Supplemental Digital Content is available in the text This experimental study was conducted with completely randomized design.Genome-wide association studies (GWAS) have revealed a large number of genetic associations of nucleotide sequence variants with susceptibility to multiple sclerosis (MS). Nevertheless, studies to identify the functional relevance of these variants lag far behind identification of the GWAS signals. Expression quantitative trait loci (eQTLs) analysis and promoter activity analysis with the variants filtered by GWAS were conducted to identify their functional alleles and haplotypes. The promoter activity was assayed with reporter constructs containing variants at 8 MS GWAS signals resulted from 18 GWAS.CYP24A1, CYP27B1, SYK, RAD21L1, PVR, ODF3B, and RGS14 genes (P < 0.05). The transcriptional regulations of sequence variants were also found by identifications of eQTLs for their corresponding genes with lymphoblastoid cells in the current study  and with dendritic cells in a previous study .The promoter activity differed by alternative sequence variants at upstream regions of the CYP24A1, CYP27B1, SYK, RAD21L1, PVR, ODF3B, and RGS14 genes, and their variants differentially affected gene expression. This might result in their associations with MS susceptibility in previous GWAS. Further functional studies are required to understand the process of transcriptional regulation of the variants identified in the current study and the mechanisms underlying susceptibility to MS.This study identified regulatory nucleotide sequences in the promoters of the  GWAS not only identified associations of MS with common sequence variants outside the MHC region, but also confirmed the genetic contribution of the genes within the MHC region.4 Only a small proportion of the variants identified by GWAS were located in coding regions, and the functional relevance of most GWAS signals remains unknown. Thus, the functions of the variants in the GWAS signals should be determined to understand the mechanisms underlying development of MS. In this study, we investigated the differences in transcription activity resulting from nucleotide substitution in promoter sequences identified by GWAS.Multiple sclerosis (MS) is a complex inflammatory disease of the central nervous system that is accompanied by neurological impairment and disability. A number of linkage and association analyses found that the major histocompatibility complex (MHC) encoding the human leucocyte antigen genes on chromosome 6 confers a genetic influence on susceptibility to MS.http://www.genome.gov/page.cfm?pageid=26525384&clearquery=1#result_table) and confirmed in the publications in which they were originally described. Genetic association signals were obtained from 18 GWAS for susceptibility to MS. We filtered the variants located within a region −2–0 Kb upstream of every gene encoding protein in order to examine the promoter activity. Linkage disequilibrium (LD) estimates were obtained with the variants within 2 Kb upstream of the transcription start site. We used genotypes of 379 Europeans at the variants provided by the 1000 Genomes Project (http://browser.1000genomes.org/). LD blocks were constructed for the variants associated with MS and variants linked to them using the algorithm of Gabriel et al5 (Haploview version 4.2) in which the block was defined according to the 95% confidence interval of the D′ value for pairwise LD between the variants with minor allele frequency > 0.05.Candidate promoter nucleotide variants for association with MS were selected from the GWAS catalog . They calculated expression level of each corresponding gene as the sum of all transcript reads per kilobase per million (RPKMs) using lymphoblastoid cell lines of 373 European individuals from the 1000 Genomes Project.6 The corresponding genotypes at the selected variants of the GWAS signals were obtained based on 1000 Genome Phase 1 (ftp://ftp.ebi.ac.uk/pub/databases/microarray/data/experiment/GEUV/E-GEUV-1/genotypes/). We identified eQTL using the simple linear regression model implemented in PLINK (v1.07). In order to reduce spurious associations produced by multiple testing, the Bonferroni correction was applied with the number of tested genes because of the linkage of the tested variants within each LD block.We examined transcriptional regulation of the selected variants at GWAS signals by eQTL analysis. This analysis helped identify variants as http://links.lww.com/MD/A104. Restriction enzyme sites  were also added to each set of primers to facilitate posterior cloning of the PCR products  to release the fragments containing the promoter of each gene. The released fragments were subcloned upstream of the firefly luciferase reporter gene in the pGL3-Basic vector (Promega) using T4 DNA Ligase (Promega). Site-directed mutagenesis was performed to substitute appropriate nucleotide variants . The sequences of all inserts were verified by direct sequencing.Luciferase reporter was constructed with the promoter variants as shown in Figure 2. Cells (1.6 × 105) were seeded 24 hour prior to transfection in 6-well plates and transfected at approximately 60% to 70% confluence. Cells in each well were cotransfected with 1.2 μg pGL3-basic firefly luciferase reporter vector (Promega) containing the reporter construct and 24 ng pRL-CMV Renilla luciferase reporter vector (Promega) using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Transfected cells were incubated for 24 hour at 37°C and 5% CO2. The amount of firefly and Renilla luciferases was measured sequentially from a single aliquot of cell lysate using the Dual-Glo Luciferase Assay system (Promega) on a CentroPRO LB962 luminometer . Transfection efficiency was controlled by screening experiments with the amount of Renilla luciferase within the range of ±30%. Firefly luciferase activity of each individual transfection was normalized to Renilla luciferase activity to correct for the difference in transfection efficiency. Promoter activity was expressed relative to that of the pGL3-Basic vector without the promoter sequence. All assays were conducted in triplicate, and at least 3 independent experiments were performed for each reporter gene construct.We selected the human embryonic kidney 293 (HEK293) cell line for luciferase reporter assay because the genes examined in the current study are all expressed in the human kidney. The HEK293 cell has been widely used in transient transfection for promoter activity experiments using plasmid vector because of its high transfection efficiency. HEK293 cells were cultured in Dulbecco's modified Eagle medium  supplemented with 10% (v/v) heat-inactivated fetal bovine serum , 100 units/mL Penicillin, and 100 μg/mL Streptomycin at 37°C in an atmosphere containing 5% CO7 Similarity of the query locus was scored by comparing with the positional weight matrix for each transcription factor, and the matrix was estimated based on real data by TRANSFAC® version 8.3. We employed a similarity threshold value of 0.85. Changes in the predicted TFBS by allelic substitution were examined.Putative transcription factor binding sites (TFBS) at the promoter loci were predicted using PROMO version 3.0.2.Statistical difference in luciferase activity among haplotypes was determined by one-way analysis of variance. Post hoc pairwise comparisons were performed with the false positive threshold of 0.05. We employed Tukey's honest significant difference test to compare all possible pairs of luciferase activity. All the statistical tests were conducted using the Statistical Package for the Social Sciences version 20 .http://links.lww.com/MD/A104) and 91 intergenic (Supplementary http://links.lww.com/MD/A104) regions. Most variants were in regulatory regions, with 5 variants located in translated regions. Nine signals were located within the region −2–0 Kb upstream of genes. One signal was located in upstream regions of the genes encoding a microRNA (hsa-mir-548ac). Eight signals were promoter sequence variants of the genes encoding CYP24A1 (MIM 126065), CYP27B1 (MIM 609506), SYK (MIM 600085), RAD21L1, CXCR5 (MIM 601613), PVR (MIM 173850), ODF3B, and RGS14 (MIM 602513) proteins and were selected to examine their promoter activity. The GWAS signals were not in strong LD with any variants from transcription start site to +1 Kb of each gene. The odds ratio estimates for their associations with MS are summarized in Table 10We obtained 192 GWAS signals resulting from 18 GWAS for susceptibility to MS. The signals were located in 101 intragenic  because the expression data of CYP24A1 and RAD21L1 were unavailable. The expression levels of SYK, ODF3B, and RGS14 were significantly affected by the variants of the GWAS signals (Table P < 8.33 × 10−3). Marginal associations were observed between PVR expression and its promoter variants (P < 0.05). The risk alleles resulted from GWAS studies increased the expression of SYK, RGS14, and PVR but decreased the expression of ODF2B.The eQTLs were identified by testing association with the expression level of 6 genes . In particular, the expression of CYP27B1-H1, SYK-A, and RAD21L1-T was greater than 2-fold higher than that of CYP27B1-H2, SYK-G, and RAD21L1-C, respectively.Differences in luciferase activity were found with reporter constructs for all genes, with the exception of CYP27B1 revealed that the alteration in expression was the result of a nucleotide substitution at 1 SNP (rs3782130) within the LD block .Luciferase activity differed significantly with only a single nucleotide substitution for reporter constructs of all 7 genes. Differential luciferase activity of 1 alternative allele was observed with natural haplotype constructs for each of 6 genes Figure . Luciferhttp://links.lww.com/MD/A104). In particular, the variants of CYP27B1, SYK, and RAD21L1 that were found to be largely different in promoter activity were also predicted to have largely different TFBS by allele substitution. No different TFBS were predicted by nucleotide substitution of only rs140522 (Supplementary http://links.lww.com/MD/A104).In silico prediction showed allele-specific TFBS at the nucleotide variants examined in the current study , and 4 of the 6 SNPs shared functions with their linked SNPs . One SNP was linked to a causal SNP (RGS14). The transcriptional regulations of the variants were supported by the eQTL analysis. The promoter variants were identified as eQTLs for the expression of the genes encoding SYK, ODF3B, RGS14, and PVR in lymphoblastoid cells. The directions of allelic effects from the promoter assay were concurred with those from the eQTL analysis. The promoter variant of CYP27B1 was reported as an eQTL from the previous study with human dendritic cells .11This study was successful in identifying functional sequence variants in 7 promoter regions. Six SNPs associated with MS were causal variants .Surprisingly, transcriptional activity was altered by a single nucleotide substitution. In particular, the G allele of the 2 D)) in immune and central nervous system cells, and CYP24A1 catalyzes the conversion of active vitamin D to the inactive form 2 D).13 Because vitamin D is an important environmental factor in treating MS, CYP27B1 and CYP24A1 have been known to be factors in susceptibility to MS, based on control of vitamin D level.14 SYK is a tyrosine kinase involved in signal transduction in a variety of immune cells,15 and aberrant regulation of SYK is associated with autoimmune diseases, such as rheumatoid arthritis16 and lupus17. PVR regulates check points to activate T cells in the central nervous system.18RGS14 and ODF3B were identified as TNF-κB p65 target genes in human B lymphoblastoid cells using ChIP-Seq.19The identified variants may influence transcription of genes that play important roles in the immune system and, thus, in MS. CYP27B1 produces active vitamin D 20 and vascular dementia.21Inflammation is another potential function of the genes that may predispose to MS. For example, CYP27B1 and CYP24A1 perturb active vitamin D level and, thus, enhance production of proinflammatory cytokines and chemokines. SYK is essential for the transmission of inflammatory signals of various cytokines and influences pathophysiology of inflammatory diseases, such as atherosclerosis22 The TFBS encompassing rs2585427 upstream of the CYP24A1 gene was identified as binding to E2F-1 in HeLa-S3 cells. Difference resulting from allele substitution, however, remains unknown. Promoter activity of the variants examined in the current study was also supported by various evidences of regulatory functions provided by RegulomeDB.23 For example, most of the variants turned out to have open chromatin structure as shown in Supplementary http://links.lww.com/MD/A104, which indicates potential regulatory sequences.Specific mechanisms underlying the different promoter activities observed in this study are not known. Nevertheless, we suspect that nucleotide substitutions would change TFBS in a variety of ways. Our in silico prediction showed that most alternative alleles examined in this study potentially induce changes in TF binding. Further research is needed to identify genuine TF profiles by allelic substitutions. We found that a predicted site was a true TFBS using ChIP-Seq data provided by ENCODE consortium.CYP24A1, CYP27B1, SYK, RAD21L1, PVR, ODF3B, and RGS14 genes. This is the first study to identify functional nucleotide variants associated with many GWAS signals for MS on a large scale. The results suggest that alternative single nucleotides and/or haplotypes of the sequence variants may produce modified TFBS. We suspect that the promoter GWAS signals for MS might be the result of different transcriptional activity. Further functional studies are needed to identify the mechanisms underlying the association with development of MS. Functional studies are also required for MS GWAS signals in regions other than the promoter to simultaneously apply the genetic risk/protective factors for MS to personalized medicine.We found differential transcriptional activity in promoter regions of the


