone variants and 5–20% gel for other proteins) and transferred to a nitrocellulose membrane or polyvinylidene fluoride membrane with Trans-Blot Turbo Transfer System . The membrane was blocked for 1 h in 5% (w/v) skim milk in Tris-buffered saline containing 0.05% (v/v) Tween 20 (TBST), then incubated with primary antibodies in Hikari Solution A  followed by incubation with horseradish peroxidase-labeled secondary antibodies and detection using the Chemi-Lumi One Ultra . The primary antibodies used included rabbit anti-Hsp90 , anti-H2B  and an oligo dT primer, as previously described . qPCR wa7 cells were fixed with 0.5% formaldehyde.Embryonic tissues were collected as described previously . For ChI7 cells) were cross-linked in 0.5% formaldehyde and suspended in ChIP buffer . The samples were incubated for 15 min on ice, sonicated for 5 s three times at 70% maximum amplitude with a Sonicator  and digested with micrococcal nuclease  and Ribonuclease A  at 37°C for 40 min. The digested samples were centrifuged at 15 000 × g for 10 min. Supernatant containing 4–8 μg DNA was incubated with a rat monoclonal antibody against H3.3   , H3K27meSample preparation from GFP-fused histone H3.1 and H3.3 expressing cells and C2C12 wild-type (WT) cells or embryonic tissues was performed as described above. The ChIP library was prepared with the Illumina protocol and sequenced on Illumina HiSeq-1500 sequencers. The sequence reads for H3.3, H3K4me3, H3K27me3, GFP and Input were aligned to the reference mouse genome  using Bowtie 2 software . Unique SI) are defined as follows:To analyze the data from ChIP-seq, we counted the number of ChIP-seq unique tags in each window (for each figure) and normalized as reads per kb per million . Signal intensities . Each Z-score was computed for each gene with GFP-incorporation intensity signals. Histone modification densities were calculated in windows around ± 2 kb a TSS. These data are listed in Supplementary Tables S4 and S5.To estimate the effect of the observed histone modification state affected by the expression of the histone H3 variant, we used a SI  was calculated in 2 kb windows using 1 kb intervals along the mouse genome, i.e. SIs with 50% overlapping 1 kb windows. Forelimb- and hindlimb-specific genes were selected from a previous report , s report .SIs of both H3K4me3 and H3K27me3 were calculated in ± 2 kb windows within a TSS of each tissue-specific gene. φ ranges from 0 to 180° (0° corresponds to only H3K4me3 and 180° corresponds to only H3K27me3). To assume a negative SI as no-signal, we filtered SI using the function +(x) = x when x has positive value or +(x) = 0 when x has negative value. In the case where SI of H3K4me3 has no signal and the SI of H3K27me3 is positive, i.e. +{(SI)H3K4me3} = 0 and +{(SI)H3K27me3} > 0, then φ = 180°. The condition of both SIs of H3K4me3 and H3K27me3 having no-signal was excluded from our analysis.For bivalent modification analysis, the relative ratio of H3K4me3 and H3K27me3 histone modifications was expressed as the angle,http://bioinf.xmu.edu.cn:8080/databases/TiSGeD/index.html) (Tissue-specific genes were selected from the tissue-specific genes database (ex.html) . SKM genex.html) . These dTotal RNA at the growth and differentiated (i.e. post-differentiation) states for C2C12 cells were obtained as described above. Library preparation and sequence analysis were performed by a protocol described previously .Immunoprecipitation (IP) was performed as described . Cleared3COOK, 30 mM KCl, 10 mM Na2HPO4, 1 mM DTT, 1 mM MgCl2, 1 mM ATP, 0.1% Triton X-100 and protease inhibitor cocktail; Nacalai Tesque Inc.). Lysates were incubated for 10 min on ice and then centrifuged at 1000 × g for 5 min. The pellets were resuspended in IP buffer . The samples were sonicated for 1 s three times and digested to lengths corresponding to mono-, di- and tri-nucleosomes with micrococcal nuclease  and Ribonuclease A  at 37°C for 30 min. To stop the digestion reaction, ethylenediaminetetraacetic acid (EDTA) was added to the samples at a final concentration of 5 mM. 5M NaCl was then added to achieve a concentration of 0.5 M and the samples were transferred to a spin column including hydroxyapatite resin  pre-washed with hydroxyapatite buffer 1 . The chromatin/hydroxyapatite mixture was incubated on a rotator for 30 min at 4°C and washed twice with HAP1B and hydroxyapatite buffer 2 . The purified nucleosomes were eluted with hydroxyapatite (HAP) elution buffer . The nucleosomes were diluted 10 fold with IP buffer and rocked with 4 μg of rat monoclonal antibody against GFP  or mouse monoclonal antibody against H3K4me3  were purchased from Dharmacon: Hira (ON-TARGETplus SMARTpool #L-043074), Daxx (ON-TARGETplus SMARTpool #L-044803), H3f3a (ON-TARGETplus SMARTpool #L-042679), H3f3b (ON-TARGETplus