Mycobacterium tuberculosis, is found in many bacteria, including Thermus species, but absent in Escherichia coli. To delineate the molecular mechanism of CarD, we determined crystal structures of Thermus transcription initiation complexes containing CarD. The structures show CarD interacts with the unique DNA topology presented by the upstream double-stranded/single-stranded DNA junction of the transcription bubble. We confirm that our structures correspond to functional activation complexes, and extend our understanding of the role of a conserved CarD Trp residue that serves as a minor groove wedge, preventing collapse of the transcription bubble to stabilize the transcription initiation complex. Unlike E. coli RNAP, many bacterial RNAPs form unstable promoter complexes, explaining the need for CarD.A key point to regulate gene expression is at transcription initiation, and activators play a major role. CarD, an essential activator in DOI:http://dx.doi.org/10.7554/eLife.08505.001 Inside cells, molecules of double-stranded DNA encode the instructions needed to make proteins. To make a protein, the two strands of DNA that make up a gene are separated and one strand acts as a template to make molecules of messenger ribonucleic acid (or mRNA for short). This process is called transcription. The mRNA is then used as a template to assemble the protein. An enzyme called RNA polymerase carries out transcription and is found in all cells ranging from bacteria to humans and other animals.Bacteria have the simplest form of RNA polymerase and provide an excellent system to study how it controls transcription. It is made up of several proteins that work together to make RNA using DNA as a template. However, it requires the help of another protein called sigma factor to direct it to regions of DNA called promoters, which are just before the start of the gene. When RNA polymerase and the sigma factor interact the resulting group of proteins is known as the RNA polymerase ‘holoenzyme’.Transcription takes place in several stages. To start with, the RNA polymerase holoenzyme locates and binds to promoter DNA. Next, it separates the two strands of DNA and exposes a portion of the template strand. At this point, the DNA and the holoenzyme are said to be in an ‘open promoter complex’ and the section of promoter DNA that is within it is known as a ‘transcription bubble’. Another protein called CarD helps to speed up transcription but it is not clear how this stage of the process works.Bae et al. have now used X-ray crystallography to reveal the structure of CarD bound to the RNA polymerase holoenyzme and a DNA promoter. The structures show that one part of CarD interacts with the DNA at the start of the transcription bubble, and another part binds to the RNA polymerase. CarD fits between the two strands of DNA in the promoter, like a wedge, to keep the strands apart. Therefore, CarD stabilizes the open promoter complex and prevents the transcription bubble from collapsing.These findings reveal a previously unseen mechanism involved in activating transcription and will guide further experiments probing the role of CarD in living cells. Another study by Bae, Feklistov et al.—which involves some of the same researchers as this study—reveals that the sigma factor also binds to DNA at the start of the transcription bubble. The general principles outlined by these studies may help to identify other proteins that regulate transcription.DOI:http://dx.doi.org/10.7554/eLife.08505.002 Escherichia coli (Eco) as a model system inform most of our understanding of how bacteria control transcription initiation. First, dissociable promoter specificity subunits, σ factors, direct the catalytic core of the RNA polymerase (RNAP) to promoter DNA sites and play a key role in unwinding the DNA duplex to create the transcription bubble in the RNAP holoenzyme open promoter complex (RPo) (Decades of research using ex (RPo) . Second,ex (RPo) .4 domain positioned at the −35 element, or both , is a transcriptional activator widely distributed among bacterial species, including Thermus species  transcription in  species , but is ies -RID  transcription initiation complexes (RPo) -RID , and a hRCF)-RID  (correspRCF)-RID , which iRCF)-RID . The disechanism . To resoes (RPo)  containies (RPo) . The strTth 23S  or full RPo crystals  to interact with the DNA  seen in the mus CarD  and CarDmus CarD  structur the DNA . By main β1-lobe   minor groove side of the DNA  bases within the transcription bubble  make direct contacts with the promoter DNA . The twoThe peptide backbone nitrogen of CarD-L124, at the N-terminal end of α5, closely approaches the backbone phosphate oxygen of the template strand (t-strand) at the −14 position [−14(t)] , possiblFo − Fc map calculated from coordinates in which CarD-W86 had been mutated to Ala (W86 is conserved among greater than 95% of CarD proteins  and was d to Ala .Tth CarD-W86A or Mtb CarD-W85A) greatly reduced the activation efficiency of both Tth and Mtb CarD (Tth 23S promoter (Previous work showed that substitution of the bulky CarD-W86 side chain by Ala (Mtb CarD . To furtpromoter . All of −12(nt) O2 . The −12 T/A base pair is a conserved part of the promoter −10 element (−12(nt) contributes to CarD activity.The position of the W86 side chain Nε allows it to interact with T2(nt) O2 . Since t2(nt) O2 , we muta element , and, as element   O2 atom. We tentatively suggest that the reduced activation efficiency of the CarD-W86F mutant is primarily due to the loss of the minor groove polar interaction with T−12(nt).Note that the CarD W86F substitution results in an approximately twofold loss in CarD fold activation . The PheMtb CarD in complex with an Mtb RNAP β-subunit fragment that includes the β1-lobe shows a relative orientation of the CarD-RID/CarD-CTD domains very different from the one in our Tth CarD structures, despite high sequence and structural similarity within the domains  (right). On the other hand, the corresponding positions in the Mtb CarD structure (P12/G99) are 24 Å apart (left) (Tth CarD conformation (thus locking the domain orientation), but not the Mtb CarD conformation . Non-reducing SDS polyacrylamide gel electrophoresis and liquid chromatography mass spectrometry confirmed that under oxidizing conditions, the CarD-RID and CarD-CTD were disulfide crosslinked in greater than 98% of CarD2C, while under reducing conditions, no disulfide bond was present in >99% of CarD2C  , the aveed here) , right.  Å apart , left (Gt (left) . Cys subormation . We engif CarD2C . We testf CarD2C  on the Mype CarD . The obsype CarD  mM DTT ionserved ; on thisn bubble , represeTth CarD increased the half-life (t1/2) of the Thermus RPo ∼threefold on each promoter  are closely linked. We hypothesized that CarD may increase the lifetime of RPo by preventing transcription bubble collapse. To test this hypothesis, we determined the effect of CarD on the lifetime of promoter complexes on a synthetic promoter template based on the 23S sequence and compared it with the same synthetic template but with a non-complementary transcription bubble (from −11 to +2) unable to collapse . On the fivefold . On the fivefold , 23S_Bubcollapse . Additiocollapse . We thusMtb CarD , is to sMtb that is also widely distributed among bacterial species, including Thermus species but not found in Eco (Tth CarD with a Taq transcription initiaton complex (−12(t) (−12(t) is important for full CarD function  Car (−12(t) . We showfunction ; (3) We n bubble ; (4) We n bubble , resolviEco has served as a model organism for the study of many cellular processes over the last few decades, including transcription. Eco RNAP forms unusually stable RPo and Eco lacks CarD, while RNAPs shown to form relatively unstable RPo come from bacteria that harbor CarD  (−12(t) is present at most σA promoters   may moduromoters , and Carromoters .Tth CarD/RPo complexes presented here reveal how the widely distributed transcription factor CarD interacts with RPo to activate initiation. The CarD-RID/RNAP β1-lobe protein/protein interaction positions the CarD-CTD to interact with the upstream edge of the transcription bubble in a functionally relevant pose that does not allosterically alter the structure of the transcription bubble nor RNAP holoenzyme/promoter interactions  O2 presented in the highly distorted minor groove (Eco (the focus of most mechanistic transcription studies) since Eco RNAP forms relatively stable complexes on most promoters   beamline X29. Data were integrated and scaled using HKL2000 . The difoscale/) , resultioscale/) .Tth CarD/Taq β1-lobe complex  was used to calculate intermolecular buried surface areas  cells  and transformants were grown at 37°C in Luria–Bertani media containing ampicillin (200 µg/ml) and kanamycin (50 µg/ml). At an A600nm between 0.6–0.8, the cultures were supplemented with isopropyl-β,D-thiogalactopyranoside  to induce protein expression for 4 hr at 30°C. The cells were then spun down by centrifugation and resuspended in buffer A  glycerol, 1 mM β-mercaptoethanol). The cells were lysed using a continuous-flow homogenizer  and then centrifuged to remove insoluble debris. The clarified cell lysate containing overexpressed (His)10Sumo-Tth CarD was first applied to a Ni2+-charged HiTrap column  that was equilibrated with buffer A, followed by a wash with five column volumes (cv) of buffer A + 25 mM imidazole. Subsequently, clarified cell lysate containing overexpressed Taq β1 was injected into the same column to form a complex with the immobilized (His)10Sumo Tth CarD. The column was washed with five column volumes (cv) of buffer A + 25 mM imidazole and 5 cv buffer A + 40 mM imidazole. The complex bound to the column was eluted with buffer A  +  250 mM imidazole. After overnight cleavage with UlpI protease  to remove the (His)10Sumo-tag from Tth CarD and dialysis against buffer A + 25 mM imidazole, a subtractive Ni2+-chelating chromatographic step removed uncleaved (His)10Sumo-Tth CarD and the cleaved (His)10Sumo-tag. The sample was concentrated and injected on a Superdex 75 gel filtration column  that was equilibrated with GF buffer  glycerol). Fractions containing purified Tth CarD/Taq β1 complex were pooled and concentrated to 15 mg/ml by centrifugal filtration. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and Coomassie blue staining were used to analyze the purity of the complex.The plasmids pET21a Taqβ1  and pETs with 1 μl of crystallization solution  ethylene glycol) and incubating over a well containing crystallization solution at 22°C. Large crystals (0.5 mm) grew within 1 day. The crystals were directly frozen in liquid nitrogen for data collection.X-ray diffraction data were collected at Brookhaven National Laboratory NSLS beamline X29. Data were integrated and scaled using HKL2000  Table 1Table 1.Taq β1-lobe . Twinning was identified by Xtriage in PHENIX . The final model was obtained after twinning refinement using PHENIX.Initial electron density maps were calculated by molecular replacement using Phaser  from staMtbrrnAP3 (−60 to +15 of the endogenous promoter sequence) was prepared as described (Tthrrn23S (−68 to +15 of the endogenous promoter sequence)   was prepequence) . Artificequence) . The pur4 footprinting on the Mtb rrnAP3 promoter  with 100 mM NaCl for the AP3 promoter or 100 mM K-glutamate for the 23S promoter, at 65°C. Core RNAP (200 nM) and σA (1 μM) were combined and incubated at 65°C for 5 min to form holoenzyme. CarD  was then added to the holoenzyme and incubated for an additional 5 min. Next, promoter DNA (10 nM) was added and RPo was allowed to form for 15 min at 65°C. Abortive transcription was initiated by the addition of an NTP mix containing the initiating dinucleotide , the next NTP  and 2 μM of FC-bubble competitor DNA when used  was also made using site-directed mutagenesis but was subjected to two additional purification steps. Tandem Q-sepharose column chromatography  was used to remove inter-molecular cross-linked CarD. Sample was first applied on a 5 ml column and eluted using a NaCl gradient from 200 mM to 1 M over 20 column volumes (cv). The purest fractions were combined and reapplied to a second 5 ml Q column and eluted using a NaCl gradient from 100 mM to 1 M over 40 cv. This purification yielded >95% intra-molecular cross-linked CarD as verified by non-reducing SDS-PAGE  and purified using the same procedure as wild-type CarD . Mtb CarSDS-PAGE  and liquTth CarD/Taq holoenzyme/us-fork (− 12 bp) complex), 4XLR (Tth CarD/Taq RPo), and 4XAX (Tth CarD/Taq β1-lobe).The structure factor files and X-ray crystallographic coordinates have been deposited in the Protein Data Bank under ID codes 4XLS ( review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent  and three reviewers, one of whom is a member of our Board of Reviewing Editors.Thank you for submitting your work entitled “CarD uses a minor groove wedge mechanism to stabilize the RNA polymerase open promoter complex” for peer review at The following individuals responsible for the peer review of your submission have agreed to reveal their identity: Stephen Harrison (Reviewing Editor and peer reviewer) and Carol Gross (peer reviewer).The reviewers have discussed the reviews with one another, and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Thermus aquaticus RNAP with the CarD transcription factor  shows that its C-terminal domain contacts the upstream fork. A hinge between the CTD and the domain that binds the β1-lobe allows the former to dock into the fork. Various biochemical measurements support the interpretation that CarD increases the RPo lifetime. This work rationalizes divergent structures in the literature: by using disulfide bond formation they validate that Tth CarD structure in the open complex reported here, accurately reflects that of the active form of Mycobacterium tuberculosis CarD. The authors further validate the structure by showing that: (a) KMnO4 footprinting indicates that DNA structure is unchanged but that the open complex is more stable, (b) CarD has no stimulatory effect on the open bubble construct and (c) the T is preferred at the -12 non-template position, over any other base as surmised from suggested contact between the crucial W86 residue. The authors also explore the effects of substituting other aromatics or branched chain amino acids at the Cα position. That some of this characterization has already been performed with the mycobacterial enzyme provides evidence for the generality of the mode of action of CarD.The structure of an open complex of Essential revisions:1) Throughout the study, Bae et al. use a total of three promoters . It is not always clear why a certain promoter was used for a certain experiment e.g. . The aut2) The abortive transcription assays  only sho3) To increase clarity of the overall presentation, the authors should provide an additional  figure showing a protein sequence alignment of CarD from different organisms, to support the claim that CarD residue W86 is universally conserved.4) In the same vein, it would be good to see the distribution of CarD. Is it present in all gram positives (+ mycobacteria) and absent in all gram negatives?4 footprinting assay (4 accessible thymine bases. The wording should be changed to reflect this distinction.5) The authors claim that CarD “does not alter the transcription bubble” by providing data from a KMnOng assay . The aut6) The polymerase element β1-lobe is not defined in the manuscript. Which residues does it contain? Do we understand correctly that this domain corresponds to the eukaryotic RNAP II protrusion domain? Please clarify in the text, because the eukaryotic RNAP II Rpb2 “lobe” flanks the downstream cleft and thus is different from the bacterial “lobe” – the nomenclature can lead to confusion.7) We recommend that the authors acknowledge that the CarD mechanism could be more complex and affect additional steps in the RPo formation.-12 (nt) interaction? For example, phenylalanine activates significantly-would it be expected to make the same contact, or to change the base preference? If so, would it be informative to look at other bases in the T-12 (nt) position?8) Is it possible to further probe the W86-T9) CarD was originally described as an rRNA regulator in response to nutrient deprivation, mediating the stringent response. Now that we know the mode of action of CarD, it would be good to return to that phenotype, and describe how it might fit in.10) There is considerable redundancy, both within this paper and with the accompanying paper on RPo. The authors should simply cross-reference and shorten the present manuscript to eliminate internal redundancy. Essential revisions:1) Throughout the study, Bae et al. use a total of three promoters . It is not always clear why a certain promoter was used for a certain experiment (e.g.). The authors make use of the 23S and the AP3 promoter for their biochemical assays, but do not provide any biochemical data for the full con promoter that was used for the structural studies. The authors should explain why the full con promoter (without the mismatch bubble) wasn't used in addition to the 23S and AP3 promoter to validate the structural results. For easier comparison of the different promoters , please provide one figure with alignments of the different promoters.Eco RNAP σS-holoenzyme, but subsequent analysis indicated the sequence appeared to be optimized for binding σ70-holoenzyme as well (Eco σ70 and Taq σA), the sequence is almost certainly optimized for binding Taq σA -holoenzyme as well. We use this sequence for many of our structural studies to achieve high-affinity, homogeneous complexes important for crystallization. The promoter is extremely active in abortive initiation assays but is actually a poor promoter in vitro and in vivo in run-off assays since there is an issue with promoter escape (RNAP is bound too tightly to the promoter). Because of these properties, the promoter is unlikely to be regulated  and we generally don’t use it for transcription assays, etc.The full con promoter sequence was derived from an in vitro evolution (SELEX) protocol optimizing for binding to  as well . BecauseMtb rRNA promoter – its regulation by Mtb CarD has already been extensively studied  For the KMnO4 assays , we showb) We have included a paragraph (end of the Introduction) explaining the use of the three promoter sequences.Mtb AP3 for Mtb AP3 for this analysis since Tth EσA was much more active on this promoter than on Tth 23S, allowing us to analyze the much weaker activity of the mutant promoters”.c) We have added a sentence explaining the use of Mtb CarD2C under oxidizing and reducing conditions), we used a mycobacterial transcription system (Mtb AP3 promoter (see subsection “The Thermus CarD/RNAP initiation complex structures represent the active conformation of CarD”).d) For n system  so we usWe have included an alignment of all three promoters .2) The abortive transcription assays  only show the trinucleotide transcription product. Were there any further run-off products observed due to mismatch incorporation (“leaky transcription”)? We urge the authors to provide the full gel or to state whether they detected mismatch incorporation.32P-NTP . The dominant products were the expected products . We have included the full, annotated gels in the supplementary figures (Abortive initiation assays were initiated using specific dinucleotide primers in the presence of only the next α- figures .3) To increase clarity of the overall presentation, the authors should provide an additional  figure showing a protein sequence alignment of CarD from different organisms, to support the claim that CarD residue W86 is universally conserved.http://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch&BLAST_SPEC=MicrobialGenomes) and discovered that CarD is even more widely distributed than originally described , occurring in 11 of 26 bacteria phyla searched. We now include a table documenting our findings (Supplementary file 1). We also include an alignment of CarD from 11 major subgroups, mostly grouped by phylum, but also dividing the proteobacteria in subgroups to show diversity within that phylumWe have previously published a CarD alignment  based on. We also4) In the same vein, it would be good to see the distribution of CarD. Is it present in all gram positives (+ mycobacteria) and absent in all gram negatives?Please see response to point 3.5) The authors claim that CarD “does not alter the transcription bubble” by providing data from a KMnO4footprinting assay . The authors should clarify: a potassium permanganate footprinting assay provides information about the general accessibility of a thymine base, but it does not provide information about the CarD dependent conformational differences (e.g. base flipping) of KMnO4accessible thymine bases. The wording should be changed to reflect this distinction.4 footprinting data (4 footprinting data (4 reactivity of thymine (T) bases within the transcription bubble is identical in the presence or absence of CarD (4 footprinting data. This paragraph (subsection “The CarD-CTD interacts with the upstream ds/ss junction of the transcription bubble”), and this statement in particular, seems sufficiently clear and we have not revised the text significantly here.The claim that CarD “does not alter the transcription bubble” is primarily based on comparison of the RPo structure (accompanying paper) and the CarD/RPo structure, as illustrated in ing data . The KMning data , obtaine6) The polymerase element β1-lobe is not defined in the manuscript. Which residues does it contain? Do we understand correctly that this domain corresponds to the eukaryotic RNAP II protrusion domain? Please clarify in the text, because the eukaryotic RNAP II Rpb2 “lobe” flanks the downstream cleft and thus is different from the bacterial “lobe” – the nomenclature can lead to confusion.We have clarified that the bacterial RNAP β1-lobe corresponds to the eukaryotic RNAP II Rbp2 protrusion domain (in the fourth paragraph of the Introduction) and we have denoted the residues in the legend to 7) We recommend that the authors acknowledge that the CarD mechanism could be more complex and affect additional steps in the RPo formation.We agree with the reviewers and have added that CarD could potentially affect other steps in the transcription initiation steps (please see the Discussion).8) Is it possible to further probe the W86-T-12(nt) interaction? For example, phenylalanine activates significantly-would it be expected to make the same contact, or to change the base preference? If so, would it be informative to look at other bases in the T-12(nt) position?We probed the interaction by mutating W86 to other hydrophobic residues and showed that activation is considerably diminished but not abolished. An F (Phe) substitution shows partial activation (∼1.6 fold versus 3 fold by wild-type CarD). We hypothesize this is due to the loss of contact between the Nԑ of W86 and O2 of the thymine base. An F substitution is able to activate better than the branched or smaller hydrophobic residues because it is more similar structurally to a W. We find substituting the -12T with other bases also diminishes activation to the same extent as an F substitution (∼1.25 to 1.7 versus 3.2 fold), supporting our hypothesis that the loss of interaction between W86 and the -12T reduces the fold- activation roughly 2 fold. We have added this observation to the manuscript (in the subsection “Role of a conserved CarD Trp residue in CarD function”).9) CarD was originally described as an rRNA regulator in response to nutrient deprivation, mediating the stringent response. Now that we know the mode of action of CarD, it would be good to return to that phenotype, and describe how it might fit in.CarD was indeed originally described as a rRNA regulator in response to nutrient deprivation . Subsequ10) There is considerable redundancy, both within this paper and with the accompanying paper on RPo. The authors should simply cross-reference and shorten the present manuscript to eliminate internal redundancy.We have removed significant redundancy with the accompanying paper, mainly in the Materials and methods.