SMARTpool #L-042835) and non-targeting (ON-TARGETplus SMARTpool #D-001810).Knockdown of eviously . siRNA ft-test.Statistical significance in RT-qPCR and ChIP-qPCR data were evaluated using the Welch We previously showed that H3.3 is incorporated into myogenic genes prior to differentiation . To addr(Dox-) or presence of Dox (Dox+) for two days  were lower in cells expressing GFP-H3.1 compared to Dox- cells after differentiation was induced . These data indicate that the forced expression of H3.1 had an inhibitory effect on myogenic potential.The myogenic potential in GFP-H3.1 expressing cells were evaluated by immunostaining with myogenic markers, myogenin (Myog) and myosin heavy chain MHC), after the induction of differentiation in the absence s Figure . Myog was Figure  and C. M, after ts Figure . Furtherd Figure . In contd Figure -C. The pd Figure  and the d Figure . The expSince H3.3 is incorporated into the promoter regions of SKM genes before differentiation , we consTo investigate whether GFP-H3.1 is incorporated into nucleosomes surrounding those containing H3.3, we performed ChIP-western analysis. Chromatin fractions prepared from GFP-H3.1 were treated with micrococcal nuclease to release mono- to tri-nucleosomes, which were then purified with HAP . IP of HAP-purified nucleosomes (HAP-IP) with anti-GFP antibodies was then performed to determine which H3 variants were present in nucleosomes containing GFP-H3.1. Western blotting using specific antibodies directed against H3.1 (which reacts with H3.1 and H3.2) and H3.3 showed that H3.1/H3.2 and H3.3 were associated with GFP-H3.1, whereas H3.3 was preferentially associated with GFP-H3.3 Figure . These dZ-score). In GFP-H3.1 expressing cells, SKM genes showed higher H3K27me3 levels than the average of all genes (Z = 0), while in WT cells, the H3K27me3 level was lower . Nucleosomes containing GFP-H3.3 were prepared as controls. Modifications of H3K4me3 and H3K27me3 on H3 were also found by reciprocal HAP-IP using anti-GFP and immunoblotting with modification-specific antibodies . H3K4me3 had similar levels of GFP-H3.1 and GFP-H3.3 while H3K27me3 levels were higher in GFP-H3.1. Furthermore, GFP-H3.1-expressing cells contained nucleosomes that were modified by H3K27me3 preferentially . These data suggest that the selective formation of the bivariant state on SKM gene loci at growth state caused an increase in H3K27me3 and decrease in H3K4me3 levels. As a result, SKM gene loci may have lost their lineage potential and no longer expressed SKM genes during differentiation.Acta1 and Ckm) in GFP-H3.1-expressing cells compared with WT cells, but unchanged in either HK (Eef1a1) or silent (Neurod6) gene loci  and a HK gene (Eef1a1) in GFP-H3.1 expressing cells at growth state  , which can affect the expression of downstream SKM genes, we used H3.3 knockdown for further analysis.To verify the specific role of H3.3 incorporation, we inhibited H3.3 incorporation by knockdown of a histone H3.3 chaperone as well H3.3 itself . Hira has been reported to mediate H3.3 incorporation into chromatin in various cells including C2C12 cells ,40–43. DEef1a1 and Neurod6 did not change significantly. H3.3 was less incorporated in the HK and SKM genes in H3.3 KD cells  were increased during differentiation . In Chd2 KD cells, both H3K4me3 and H3K27me3 levels in SKM genes were lower than in WT cells at growth state and remained unchanged after differentiation. Chd2 KD did not affect the levels of H3K4me3 in the HK gene (Eef1a1) or H3K27me3 in the CBL gene (Neurod6), consistent with locus-specific H3.3 incorporation mediated by Chd2. Similar results were obtained by MyoD KD . Thus, the inhibition of H3.3 incorporation affected both H3K4me3 and H3K27me3, a phenotype more severe than that observed by GFP-H3.1 overexpression, which only enhanced H3K27me3.In H3.3-knockdown (H3.3 KD) cells at growth state, the levels of H3K4me3 and H3K27me3 in SKM genes were significantly decreased compared to control cells Figure . In conts Figure . These rs Figure . It has s Figure . In WT cActa1 and Ckm) under differentiation conditions  or mutant H3.3, in which serine 31 was substituted to alanine (H3.3 S31A). Analysis using HAP-purified nucleosomes revealed that GFP-H3.1 A31S and GFP-H3.3 S31A were incorporated, perhaps by specific chaperones –49, at tin vivo, we performed ChIP-seq for H3.3, H3K4me3 and H3K27me3 using mouse embryonic tissues. We used the trunk at E10.5 (where myogenic genes are committed but not expressed), the hindlimb (myogenic genes are expressed) and liver (myogenic genes are silent) at E14.5  and forelimb  . H3.3 was also present at the TSS of these genes in both tissues. H3K4me3 in hindlimb genes became more abundant in E14.5 hindlimb tissue than that in E10.5 trunk tissue. In contrast, H3K4me3 and H3K27me3 were