Catharanthus roseus plant is the source of many valuable terpenoid indole alkaloids (TIAs), including the anticancer compounds vinblastine and vincristine. Transcription factors (TFs) are promising metabolic engineering targets due to their ability to regulate multiple biosynthetic pathway genes. To increase TIA biosynthesis, we elicited the TIA transcriptional activators (ORCAs and other unidentified TFs) with the plant hormone, methyl jasmonate (MJ), while simultaneously silencing the expression of the transcriptional repressor ZCT1. To silence ZCT1, we developed transgenic hairy root cultures of C. roseus that expressed an estrogen-inducible Zct1 hairpin for activating RNA interference. The presence of 17β-estradiol (5μM) effectively depleted Zct1 in hairy root cultures elicited with MJ dosages that either optimize or inhibit TIA production (250 or 1000μM). However, silencing Zct1 was not sufficient to increase TIA production or the expression of the TIA biosynthetic genes , illustrating the tight regulation of TIA biosynthesis. The repression of the TIA biosynthetic genes at the inhibitory MJ dosage does not appear to be solely regulated by ZCT1. For instance, while Zct1 and Zct2 levels decreased through activating the Zct1 hairpin, Zct3 levels remained elevated. Since ZCT repressors have redundant yet distinct functions, silencing all three ZCTs may be necessary to relieve their repression of alkaloid biosynthesis.The  Catharanthus roseus plant is the source of many valuable terpenoid indole alkaloids (TIAs), including the anticancer compounds vinblastine and vincristine. Despite the low levels of these compounds in C. roseus (0.0002 wt%), these pharmaceuticals continue to be used for cancer treatments [C. roseus cultures. In particular, C. roseus hairy root cultures are model systems for studying the production of TIAs due to their genetic and biochemical stability, and fast growth in hormone-free media.The eatments . The streus 0.000 wt%, theAsα, Tdc, Asα + Tdc, Asα + Asβ, or Asα + Asβ + Tdc; see Dxs and G10h, also known as G8o) did not considerably increase TIA levels [TIAs are the condensation products of two precursor pathways. Tryptamine from the indole pathway and secologanin from the terpenoid pathway condense to form strictosidine, the backbone of TIAs . InitialA levels .C. roseus have focused on engineering the transcriptional regulation of TIAs through TFs [Transcription factors (TFs) are promising metabolic engineering targets due to their ability to regulate multiple biosynthetic pathway genes . Therefoough TFs ,18. The ough TFs ,19–25 anough TFs ,26–28. Jough TFs .Catharanthus AP2/ERF domain) transcription factors, including ORCA2 and ORCA3, are well-known transcriptional activators of several biosynthetic genes in C. roseus  [G10h was not expressed, TIA production did not increase except upon addition of loganin. When Orca3 was overexpressed in C. roseus hairy roots, TIA levels did not significantly increase (even with loganin addition) despite increased expression of several biosynthetic genes  [Orca2 in hairy roots increased the levels of certain metabolites  while other metabolites decreased  [Str, T16h, and D4h). Despite the increases in biosynthetic gene expression upon Orca overexpression, elicitation with JA alone produced a larger increase on TIA levels [The ORCA (octadecanoid-responsive . roseus . The oveand D4h) . Since Gand Str) . Similarericine)  even witA levels , since sA levels . These rA levels . The ove2/His2-type (transcription factor IIIA-type) zinc finger protein family, ZCT1, ZCT2, and ZCT3, are transcriptional repressors that inhibit the expression of Tdc and Str [Tdc and Str by ORCAs [C. roseus hairy roots, optimum MJ dosages favored high transcript levels of Orca to Zct while inhibitory MJ dosages repressed TIA levels and induced high transcript levels of Zct to Orca [Zcts, Zct1 responded most strongly to increased MJ. Furthermore, overexpression of Orca2 [Orca3 [Zcts, especially Zct1 and Zct2. Therefore, our strategy to enhance TIA biosynthesis involves MJ to induce TIA genes through ORCAs and other unidentified TFs while simultaneously silencing ZCT1 to counter its activation by MJ and ORCA.Three members of the Cys and Str . ZCT proby ORCAs . In C. r to Orca . While Mof Orca2  or Orca32 [Orca3 ,30 activZct1 in C. roseus hairy roots elicited with MJ. We employed a previously characterized estradiol-inducible system [Zct1 silencing, and analyzed the effect of Zct1 silencing on TIA production. To further understand the regulation of TIAs in C. roseus, we monitored the expression of biosynthetic genes and other transcription factors. Zct1 silencing did not affect TIA biosynthesis or the expression of biosynthetic genes and transcription factors, illustrating the tight regulation of TIA biosynthesis.In this study, we explored the effect of silencing e system  to succeArabidopsis actin-11 gene with a multiple cloning site (MCS) on each side of the intron. Two 163bp fragments of Zct1 (Genbank accession AJ632082) were amplified from C. roseus cDNA using primers containing restriction sites and cloned into each of the MCSs to generate a hairpin in the pSK-Int vector (Zct1 hairpin (Zct1hp) was subsequently removed from pSK-Int and cloned into the pER8 backbone using restriction cloning (XhoI-SpeI) (pER8-GFP and pSK-Int were obtained from Dr. Nam-Hai Chua (The Rockefeller University). The pSK-Int vector is an intermediate cloning plasmid for generating hairpin RNAi constructs. It contains the third intron from the t vector . The ZctoI-SpeI) . A GFP-hoI-SpeI) . The GFPEscherichia coli competent cells were used for cloning . E. coli was grown in LB media  at 37°C overnight at 250 rpm when in liquid culture. The pER8-Zct1hp and pER8-GFPhp constructs were electroporated into Agrobacterium rhizogenes R1000 (ATCC 43056) as previously described, except LB media was used instead of YM [DH5α ad of YM . R1000 wC. roseus seedlings were germinated from seeds , grown aseptically, and transformed with A. rhizogenes R1000 containing pER8-Zct1hp, as previously described [Agrobacterium-mediated transformation method and estrogen-inducible construct [escribed . Of the onstruct .C. roseus hairy roots [Of these 144 transgenic Zct1hp lines, 26 well-growing lines were originally adapted to liquid culture, of which 8 lines survived long-term subculture in liquid (over 16 subcultures at the time of publication). Of the 71 transgenic GFPhp lines, 36 were originally adapted to liquid culture, of which 10 have survived long-term subculture in liquid (6–7 subcultures at the time of publication) and were subcultured every 28-days and maintained as previously reported [ry roots .C. roseus hairy root cultures generated by transformation with A. rhizogenes strain 15834 without any additional plasmids) and transgenic C. roseus roots using the CTAB method as previously described [Rps9 (the housekeeping gene), LexA, hygR, virD, and rolC , LexA , hygR (hygromycin-resistance gene used for selection), and rolC , which indicates the successful elimination of Agrobacterium and confirms the transgenes are not due to any contaminating Agrobacterium.All transgenic Zct1hp and GFPhp lines have C. roseus hairy roots were induced with 5μM 17β-estradiol (Fisher Scientific) on day 26 . Previously, induction with 5μM 17β-estradiol showed strong and tightly regulated expression of GFP in control transgenic C. roseus hairy roots [To express the silencing hairpin (Zct1hp or GFPhp), transgenic ry roots . Stock sC. roseus hairy roots were blotted and flash-frozen in LN2.Cultures were elicited with MJ  24 h after induction with 17β-estradiol. Stock solutions were prepared in ethanol  and added to 50mL of culture media to achieve final concentrations of 250μM or 1000μM MJ. Cultures were harvested 8, 24, and 48 h after MJ addition for mRNA analysis or after 3, 5, and 7 days for TIA metabolite analysis. C. roseus hairy root cultures as previously described [TIAs were extracted from transgenic escribed . In shorC. roseus hairy root extracts were analyzed through HPLC  and separated using a reversed-phase C18 column .TIA levels in the The HPLC mobile phases for TIA separation were: 99.9% water with 0.1% (v/v) formic acid as the aqueous phase, and 99.9% acetonitrile with 0.1% (v/v) formic acid as the organic phase. The protocol consisted of the following steps: 1) 90% aqueous and 10% organic as the initial condition, 2) gradient to 70% aqueous and 30% organic over 20 minutes, 3) gradient to 100% organic over 8 minutes, 4) gradient to 90% aqueous and 10% organic over 10 minutes, and 5) isocratically at 90% aqueous and 10% organic for 20 minutes to equilibrate the column for the next injection. All the flow rates were maintained at 1.0 mL/min.Strictosidine , ajmalicine , serpentine , tabersonine-like peak 5 (329 nm), tabersonine-like peak 6 (329 nm), and tabersonine  were monitored at the respective wavelengths and quantified with calibration curves using pure standards or Beer’s Law correlations (for strictosidine). The UV absorbance spectrum of Peak 5 and 6 were similar to that of tabersonine . MS analC. roseus hairy root cultures by qPCR. mRNA was extracted from frozen hairy root cultures using the RNAzol®RT (Molecular Research Center) method and quantified using a NanoDrop . Extracted RNA was treated with DNase to remove genomic DNA, and cDNA was synthesized from the mRNA .Transcript levels of transcription factor and TIA biosynthetic genes were monitored in the transgenic Orca2, Orca3, Zct1, Zct2, and Zct3) and TIA biosynthetic genes  were monitored using the primers listed in 2 Real-TimeTM SYBR Green/ROX PCR master mix (SABiosciences) and QuantStudio 6 (Applied Biosystems) using the thermocycler protocol previously described [Transcription factor genes  as a control for the effects of RNAi (referred to as GFPhp in this text).The estrogen-inducible XVE system is made up of an artificial XVE transcription factor containing the DNA-binding domain of the bacterial repressor Lereceptor . The XVEry roots . TherefoOf the 428 Zct1hp hairy roots generated, 144 survived two rounds of selection on hygromycin and 26 of these were transferred to liquid media. Of the 255 GFPhp roots generated, 71 survived two rounds of hygromycin selection and 36 of these were transferred to liquid media . We veriZct1 silencing under estradiol induction in transgenic C. roseus hairy root cultures through qPCR. 17β-estradiol (5μM) was added to three individual Zct1hp lines  for 24 h since estrogen-inducible gene expression was high at this concentration and timepoint [Zct1 silencing varied between the three estradiol-induced Zct1hp lines, with the Zct1 levels ranging from 0.35- to 0.9-fold of their untreated controls  was first added to Zct1hp lines for 24 h, followed by the addition of either 250μM or 1000μM MJ for 8, 24, and 48 h [Zct1 expression was monitored through qPCR.MJ induces TIA production through the action of several transcriptional activators  ,20,23,25oduction . Therefoand 48 h ,31. Zct1Zct1 in Zct1hp-38 line, and this decrease was sustained over 48 h , tabersonine (p = 0.01), and representative tabersonine-like compounds , but no further increases occurred with both 17β-estradiol and 25003B0043M MJ  was added to Zct1hp-38 for 24 h, followed by 250μM or 1000μM MJ for 8, 24, and 48 h [G10h, Tdc, and Str was monitored by qPCR.To understand why TIA levels did not further increase with  and Str  using inand 48 h . The expG10h, Tdc, and Str increased upon treatment with 250μM MJ, but did not increase further upon treatment with 17β-estradiol and 250μM MJ , suggesting that other transcription factors induced by MJ or other unidentified mechanisms contribute to the repression of TIAs.TIA gene expression was not increased upon Zct1 was successfully silenced, expression of TIA genes regulated by Zct1 was unchanged  was added to Zct1hp-38 for 24 h followed by the addition of 250μM or 1000μM MJ for 8, 24, and 48 h. We monitored Orca2 and Orca3, transcriptional activators of Tdc and Str, and Zct2 and Zct3, other transcriptional repressors of Tdc and Str, through qPCR.Although nchanged . To inveOrca2 and Orca3 by 7 and 33-fold after 48 h, respectively, while 1000μM MJ lowered Orca2 and Orca3 to 2 and 12-fold  levels remained low through silencing, but Zct3 levels remained elevated. Increases in TIA production may still have been limited due to high basal levels of ZCT3 (and ZCT2). Basal levels of Zct  were more abundant than basal levels of Orca  in the hairy root cultures  than ZCT3 (27 kDa), which also has a longer spacer between its zinc finger motifs [completely silenced, the non-silenced ZCT proteins may still be limiting TIA gene expression in the absence of ZCT1. Also, since the ZCT repressors have distinct functions, silencing all three ZCTs simultaneously may be necessary to relieve their repression on alkaloid biosynthesis.Although the three ZCT proteins are thought to be functionally similar in repressing r motifs . Recentlr motifs . These fC. roseus hairy roots with inducible Zct1 silencing through RNAi. Upon induction with 17β-estradiol, Zct1 induction by MJ at both low (250μM) and high (1000μM) concentrations was considerably reduced. Despite these low levels of Zct1, MJ-elicited TIA production and TIA gene expression  did not further increase in Zct1 silenced cultures. Due to high homology, levels of Zct2 (but not Zct3) were also silenced in Zct1 silenced lines under MJ-elicited conditions. The ZCT repressors are thought to play overlapping but distinct functions in C. roseus. Since basal levels of Zct2 and Zct3 were high, silencing the expression of all three ZCTs simultaneously may be necessary to eliminate the repression by the ZCTs and to therefore increase TIA production.In summary, we successfully developed transgenic S1 Fig(TIFF)Click here for additional data file.S2 FigRps9 (housekeeping gene), LexA, hygR, virD2 (Agrobacterium control), and rolC (hairy root control) genomic DNA was amplified in 10 Zct1hp and 9 GFPhp transgenic lines by PCR. WT = wild-type hairy roots, R1000 = A. rhizogenes containing pER8-Zct1hp or pER8-GFPhp plasmid, NT = no template control.(TIFF)Click here for additional data file.S3 FigHPLC traces at 329 and 254 nm, and UV spectra of the associated peaks.(TIFF)Click here for additional data file.S4 Fig17β-estradiol (5μM) was added for 24 h, then 250μM MJ was added for the time specified. Error bars represent standard deviations of qPCR triplicates.(TIFF)Click here for additional data file.S5 Fig17β-estradiol (5μM) was added for 24 h, then 250μM or 1000μM MJ was added for the time specified (3 and 7 d). The TIA were separated by HPLC and quantified by UV absorbance. Error bars represent standard deviations between two biological replicates.(TIFF)Click here for additional data file.S6 Fig17β-estradiol (5μM) was added for 24 h, then 250μM MJ was added for the time specified (8 and 24 h). Error bars represent standard deviations of qPCR triplicates.(TIFF)Click here for additional data file.S7 Fig17β-estradiol (5μM) was added for 24 h, then 250μM MJ was added for the time specified (8 and 48 h). Error bars represent standard deviations of qPCR triplicates.(TIFF)Click here for additional data file.S8 Fig17β-estradiol (5μM) was added for 24 h, then 250μM MJ was added for the time specified (8 and 24 h). Error bars represent standard deviations of qPCR triplicates.(TIFF)Click here for additional data file.S9 Fig17β-estradiol (5μM) was added for 24 h, then 250μM MJ was added for the time specified (8 and 48 h). Error bars represent standard deviations of qPCR triplicates.(TIFF)Click here for additional data file.S10 FigRps9 (housekeeping gene) is higher than the transcript level of the specific gene monitored. ΔCt of 1 represents a 2-fold difference in transcript levels. Error bars represent standard deviation of three transgenic lines. Statistical significance calculated used Student’s t-test; * denotes p< 0.05.Negative values of ΔCt mean that the transcript level of (TIFF)Click here for additional data file.S1 TableASα = anthranilate synthase α subunit; ASβ = anthranilate synthase β subunit; TDC = tryptophan decarboxylase; DXS = 1-deoxy-D-xylulose-synthase; G10H = geraniol-10-hydroxylase; DAT = deacetylvindoline 4-O-acetyltransferase.(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table*primer sequences were adapted from . **prime(DOCX)Click here for additional data file.S4 TableOrca and Zct primers were previously described in [Tdc and G10h primers were previously described in [Str primers are newly designed.ribed in . Tdc andribed in . Str pri(DOCX)Click here for additional data file.