decreased, albeit slightly, in forelimb-specific genes. In the liver, H3K27me3 was highly observed in both gene groups and little H3.3 and H3K4me3 were detected.Next, we focused on gene sets specifically expressed in the hindlimb or forelimb at E14.5  to assesWe also analyzed the correlation of the bivalent state and H3.3 incorporation. For an overview of the colocalization, every 200 bp window (close to the mono-nucleosome size) over the entire genome was binarized into ‘enriched’ or ‘depleted’ based on the ratio of the ChIP signal to input. If the ChIP/input ratio in a region was >0 or <0, the region was scored as ‘enriched’ or ‘depleted’, respectively. In this analysis, 63, 74 and 70% of the genome in the trunk, hindlimb and liver, respectively, was scored as H3.3 enriched. We then determined whether either or both H3K4me3 and H3K27me3 were also enriched on H3.3-enriched or -depleted regions, finding the bivalent state in H3.3-enriched regions was 47, 60 and 30% in the trunk, hindlimb and liver, respectively, which was substantially higher than values in H3.3-depleted regions  Figure . These rTo examine the relationship between H3.3, H3K4me3 and H3K27me3 modifications in detail, ChIP-seq signals within ± 2 kb of the TSS of each set of genes  were evaluated quantitatively. The H3.3 signal intensity was plotted against the relative ratio of H3K4me3 and H3K27me3 levels in each gene Figure . In the In a previous study, we showed that the histone H3.3 variant is incorporated into SKM genes prior to muscle cell differentiation . Here, tIn bivalent mono-nucleosomes, H3K4me3 and H3K27me3 are present in different H3 tails . The forAmong histone H3.3 chaperones, Hira KD affected the myogenic gene expression, including MyoD. This result is probably due to the global suppression of H3.3 incorporation, consistent with its general role in H3.3 chromatin assembly . In contWe showed that replacement of H3.3 with H3.1 inhibited the expression of SKM genes prior to transcription, but had no effect on HK genes, including no modifications to H3K4me3, H3K27me3 or even transcription. These results suggested that once genes are actively transcribed, the choice of the histone variant around the promoter regions has little effect on the histone modification or transcription state.Previously, H3.3 knockdown in mouse ES cells caused a decrease in H3K27me3 levels but no change in H3K4me3 levels . In contChIP-seq and RNA-seq data were deposited into the DNA Data Bank of Japan database .Supplementary Data are available at NAR Online.


Efficient pre-replication complex (pre-RC) formation on chromatin templates is crucial for the maintenance of genome integrity. However, the regulation of chromatin dynamics during this process has remained elusive. We found that a conserved protein, GRWD1 (glutamate-rich WD40 repeat containing 1), binds to two representative replication origins specifically during G1 phase in a CDC6- and Cdt1-dependent manner, and that depletion of GRWD1 reduces loading of MCM but not CDC6 and Cdt1. Furthermore, chromatin immunoprecipitation coupled with high-throughput sequencing (Seq) revealed significant genome-wide co-localization of GRWD1 with CDC6. We found that GRWD1 has histone-binding activity. To investigate the effect of GRWD1 on chromatin architecture, we used formaldehyde-assisted isolation of regulatory elements (FAIRE)-seq or FAIRE-quantitative PCR analyses, and the results suggest that GRWD1 regulates chromatin openness at specific chromatin locations. Taken together, these findings suggest that GRWD1 may be a novel histone-binding protein that regulates chromatin dynamics and MCM loading at replication origins. DNA replication is of fundamental importance in cells and is strictly regulated to occur precisely once per cell cycle. An essential step in DNA replication is the formation of a pre-replication complex (pre-RC) by the ORC, CDC6, Cdt1 and MCM helicase complexes at a replication origin during the low Cdk period. After activation of the MCM helicase by Cdk, reassembly of the pre-RC is strictly prohibited to prevent rereplication . In humaTheoretically, loading of two MCM complexes at one replicon would be sufficient for replication. However, it has been demonstrated that excess MCM loading is crucial for maintenance of genome integrity. For example, although cells with depleted MCM replicate at normal rates, they are hypersensitive to replicative stress and defective in Rad17-dependent ATR-mediated replication checkpoint activation . MoreoveThe assembly of chromosomal DNA and histones into nucleosomes is the most fundamental step in eukaryotic chromatin structure. The nucleosome structure generally restricts access by various factors that facilitate a variety of DNA-templated processes. Therefore, the deposition, remodeling and