Intrinsically disordered proteins/regions (IDPs/IDRs) are proteins or peptide segments that fail to form stable 3-dimensional structures in the absence of partner proteins. They are abundant in eukaryotic proteomes and are often associated with human diseases, but their biological functions have been elusive to study. In this study, we report the identification of a tin(IV) oxochloride-derived cluster that binds an evolutionarily conserved IDR within the metazoan TFIID transcription complex. Binding arrests an isomerization of promoter-bound TFIID that is required for the engagement of Pol II during the first (de novo) round of transcription initiation. However, the specific chemical probe does not affect reinitiation, which requires the re-entry of Pol II, thus, mechanistically distinguishing these two modes of transcription initiation. This work also suggests a new avenue for targeting the elusive IDRs by harnessing certain features of metal-based complexes for mechanistic studies, and for the development of novel pharmaceutical interventions.DOI:http://dx.doi.org/10.7554/eLife.07777.001 DNA contains instructions to make all the proteins and other molecules that drive essential processes in cells. To issue such specific sets of instructions, a section of DNA—called a gene—is first copied to make molecules of messenger ribonucleic acid (or mRNA for short) in a process called transcription. This process is tightly regulated in all living organisms so that only a subset of genes are actively transcribed at any time in a given cell.A group or ‘complex’ of proteins called TFIID plays an essential role in starting the transcription of genes that encode proteins in humans and other eukaryotic organisms. However, it is tricky to study how TFIID works because mutant cells that are missing individual components of the complex are unable to properly transcribe the required genes and soon die. Consequently, many studies of TFIID have used purified proteins in artificial systems where it is possible to examine particular aspects of TFIID activity in depth.Here, Zhang et al. used a combination of chemistry, biochemistry, and molecular biology techniques to identify a new molecule that can selectively bind to the TFIID complex. In an artificial system containing purified proteins and other molecules, this molecule ‘locks’ TFIID onto DNA and prevents a change in shape that is required for transcription to start. The experiments show that this rearrangement is only required to make the first mRNA copy of a gene because the molecule had no effect on initiating further rounds of transcription on the same DNA.Zhang et al.'s findings reveal that TFIID is very dynamic in controlling transcription, and that subsequent rounds of transcription follow a different path to make mRNAs. The next steps are to use new techniques such as single-molecule imaging to directly visualize the molecules involved in transcription, and to use the new molecule to block the start of transcription in living cells.DOI:http://dx.doi.org/10.7554/eLife.07777.002 Intrinsically disordered proteins/regions (IDPs/IDRs) constitute a significant fraction of the metazoan proteome . By virtIDRs are typically composed of low-complexity sequences and are often rich in polar amino acid residues, making them challenging targets for intervention by conventional small-molecule inhibitors, which often require stable hydrophobic-binding pockets . HoweverTranscription initiation by eukaryotic RNA polymerase II (Pol II) is a highly regulated process requiring the coordinated actions of Pol II and a group of general transcription factors  . Of all TFIID is a protein complex composed of the TATA-binding protein (TBP) and ∼14 TBP-associated factors (TAFs) . TBP recIn a search for small molecule compounds to selectively perturb TFIID function, we identified a tin-based metal cluster as a specific binder and modulator targeting an IDR within the TAF2 subunit of metazoan TFIID. By virtue of its specificity for interfering with the first round of transcription initiation, this metal cluster compound serves as a useful tool for studying the role of TFIID in both transcription initiation and reinitiation. This non-POM metal cluster revealed a novel mode of interaction with a low-complexity protein domain, demonstrating the feasibility of using metal-based compounds to selectively target IDRs.1, ChemDiv 7241-4207) that reproducibly and selectively bound to both Drosophila and human TFIID  inhibited both Drosophila and human TFIID-directed transcription, but not transcription directed by TBP  cad by TBP , confirmd by TBP . Taken t1 in-house and were surprised to find that the resynthesized compound was completely inactive in the transcription assay (2) with varying levels of inhibitory activity (2) added as an anti-oxidant in the final recrystallization step in a subset of the commercially supplied samples  C-terminal fragment (residues [1125–1221]) bound selectively to the original tin-oxochloride containing sample in the arrayed library (Drosophila and human TAF2 (Identification of tin(IV) oxochloride led us to consider presumptive targets that should be histidine-rich domains of phylogenetically conserved TAF subunits. Testing this hypothesis, we found that a GST fusion of the histidine-rich  library . The tarman TAF2   oxochloride cluster as responsible for binding to the GST-dTAF2 (1125–1221) fragment, we carried out an independent surface plasmon resonance assay . In thisPrevious reports indicated that TAF2 can directly interact with DNA and is required for the recognition of the Inr element by TFIID . In addiThese results led us to re-examine the finding in We thus hypothesized that there is some sort of synergy between DNA and the inhibitor in binding to the TAF2-disordered region, and further examined this interaction in the context of holo-TFIID complex using a DNase I footprinting assay. We found the holo-TFIID protects an extended area of the promoter DNA from DNase I digestion, as previously reported on the super core promoter (SCP1)  Figure . The sigIt has generally been accepted that TFIID-promoter binding is a rate-limiting step in transcription initiation, and overall affinity of TFIID for promoter DNA correlates with transcription activity . It is i32P labeled guanosine triphosphate  in the reaction. In this assay, a productive PIC would lead to the synthesis an ApG dinucleotide that is detected by autoradiography. Indeed, we observed inhibition of ApG synthesis  during transcription targeted by the inhibitor, we examined the synthesis of the very first dinucleotide during initiation, using only adenosine triphossphate  and alpha-ynthesis , suggestTo test which step during PIC assembly is affected by the inhibitor, we next performed a series of experiments in which DNA templates were pre-incubated with only a subset of protein factors  to mainInterestingly, pre-incubation of TFIID or TFIID together with TFIIB with the DNA template led to a more severe inhibition  , and theTo examine the effect of the chemical inhibitor on this isomerization step, we used suboptimal levels of TFIID to first assemble a TFIID-TFIIB sub-complex at the promoter. This sub-complex gives a weak footprint   and theyThe tight correlation between Pol II engagement and inhibitor sensitivity does not rule out the possibility of a mutual steric exclusion between inhibitor binding to the TAF2 IDR and Pol II engagement at the promoter, which may be independent of the conformational isomerization. Because Pol II and TFIIF are thought to leave the promoter during the elongation phase after de novo transcription initiation, while TFIID may be retained at the promoter as part of a reinitiation scaffold , we reasDrosophila embryo nuclear extracts and a dual template assay system, Kadonaga has demonstrated that each round of transcription requires complete assembly and disassembly of the PIC. Thus, at least in vitro, under certain transcription conditions, there is no commitment of any limiting transcription factor to the initial template to form a pre-licensed reinitiation scaffold , indicating the detection of one de novo round of transcription initiation plus ∼2 rounds of reinitiation. In the absence of Sarkosyl treatment, the presence and pre-incubation of DNA 1 with the protein factors still severely compromised (by 12–16-folds) transcription from DNA 2 . As expected, this template commitment under multi-round transcription conditions can also be partially alleviated by the addition of extra TFIID (by ∼sixfold) (lanes 16 and 19). These findings suggest that commitment of the limiting factor (including TFIID) to DNA 1 during de novo transcription initiation mostly persists through multiple rounds of reinitiation. Therefore, as reported with other human transcription systems, our highly purified system also involves a stable reinitiation scaffold of which TFIID is likely a critical component.To probe the functional states of TFIID (particularly that of the TAF2 IDR) during transcription reinitiation, we treated the reaction with the inhibitor either before or after PIC assembly, either under the condition that would allow multiple rounds of transcription or under Sarkosyl treatment condition limiting transcription to the first, de novo round . As expe32P labeled CTP as a substrate, this system will allow the first Pol II molecule to synthesize a nascent transcript corresponding to the length of the G-less cassette, followed by progressively shorter transcripts that result from reinitiation  oxochloride-derived inhibitor turned out to be a newly identified TAF2 DNA-binding domain that likely becomes structured upon DNA binding, perhaps reminiscent of basic domains in the leucine zipper family of activators . On the One possible explanation for the inhibition mechanism is an over-stabilization of TAF2 IDR-DNA interaction by the inhibitor. In this model, the inhibitor-stabilized TAF2 IDR is an impediment to full Pol II engagement. We postulate that this Pol II-dependent isomerization is a multi-step process that engages an extended region of the promoter DNA that gets blocked at the Inr by the inhibitor. This model is consistent with our DNase I footprinting results. Alternatively, if the TAF2 IDR is an active helper for Pol II to overcome the barrier posed by other bound factors (like TFIID) to interact with the upstream DNA and the associated protein factors (such as TFIIB), interference of TAF2 IDR function could also lead to a partially impeded Pol II engagement. This is reasonable because IDRs are known to interact with multiple partners with diverse functions . Based oGiven that IDRs are highly sensitive to their local molecular environment , it is pThe tin-based chemical reported here is the first known reagent capable of directly and mechanistically distinguishing the functional states of TFIID during initiation and reinitiation and discriminating between these two modes of transcription. Reinitiation is likely an important aspect of transcription regulation in vivo, possibly related to ‘bursting’ , but itsWe should point out that de novo initiation and reinitiation may not necessarily represent mechanistically distinct pathways under certain conditions in vitro. Numerous possibilities have been proposed by researchers for the apparent differences in observing disengagement of PIC components at each round  vs retenAlthough the newly identified compound may present complications for direct in vivo applications, this reagent revealed an intriguing possibility of specifically blocking de novo transcription initiation without affecting reinitiation via targeting of TFIID. Interestingly, the canonical TFIID complex has been reported in several cases to be dispensable for ongoing transcription in terminally differentiated, non-dividing cells . AccordiOur unbiased de novo identification of a tin-based metal cluster as a selective transcription inhibitor provides an example of a potential category of metal-based biologically active compounds that is distinct from the conventional transition metal-based POMs . The criDrosophila melanogaster embryos was prepared as described , and (His)6-Halo fusion proteins was purified by Ni-NTA agarose resin  to ∼>90% purity in SDS-PAGE Coomassie brilliant blue-stained gels. Nuclease treatment of recombinant protein for small-molecule microarray binding was carried out on beads before elution with 0.5 U/µl DNase I  and 10 ng/µl RNase  at room temperature with mixing for 1 hr in a buffer containing 50 mM Tris pH 8.0, 5 mM MgCl2, 2.5 mM CaCl2, 5% glycerol, and 1 mM dithiothreitol (DTT).Recombinant