eviction of nucleosomes are important for almost all DNA-related operations. In transcription, chromatin-remodeling proteins, histone chaperones and histone-modifying enzymes are thought to synergistically stimulate the reaction. The situation may be similar for efficient pre-RC formation on chromatin. In this regard, it has been reported that HBO1 enhances licensing through its acetylation activity –14. In aWe previously identified GRWD1 (glutamate-rich WD40 repeat containing 1) as a novel Cdt1-binding protein . GRWD1 iPreparation of cross-linked chromatin was carried out essentially as described for ChIP-qPCR. However, MNase treatment  was added after sonication. The ChIP library was prepared with the Illumina protocol and sequencing analysis was performed using the Genome Analyzer GAIIx (Illumina KK). Sequencing for the MCM7 and HA-GRWD1 ChIP analysis was performed using the HiSeq1500 (Illumina KK). The sequence reads were aligned to the human genome (hg19) using Bowtie software . Peak detection and identification of the binding sites were obtained by correcting from Input DNA, using MACS2 broad peak detection. Genomic annotations including transcription start sites (TSSs) and transcription end sites (TESs) were determined using RefSeq. Co-localization analyses of ChIP-Seq data were obtained as described previously ,26.To estimate the ChIP-seq signals, we utilized the moving-average method in which the number of mapped reads was calculated every 0.1 kb interval with 1 kb window size. In addition, read numbers of the ChIP samples were normalized using Reads Per Million (RPM) , and furHeLa cells were cross-linked with 1% formaldehyde for 5 min at room temperature. Glycine was added to a final concentration of 125 mM, and the cells were rinsed with cold PBS and harvested. Cells were then lysed with 2% TX-100, 1% SDS, 100 mM NaCl, 1 mM EDTA and 10 mM Tris-HCl (pH 8.0) containing a protease inhibitor cocktail and sonicated for 18 min (30 s on/60 s off cycles) using a Bioruptor (Diagenode) set at the highest intensity. Samples were centrifuged at 15 000 rpm for 10 min at 4°C to precipitate cellular debris. For input control samples, prior to phenol-chloroform extractions, the soluble chromatin was treated with proteinase K and incubated for 6 h at 65°C to reverse cross-linking. Chromatin samples were then subjected to two consecutive phenol-chloroform extractions. FAIRE chromatin samples recovered in the aqueous layers were then subjected to reversion of cross-linking. Finally, the DNAs were ethanol precipitated. Input and FAIRE DNAs were then analyzed by agarose gel electrophoresis followed by EtBr staining.For FAIRE-Seq, the DNAs were subjected to Seq  and data analysis was performed essentially as described above. Read numbers of the FAIRE samples were normalized by subtracting those of the input samples. FAIRE-qPCR was performed on the input and FAIRE DNAs essentially as described for ChIP-qPCR.GRWD1 is composed of 446 amino acids, including a glutamate-rich acidic domain and a WD40 repeat domain Figure . A similWe raised rabbit anti-human GRWD1 antibodies, which recognized a ∼55 kDa band in immunoblots   and yielvice versa  and MCM4 genes. We performed chromatin immunoprecipitation (ChIP) with anti-GRWD1 antibodies on asynchronously growing cells, and assessed the presence of origin sequences by quantitative real-time PCR analysis (qPCR) . Importantly, silencing of GRWD1 decreased the ChIP signals (see below), confirming the validity of the assay. These data show that GRWD1 indeed binds to replication origins.We examined whether GRWD1 is present at two well-known replication origins near the ) Figure . We prev) Figure . Here, w) Figure . Some en) Figure . In addiWe investigated the cell-cycle dependence of GRWD1 binding using previously described chromatin samples from synchronized T98G cells . The oriLMNB2 origin was significantly enhanced upon Cdt1 overexpression in HEK293T cells  with utilizing aggregation plots  and ∼347 000 peaks of MCM7 binding   and MCM7 peaks (MCM7_w0.5_CDC6) appeared to be clustered on some specific regions, forming pre-RC-enriched domains including known origins . MACS2 broad peak caller identified ∼458 000 peaks of CDC6 binding  Figure . For acc) Figure  (Supplem) Figure . This pa) Figure . This as) Figure . Hereafts Figure . Given tntiality . In the ntiality . In Drose ∼11 kb . On the e ∼11 kb ,40–41. Ie ∼11 kb –36,38. We ∼11 kb  and F. Fe ∼11 kb . Togethelamin B2, MCM4 and c-myc origins . Consequently, ∼208 000 GRWD1 binding peaks were selected Figure  and B. Ws Figure . The firs Figure . These ds Figure . Since tIt seemed likely that GRWD1 regulates chromatin dynamics as a histone-binding protein. Therefore, we finally characterized the chromatin architecture around pre-RC sites and understand the effect of GRWD1 depletion. We here used FAIRE , a method of isolating genomic regions with depleted or coarse nucleosomes –44.lamin B2 and MCM4 origins, revealing that the signals at these regions were decreased upon GRWD1 depletion . We then investigated quantitative FAIRE signals by qPCR on lamin B2, MCM4 and c-myc loci showed prominent positive signals, indicating that these origins are in open chromatin structure . We examined quantitative ChIP signals and FAIRE signals by qPCR at these sites. Like LMNB2 and MCM4 origins, the origin sequences were significantly enriched with anti-CDC6, anti-Cdt1, anti-MCM7, anti-MCM3 and anti-GRWD1 antibodies . As is the case for LMNB2 and MCM4 origins, some enrichment of the origin-distal regions was also observed, but less efficient. Furthermore, silencing of GRWD1 decreased the GRWD1 and MCM ChIP signals, but not CDC6 and Cdt1 signals . These data indicate that pre-RCs are formed on these origins in a GRWD1-stimulated manner. We then carried out FAIRE-qPCR assay . The data indicate that the origin sites have open chromatin structure and GRWD1 plays a role in maintaining the chromatin openness, like LMNB2 and MCM4 origins.We next analyzed the genome-wide FAIRE signal profiles and compared it with that from the GRWD1-depleted cells. In a visual inspection of the data, the origin regions of e Figure . In addie Figure . The FAIn Figure . We examn Figure . Howevern Figure . As suggn Figure , the firn Figure . On avern Figure . Taken tlamin B2 and MCM4 locus origins, in a CDC6- and Cdt1-dependent manner . Therefore, it is possible that GRWD1, in cooperation with HBO1 and SNF2H, might relax histone-DNA interactions to increase the ratio of nucleosome-free DNA rather than simply evict most histones. Similar findings were observed at rDNA origins in fission yeast, where depletion of Sir2 HDAC may result in open chromatin structure, apparently without changing histone levels and positioning . In addition, GRWD1 is required for maintenance of genome integrity, because its depletion leads to DNA damage Figure . FurtherChIP-seq and FAIRE-Seq data were deposited with accession codes DRA000924, DRA001271 and DRA002539.Supplementary Data are available at NAR Online.


Vesicular stomatitis virus (VSV) is the prototype for negative sense non segmented (NNS) RNA viruses which include potent human and animal pathogens such as Rabies, Ebola and measles. The polymerases of NNS RNA viruses only initiate transcription at or near the 3′ end of their genome template. We measured the dissociation constant of VSV polymerases from their whole genome template to be 20 pM. Given this low dissociation constant, initiation and sustainability of transcription becomes nontrivial. To explore possible mechanisms, we simulated the first hour of transcription using Monte Carlo methods and show that a one-time initial dissociation of all polymerases during entry is not sufficient to sustain transcription. We further show that efficient transcription requires a sliding mechanism for non-transcribing polymerases and can be realized with different polymerase-polymerase interactions and distinct template topologies. In conclusion, we highlight a model in which collisions between transcribing and sliding non-transcribing polymerases result in release of the non-transcribing polymerases allowing for redistribution of polymerases between separate templates during transcription and suggest specific experiments to further test these mechanisms. RNA dependent RNA Polymerases tight association with their template creates an almost infinite dilution transcription machinery. Polymerases are delivered to the host cytoplasm associated with the genome template, however, they initiate transcription only at or near the 3′ end of the genome template. How these polymerases initiate and sustain transcription is completely unknown. Given the efficiency of these polymerases and their nontrivial template interactions, understanding their mechanism has both medical and nano-technological applications. Here we show that efficient transcription requires a sliding mechanism for non-transcribing polymerases and can be realized with different polymerase-polymerase interactions. Transcription is the process of polymerase driven synthesis of mRNA from the genome template. In eukaryotic cells, polymerases engage their promoters through 3D diffusion Here we focus on vesicular stomatitis virus (VSV) which is a prototype of NNS RNA viruses. VSV genomic RNA encodes the 5 viral proteins in the order 3′-N-P-M-G-L-5′ and is fully encapsidated by 1,250 copies of nucleoproteins (N) to form the genome template (N-RNA) The catalytic unit of the VSV polymerase is the L protein, 50 copies of which are packaged within the virion along a single genome template Understanding the position of