proteins were expressed in G3-BCAT plasmid  starting at position +155 (+192 for the ‘L’ DNA template) relative to the transcription start site was used. For the DNase I footprinting assay, the reverse primer was used together with a forward primer (5′ CATAACCTTATGTATCATACACATACG 3′), starting at position −153, to generate a DNA template by PCR. DNA oligonucleotide primers were custom synthesized . Both transcription and footprinting assays used wild-type SCP1-containing DNA unless otherwise specified.Small-molecule microarray manufacture and screening were carried out as described . In brie2 cluster on the microarray is likely mediated through coordinating interactions and/or local polymerization of the material, which will preserve similar surface features as proposed in Note: the retention of SnOCl6-Halo-tagged reference protein  were immobilized through Halo tag on the reference and active cell, respectively (no evidence for direct interaction between the (His)6 tag and the inhibitor was observed). The inhibitor binding assay was performed in running buffer  (HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; EDTA: ethylenediaminetetraacetic acid; DMSO: dimethyl sulfoxide) at a flow rate of 30 µl/min with 120 s of association and dissociation at 25°C. SnOCl2 or SnOCl2·pyridine was prepared at concentration of 5 µg/ml in different buffers adjusted with citrate for lower pH or supplemented with imidazole at specified concentrations.The surface plasmon resonance experiments were performed using a BIACORE T100 . GST and GST tagged dTAF2(1125–1221) were captured on reference cell and active cell, respectively, through GST capturing kit  on CM5 sensor chip . For the capturing of hTAF2 (990–1199), a HaloTag Amine(O4) Ligand  was immobilized on all flow cells using amine-coupling chemistry on CM5 sensor chip. A (His) protein ) and a  and the signals were quantified by ImageQuant TL software .For TAF2 IDR-DNA binding, GST fusions immobilized on glutathione sepharose 4B beads (∼2 mg/ml) were briefly pretreated with 100 µg/ml RNase A in PBST in the presence of complete protease inhibitor (Roche) to remove endogenous nucleic acid (predominantly RNA) from 2, 100 mM KCl, 0.1 mM EDTA, 0.01% NP40, 0.02% Tween 20, 1 mM DTT, and 100 µg/ml BSA). The chemicals or the DMSO control was dissolved with the same GTF buffer , kept on ice for a few hours, spun at 15,000 rpm for 15 min, before they were used to treat the protein factors  at specified concentrations at room temperature (23°C) for 15 min. Next, 100 ng DNA in 12.5 µl DNA buffer (0.2 µl RNasin (Promega) and 8 mM spermidine in water) was added and incubated at 30°C for 30 min for PIC assembly. NTPs were then added to 0.5 mM each to allow RNA synthesis at 30°C for 30 min. RNA products were analyzed by primer extension and 6% urea-polyacrylamide gel electrophoresis, scanned by Typhoon scanner , and quantified by ImageQuant TL software .The procedure was as described  with min32P labeled GTP, 3000 Ci/mmole, 10 mCi/ml, Perkin Elmer, Waltham, MA); (2) the transcription was stopped by incubation at 65°C for 30 min, followed by 4 U shrimp alkaline phosphatase (New England Biolabs (NEB), Ipswich, MA) treatment at 37°C for 1 hr; and (3) the end product was separated by 15% TBE-urea gel .For the dinucleotide synthesis monitoring abortive initiation, following modifications were made according to a previous report : (1) theFor the step-wise PIC assembly test shown in 32P labeled CTP, 3000 Ci/mmole, 10 mCi/ml, Perkin Elmer); (3) no GTP was included in the reaction; (4) the reaction was stopped with 100 µl of Stop Solution , further incubated at 55°C for 10 min, then extracted by phenol:chloroform:isoamylalcohol (50:49:1) and precipitated with ethanol; (5) the precipitation pellet was re-suspended in 4 µl buffer (50 mM Tris pH 7.5 and 2 mM EDTA) containing 10 U/µl RNase T1 (Thermo Fisher), digested at 37°C for 10 min, then separated by 6% polyacrylamide-urea denaturing gel.The colliding polymerase assay using G-less cassette DNA template has the following modifications based on a previous report : (1) theSarkosyl was tested in our system following previously a reported procedure . To restFor the two-DNA, template-commitment assay, DNA 1 (or DNA buffer only) was incubated with the complete set of protein factors in GTF buffer for 30 min at 30°C in a total volume of 12 µl, followed by an addition of 6 µl GTF buffer (with extra TFIID as an option) and 6 µl DNA 2. After incubation at 30°C for a specified period of time, NTPs were added and the incubation continued at 30°C for another 30 min for RNA synthesis. Sarkosyl was added to 0.1% immediately (within ∼30 s) after the addition of NTPs as an option to restrict transcription to a single, first round.32P) labeled primer starting at the +155 of the template strand. The TFIID-promoter binding , with or without the chemical at specified concentrations, at 30°C for 30 min. For the high-salt challenge experiments in 2, 50 mM KCl, 50 μM EDTA, 0.5 mM DTT, and 2.5 mM CaCl2) then immediately digested by pre-diluted DNase I  at 30°C for 60 s. The DNase I digestion was stopped with 100 µl of Stop Solution . The digestion products were purified, separated by 6% polyacrylamide-urea denaturing gel, and the image further processed as in the transcription assay.The assay is essentially as described  with mod binding  was carr2, 50 mM KCl, 0.05 mM EDTA, 0.005% NP40, 0.5 mM DTT, and 5 mM CaCl2) was added, and after 30 s, the digestion was stopped and further processed as described above.The DNase I footprinting assay monitoring conformational isomerization during PIC assembly  was carr2·pyridine solution (15 µg/ml in GTF buffer with 2% DMSO) or the control solution was added and incubated at room temperature for 15 min. Next, GTF set 2  was supplemented, together with 4.5 µl DNA buffer (supplemented with 36 ng purified yeast tRNA and 1 ng poly(dG:dC)) and the incubation was continued for another 30 min at 30°C. The final reaction mixture was subjected to digestion by 2 µl 40 mU/µl DNase I (NEB) and the end digestion product was analyzed by gel electrophoresis. The experiment in To monitor the inhibition of the conformational isomerization during PIC assembly ; ‘Otava’ stands for Otava Ltd ; ‘Princeton’ stands for Princeton Biomolecular Research Inc. ; and ‘LifeChem’ stands for Life Chemicals Inc . Nuclear magnetic resonance (NMR) and mass spectrometry were used by these commercial suppliers for quality control, which all indicated ‘>95%’ purity of the intended organic compounds . Commercial proton-induced X-ray emission elemental analysis  was carr2 was also purchased from Princeton Biomolecular Research Inc. for the track of the tin-related activity (available upon request) and used to draw the scheme in Details of in-house synthesis and analysis of the original lead (1) and analog (2) compounds are available upon request. Detailed procedure for resynthesis and purification of analog compound 2, sealed with a Teflon-lined screw cap and incubated at 83°C in a heat block . The obtained precipitates were vacuum filtered and quickly transferred into sealed flasks and dried under high vacuum. The rest of the simple organic and inorganic tin compounds were purchased from Sigma–Aldrich.Synthesis of the organic solvent coordinated SnClompounds  was acco2 could be achieved by refluxing in iso-propanol under air atmosphere for several hours, which led to potent TFIID-specific transcription inhibitory activity  in tetrahydrofuran (26 ml), or water (26 ml) at room temperature for 2 hr; (b) addition of H2O2  to solution of SnCl2 (2 g) in water (52.7 ml); (c) addition of sodium perborate (NaBO3) to aqueous solution of SnCl2; (d) addition of m-chloroperbenzoic acid (mCPBA) (58 mg) to a solution of SnCl2 (64 mg) in water (1.8 ml); (e) addition of tert-butyl hydroperoxide (TBHP) to solution of SnCl2 in dichloromethane. The oxygenation of SnCl2 in tetrahydrofuran by oxygen to produce (SnOCl2)X was reported previously  bubeviously . The oth2, SnOCl2 cluster was also prepared from tin(IV) chloride (SnCl4) , bis(trimethylsilyl)peroxide (BTSP)  was added drop wise, and the reaction was allowed to proceed for 1 hr. The volatiles were removed in vacuum and the residue was used for the activity assay, or to complex with pyridine.Independently of oxygenation of SnCl (SnCl4)  as previ (SnCl4) : to a co2·pyridine (SnOClpyridine  was preppyridine . review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent , a Reviewing Editor, and two peer reviewers.Thank you for submitting your work entitled “Chemical Perturbation of an Intrinsically Disordered Region of TFIID Distinguishes Two Modes of Transcription Initiation” for peer review at The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.The reviewers find the work interesting; however, they have problems with the initiation/reinitiation assay. Furthermore, Reviewer #2 has additional points that we believe will have to be addressed in a revised version.Reviewer #1:The authors report the discovery of a TFIID inhibitor that blocks the formation of PICs. Evidence is presented for the interaction of the compound with a low complexity region of TAF2 that unexpectedly stabilizes binding of TFIID to promoter DNA. Using a defined set of initiation factors and Pol II, with and without Sarkosyl, the authors conclude that the compound does not inhibit reinitiation. The data are of high quality and demonstrate that the metal complex does inhibit transcription in vitro, likely through TFIID, however the claim that reinitiation is not affected will require further proof. The manuscript is well written, but could be improved by eliminating unnecessary data.The dual template experiment  demonstrOverall, this is an interesting study (except for the description of the identification of the inhibitor) that has significant merit. A better demonstration that reinitiation is not inhibited by the compound would improve the significance of the conclusions that could be drawn. If reinitiation is really not affected, determining the identity of the resistance factor would be exciting.Reviewer #2:In this paper the authors report the discovery of a class of novel inhibitors of RNA polymerase II. These tin-based compounds apparently interact with an intrinsically disordered region of the TAF2 subunit of TFIID. If the inhibitors act before TFIID is stably associated with the template, transcription is very significantly reduced. The authors show that the footprint of TFIID on a TATA-containing promoter is altered near the INR in the presence of the inhibitor. They also show in a Sarkosyl-based reinitiation assay that the effects of the inhibitor appear to be confined to the initial round of transcription, with subsequent rounds unaffected. The authors conclude that this inhibitor affects the initial productive engagement of the polymerase with the template, but not the case of polymerase entering additional rounds of transcription.I am impressed with the amount of clever work that the authors performed to track down the actual inhibitor. There is clear potential here for novel mechanistic insights into PIC assembly, initiation and very early elongation through the use of these tin-based compounds. However, I have significant reservations about the paper as it now stands.First, it is critical to establish at what stage of transcription the inhibitor actually acts. Transcription in this paper was assayed by primer extension, usually with a primer around +150. Is polymerase able to begin transcription but is then blocked at some earlier step point? Given where the differences are in the footprints, it is tempting to imagine a blockade at initiation or clearance. For example, the initial downstream expansion of the transcription bubble might be blocked. Fortunately this would be easy to test – does the inhibitor have any effect in an abortive initiation assay? If the initial few bonds can be made in the presence of the inhibitor, how long can the transcripts become? If no RNA is made at all, can the transcription bubble form on inhibitor-treated complexes when ATP is added?The authors interpret the footprint alterations resulting from the inhibitor as evidence for an enhanced or stabilized binding of IID to the promoter. I don't think this interpretation is justified – TFIID binding as judged by overall protection in the footprint doesn't change significantly with the inhibitor. What has changed is the alignment of (presumably) TAF2 with the INR, which in turn changed the template accessibility to DNase over this region. The localized nature of these changes provokes