polymerases along the genome template may provide important clues as to the mechanism of transcription initiation. Polymerases remain associated with the N-RNA templates released from detergent-disrupted virions. When these purified templates are examined with immunogold electron microscopy, the polymerases are found to be almost randomly distributed along the genome template The polymerases initiate transcription either at the 3′ end of the genome template One plausible mechanism to initiate transcription is for a few polymerases to dissociate from the genome template during its delivery to the cytoplasm. The genome template is delivered to the cytoplasm after G protein facilitated fusion of viral envelope with endocytic membranes Here we constructed Monte Carlo simulations to gauge the fitness of various early transcription models based on their N-mRNA production levels after one hour of transcription. In total 20,000 simulations of early transcription were performed testing a range of polymerase dissociation rates, sliding rates, TIS binding strengths, linear and circular topologies of the genome template and two different polymerase–polymerase collision conditions. Our simulations show that the initial distribution of polymerases is not critical since the transcription machinery rapidly reaches a steady state. We identify the following mechanisms that can sustain transcription and are consistent with our measurements of the dissociation constant of polymerases from their genome template: i) Transcription through sliding facilitated polymerase release, in which polymerases are released from the template through their collision with a transcribing polymerase and engage the TIS through 3D diffusion; ii) Transcription through sliding facilitated initiation on circular templates, in which polymerases find the TIS through 1D sliding on a circular genome template; iii) Transcription through sliding and non-colliding polymerases (in which transcribing and non-transcribing polymerases can pass each other). At the end we propose that sliding of non-transcribing polymerases plays a critical role in VSV transcription. Specifically we highlight the sliding facilitated polymerase release model and discuss how the predictions of this model are consistent with low dissociation constant of polymerases under no transcription and the previously observed redistribution of polymerases between templates during transcription We measured the dissociation constant of L from the whole genome template by detergent disrupting the VSV virions and separating the N-RNA template bound with P and L proteins from the rest of the viral proteins using a glycerol cushion spin as detailed in the . The L psl and would fall off spontaneously from the genome template with the rate Koff. In the event of a collision between a transcribing and a non-transcribing polymerase, the non-transcribing polymerase would be forced off the template. Free polymerases in solution would find the template through a diffusion limited reaction and would have a binding affinity R to the TIS relative to a random binding site on the template. Neither Koff, Dsl nor R values are known experimentally, therefore we performed 4,000 simulations spanning a large range of these parameters . We report the measured N mRNA production for each condition as shown in We simulated transcription from a set of 50 polymerases on the linear genome template using the Monte Carlo rules explained in the methods. In brief, 50 polymerases were followed on the template. Polymerases would only initiate transcription from the TIS at the 3′ end. During transcription, polymerases had a 30% probability of falling off the template at the end of each gene. Non-transcribing polymerases would slide along the template with the 1D diffusion rate Doff>10−2/s) allowed for sustained transcription independent of Dsl as shown in High fall off rates (Koff <10−3/s) are incapable of sustaining transcription unless the sliding rate of the non-transcribing polymerases is increased to Dsl>103 nm2/s as shown in sl  = 104 nm2/s, Koff = 6.4×10−6/s a total of 72.8±6.2 N mRNA molecules were synthesized. Although in this model polymerases slide readily on the template, examination of polymerase histories showed that only 2% of polymerases found the TIS through sliding, whereas 98% found the TIS through 3D diffusion from solution. From the polymerases that found the TIS through 3D diffusion, 99.7% were dissociated from the template through a collision with a transcribing polymerase. In sliding facilitated polymerase release as highlighted in Low fall off rates  as shown in To test these conditions, we changed the collision rules so polymerases can bypass each other and simulated this model by exploring the sliding, dissociation and TIS binding strengths (Doff>10−2/s) we could observe sustained transcription independent of the sliding diffusion coefficient Dsl similar to results shown in As expected under high dissociation rates  and Dsl (from 10 nm2/s to 105 nm2/s) as shown in To address the issues of initial conditions further, we have measured the N mRNA production with initial conditions: i) randomly distributed polymerases and ii) asymmetrically distributed polymerases at the 5′ end. These measurements were performed on the same range of KThe Monte Carlo simulations presented here were used to quantify the transcription efficiency and sustainability of various models. Simple mechanisms like jump starting the transcription with initial dissociation of polymerases during entry did not produce sustainable transcription. The most critical issue for sustaining transcription is to have efficient mechanisms for delivery of polymerases to TIS at the 3′ end. This can be achieved either through 3D diffusion or 1D sliding on the genome template. Below we will discuss specific models presented from our results that can sustain transcription.off>10−2/s). The interesting aspect of this model is that it is insensitive to sliding rates, genome topology and/or collision conditions as shown in The most simple model that can deliver the polymerases to the 3′ initiation site through 3D diffusion is based on dissociation of non-transcribing polymerases from the genome template. In order for this model to sustain transcription, two conditions need to be satisfied. Like any other model that relies on 3D diffusion, this model requires a high relative affinity for the TIS (R>500) and a high dissociation rate of . This creates an interesting model in which immediately upon halting transcription, all the polymerases would bind back to the genome template as shown in This is the first model we tested in which polymerases would find the initiation site through 1D sliding and are capable of sustaining transcription. The advantage of this model is that it is operational even under low relative binding strength of the TIS (R = 1) as shown in If we assume that transcribing polymerases can bypass non-transcribing polymerases on the template and that non-transcribing polymerases can slide on the genome template we arrive at a model that does not require circular templates, sustains a high rate of transcription and can operate with almost no free polymerases in solution either during or under non-transcribing conditions as shown in All the mechanisms represented here and tested in the MonteCarlo simulations are inspired by previously observed mechanisms in DNA enzymes and molecular motors. It is however possible that redistribution of polymerases in NNS RNA viruses has a unique and different mechanism. One such mechanism, can be transferring of the polymerases from one section of the template to the other through collisions between template sections, such a model can be termed effectively the “Spider-Man” model. In such a model, sliding of polymerases would not be essential since polymerases can redistribute along the template through strand transfer collisions. Theoretical prediction of the spider-man model is complicated, since it depends on the three dimensional dynamics of the genome template, which is currently unknown. The spider-man model however is predicted to be heavily dependent on template self collisions and therefore it predicts a severe decrease in polymerase redistributions under stretched template geometries which can be achieved by mechanical manipulations of the template.It becomes clear when comparing the panels in Failure to detect free polymerases during transcription would make the sliding non-colliding polymerase mechanism proposed in Given the 20 pM dissociation constant measured for L with its full genome template, how can a sliding model be justified? Indeed, the dissociation constant of molecular motors from their tracks is in the low nanomolar range and the dissociation constant of the T7 RNA polymerase to its class III TIS is in the range of 40–60 nM Cellular DNA based RNA polymerases have been shown to bind directly to their promoters to initiate transcription In NNS RNA viruses, aside from initiation at the 3′ end In our model we investigated only the first one hour of transcription. The kinetics of transcription during the replication cycle beyond the first hour have been previously modeled for viral infection cycles including: kinetic modeling of viral growth in VSV Understanding how polymerases initiate and sustain transcription is attractive both as a potential antiviral target and also because these polymerases represent an almost infinite dilution transcription machinery. Understanding the mechanics of this machine therefore may open doors to nano-technological applications.Total = 300 segments along the template (n = 1 to 300). There are 50 polymerases that occupy the genome template Li (i = 1 to 50). No two polymerases can occupy the same segment simultaneously. To create a Monte Carlo simulation, we also digitized the time domain. The essential time scale for the simulation τ is the time required for transcribing one segment of the N-RNA by a polymerase. Given an average rate of transcription of 3.7 nt/s i) were tracked along the digitized genome template with incremental time τ. In each time step all the polymerases are forced to choose a reaction with the following rules:Negative stain electron microscopy of single VSV polymerases shows them as 10 nm ring-like structures which can adopt different conformations with appendages sl; 4) Collision of non-transcribing polymerases results in reflection of trajectories without dissociation from the template; 5) Reflective boundary conditions are set up at the ends of the template for non-transcribing polymerases; 6) Non-transcribing polymerases fall off the template with a rate Koff and can immediately rebind back with a rate Khopp  Transcription is initiated only if the polymerase is on the TIS; 2) Transcription follows a linear trajectory during each time step τ, with 30% fall off rate at the end of each gene junction; 3) Non-transcribing polymerases move along the genome randomly, with a step size calculated based on the mean square displacement associated with a 1D random walker with a diffusion coefficient Dte Khopp  or can son is not determined experimentally, however an upper limit is calculated based on the diffusion-limited reaction. The upper limit of a diffusion limited reaction is Kbinding based on the Kon in the simulation. To incorporate the stochastic binding within the Monte Carlo simulations, we used a Gillespie Algorithm (SSA) binding time, and will be kept in solution until the simulation time exceeds tbinding time. The polymerase then gets reassigned to an available site on the template. The parameter R, which is the ratio of binding strength between TIS and unspecific sites, determines the probability of polymerase binding to the TIS PTIS =  ) or the probability of randomly binding on the template Pnonspecific =  ) in which nTotal is the total number of available binding sites.Each polymerase that falls off the template will be assigned its own tIn the described simulations with no sliding, we could not reconcile a healthy transcription rate, which produces ∼100 N mRNA in 1 hr S1 FigureAnalysis of VSV proteins binding to RNPs. VSV proteins were sequentially extracted in low then high salt buffers after virions lysis. Soluble and RNPs-bound factors were fractionated by ultra-centrifugation on 20% glycerol cushion, and analyzed by SDS-PAGE and proteins staining. VSV: full virions; 1–10: fractions collected from top to bottom of tubes after ultra-centrifugation, respectively; RNPs: pelleted RNPs, respectively.(TIF)Click here for additional data file.S2 FigureWestern blots of varying dilutions of RNP and Fraction 1. The following volumes of Fraction 1 were loaded: 1 = 30 µl; 2 = 20 µl; 3 = 10 µl; 4 = 5 µl; 5 = 2.5 µl; 6 = 1 µl. RNPs: 1 µl from each dilution 1 = 1/1; 2 = 1/5; 3 = 1/10; 4 = 1/50; 5 = 1/100.(TIF)Click here for additional data file.S3 FigureLinear genome . Number of N mRNA after one hour of transcription versus various dissociation rates and different 1-D diffusion coefficients under different promoter strength R: B). R = 1 C). R = 500. The morphology of N-RNA template is linear and initially all 50 L are located either asymmetrically at the 5′ end of the genome (dark grey) or randomly along the template (light grey). Under R = 500, Dsl = 104 nm2/s and Koff = 10−5/s, random distribution produced 86.2±3.7 N mRNA compared to production of 72.8±7.7 with asymmetric distribution.(TIF)Click here for additional data file.S4 FigureMonte Carlo simulations of UV irradiated genome templates. Monte Carlo simulations of 50 polymerases on a linear genome template with a damage site on the end of N gene, (A) shows a representation of this model with collision rules resulting in release of non-transcribing polymerases after collisions, and also template damage rules: the transcribing polymerase is either stuck (I) or released (II) at the damage site. (B) The calculated N mRNA amounts within one hour of simulations for various sliding Dsl and dissociation Koff rates with relative TIS binding affinity R = 500. (C) Number of Polymerase that remain on the template vs Time . Green line follows template damage rule I, and grey line follows rule II.(TIF)Click here for additional data file.S1 TextSupplemental information. This file includes dissociation constant measurements and details of the Monte Carlo Simulations.(DOCX)Click here for additional data file.