questions specifically about transcription bubble formation, initiation and clearance, as I just noted. It would have been useful to see if these changes persisted even when IIB and IIF were present, since these factors partially rescue transcription from the effect of the inhibitor. Another point about the footprints – as best as I can tell the TATA box is not protected. Can the authors comment on that?Finally, a central point of the paper is the differential effect of the inhibitor on re-initiation versus the first round of transcription. I am afraid I am skeptical of the assay. The key assumption is that Sarkosyl at 0.1% prevents the assembly of new PICs and thus confines transcription to a single round. It is not widely appreciated, but Pol II PICs do not all initiate immediately once NTPs are added. I know from my own experience that, for example, transcription for 5 min with cold NTPs still allows robust new synthesis of labeled RNAs by Pol II PICs when labeled precursors are spiked in during the next 5 min. Critically, in these experiments the RNAs were very short (<20 nt), so by definition the observed transcription in the second labeling period could not have come from promoters which were already transcribed once in the initial 5 min. I suspect that the extra transcription seen when Sarkosyl is left out simply represents the failure of the otherwise late-starting complexes to initiate. This isn't simply based on my own experience – the Yudkovsky et al. paper cited here shows that TFIID-based complexes isomerize slowly to transcriptional competence, with a half time of 10-15 min. Does Sarkosyl affect that isomerization?The novel inhibitor identified here could provide important new insights into Pol II initiation mechanisms, but in my view more should be done to characterize the inhibitor's effects.eLife. Your revised article has been favorably evaluated by Jim Kadonaga (Senior Editor), a Reviewing Editor, and two peer reviewers. The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below.Thank you for resubmitting your work entitled “Chemical Perturbation of an Intrinsically Disordered Region of TFIID Distinguishes Two Modes of Transcription Initiation” for further consideration at In light of the following comment by Reviewer #2 we would request one small revision: “My biggest concern focuses on how the proposed mechanism is described. The authors assume , which are conditions that we felt were not robust and could compromise the reproducibility of our experiments. On the other hand, when added immediately after NTPs, a broad range of Sarkosyl concentrations from 0.04% to at least 0.64%, give the same result of preventing reinitiation. Therefore we chose to add 0.1% Sarkosyl within 30 seconds after NTPs addition to restrict transcription to a single round.How long after NTPs were added was the Sarkosyl added?The answer is within ∼30 seconds. This much incubation time is sufficient for the formation of the first 1∼2 phosphodiester bonds which will render much stronger Sarkosyl resistance . Under our experimental setup, this 30 min incubation time might allow some additional de novo PIC assembly  changes in the protection patterns consistent with conformational changes and “isomerization” over an extended region of the promoter that is arrested by the inhibitor . Please It would have been useful to see if these changes persisted even when IIB and IIF were present, since these factors partially rescue transcription from the effect of the inhibitor.Actually, TFIIB doesn’t rescue transcription from the inhibition, while TFIIF does so only partially . Since TAnother point about the footprints – as best as I can tell the TATA box is not protected. Can the authors comment on that? It has been previously reported that TFIID fails to efficiently protect the TATA box at the super core promoter , and that TFIIA can facilitate a structural transition to cause TATA box protection . In our transcription assays, TFIIA had no detectable effect. However, we noticed that TATA box protection became increasingly evident upon the addition of TFIIB and TFIIF, and complete protection occurred once Pol II was included in the system, which is consistent with PIC assembly in the absence of TFIIA .Finally, a central point of the paper is the differential effect of the inhibitor on re-initiation versus the first round of transcription. I am afraid I am skeptical of the assay.To address this reservation, we have now performed an independent assay to verify the occurrence of reinitiation in our system and its resistance to the inhibitor .I suspect that the extra transcription seen when Sarkosyl is left out simply represents the failure of the otherwise late-starting complexes to initiate.In addition to the independent validation of reinitiation, we have also performed titrations of Sarkosyl at different time points to compare with the original Hawley and Roeder (1985 JBC) results. We observed very similar responses to Sarkosyl as previously reported . In thatDoes Sarkosyl affect that isomerization? The TFIID-DNA complex isomerization described by Yakovchuk et al. (the Yudkovsky paper is on reinitiation)  that Pol II is recruited to the inhibited complex but somehow cannot function. However, they don't actually demonstrate that Pol II is recruited- couldn't the inhibitor work to change TFIID's conformation so that Pol II cannot join the nascent PIC? I think an argument in this direction can be made based on the right-most panel of– it would seem that the downstream (+16 through about +40) footprint changes characteristic of the complete complex still appear when Pol II is added after the inhibitor, but the TATA to +1 changes are reduced. This seems, at least to me, to suggest that Pol II is present in the inhibited complex even though it cannot access the transcription start site.We agree with Reviewer #2 and thank him/her for the cautious and accurate assessment of the potential Pol II interaction with downstream promoter DNA in the presence of the inhibitor. We have modified the corresponding Results section (see the subsection “The inhibitor arrests an isomerization step required for full Pol II engagement”) and


Illingworth et al. show that the ability of Ring1B, a core component of PRC1, to ubiquitinate histone H2A is dispensable for early mouse embryonic development and much of the gene repression activity of PRC1. Polycomb-repressive complex 1 (PRC1) and PRC2 maintain repression at many developmental genes in mouse embryonic stem cells and are required for early development. However, it is still unclear how they are targeted and how they function. We show that the ability of RING1B, a core component of PRC1, to ubiquitinate histone H2A is dispensable for early mouse embryonic development and much of the gene repression activity of PRC1. Our data support a model in which PRC1 and PRC2 reinforce each other's binding but suggest that the key functions of PRC1 lie beyond the enzymatic capabilities of RING1B. Drosophila Polycomb) whose chromodomains are able to bind H3K27me3  complexes. Polycomb-repressive complex 2 (PRC2) is responsible for trimethylation of Lys27 on histone H3 (H3K27me3) via the EZH2 or EZH1 protein subunit . CanonicH3K27me3 . PRC1 alH3K27me3 . These nHox gene repression by ectopic expression of a catalytically inactive RING1B in Ring1B-null mouse embryonic stem cells (mESCs) suggested that the repressive (and chromatin compaction) activities of canonical PRC1 may be largely independent of RING1B-mediated H2A ubiquitination  mESCs, catalytically inactive RING1B also permits development to progress much further than in Ring1B-null mice . This amino acid change has been shown to disrupt the interaction of RING1B with the E2 UBCH5C and ablates the ability of RING1B to act as an E3 ligase in vitro  and homozygous (Ring1BI53A/I53A) knock-in alleles at the endogenous Ring1B locus in E14TG2a mESCs  showed up-regulation and down-regulation, respectively. Differentially expressed genes in Ring1BI53A/I53A overlap well (41 of 55 up-regulated and 19 of 25 down-regulated) with those also showing differential expression in Ring1B−/− mESCs. Even for this small number of “nonrescued” genes, the fold change in up-regulation relative to wild type is lower in Ring1BI53A/I53A than in Ring1B−/−  revealed that RING1B is present at multiple canonical Polycomb target sites in Ring1BI53A/I53A cells, albeit at reduced levels compared with wild type . Manual inspection of normalized mapped ChIP-seq reads showed that the qPCR results were replicated in the sequencing data  increase in ChIP-seq signal in the Ring1BI53A/I53A mESCs , and occurred preferentially within gene bodies . To determine whether RING1B and H3K27me3 levels may be simply tracking transcription, we compared RING1B/H3K27me3 levels over rescued and nonrescued genes, and this revealed that the genes most up-regulated in Ring1BI53A/I53A cells do indeed have a higher loss of H3K27me3 and RING1B ChIP signal . Despite this, the abundance of sites with reduced H3K27me3/RING1B levels in Ring1BI53A/I53A cells greatly exceeds the number of differentially expressed genes, suggesting that altered expression alone is not the principal driver of these chromatin changes.In mammalian genomes, the placement of PRC2 has been suggested to occur primarily at CpG islands . The cond levels C,D. Thes3A mESCs D, sugges3A cells E,F, the Ring1BI53A/I53A. Two lines of evidence suggest that this is not the case. First, there are no obvious problems with the composition of canonical PRC1 that contains RING1BI53A  identified highly variable levels of H3K27me3 with no loss or gain of H3K27me3 as a group. Consequently, we believe that our data provide some support for the self-reinforcing recruitment model mediated in part by H2AK119ub.While our data align with the model in which PRC1 enzymatic activity can direct PRC2 recruitment, we cannot easily discount the possibility that abrogated H3K27me3 deposition is due to an alternative deficit in the function of NG1BI53A . MoreoveRing1BI53A+/ mice from heterozygous knock-in mESCs. Correct targeting was validated in embryonic day 12.5 (E12.5) embryos using the PCR strategy illustrated in Ring1BI53A/I53A embryos when compared with wild type , suggesting that the E3 ligase activity of RING1B is required for full murine embryonic development. However, in contrast to the reported embryonic lethality of Ring1B−/− by E10.5, we found that Ring1BI53A/I53A embryos could complete gastrulation and develop to E15.5, albeit at sub-Mendelian frequencies  , suggesting some defects in development of the cardiovascular system. One of these three E15.5 Ring1BI53A/I53A embryos exhibited exencephaly  C–E. No ged edema E, arrowsncephaly . It has f CDKN2A . However53A ESCs D, and soRing1B-null ESCs and support embryonic development to an extent much greater than that reported for Ring1B knockout. Our findings support the notion that loss of RING1B E3 ligase activity and the consequent loss of most H2AK119ub only partially disrupt polycomb recruitment and function, consistent with the ability of ectopically expressed catalytically inactive RING1B to maintain chromatin compaction at polycomb target loci  was generated by BAC recombineering  cloned into PL253 by gap repair. A floxed neomycin resistance cassette was then integrated into intron 3  in this gap-repaired plasmid using a mini targeting vector. The resulting plasmid was linearized and electroporated into E14TG2a ESCs  and a wild-type version of Ring1B exon 3 was modified so that a lone LoxP site was introduced into intron 2 , and a floxed neomycin resistance cassette was introduced into intron 3  using mini targeting vectors. Cre-mediated removal of the neomycin resistance cassette generated either a conditional knockout Ring1B allele with exon 3 flanked by LoxP sites or a Ring1B-null allele deleted for exon 3 that produces a transcript containing a premature STOP codon encoding a 42-amino-acid N-terminally truncated protein. Details of genotyping by PCR and cDNA sequencing are in Supplemental Table 2.Ring1B mutant mice were performed under a UK Home Office project license (PPL 60/4424) with approval from an institutional ethics committee.Generation and analysis of http://www.ncbi.nlm.nih.gov/geo) under accession number GSE69978. Details of qRT–PCR for verification of expression changes are in Supplemental Table 2. A full protocol is in the Supplemental Material.The Amino Allyl MessageAmp II with Cy3 kit  was used to produce cRNA using the manufacturer's protocol. Six-hundred nanograms of cRNA was fragmented and hybridized to a SurePrint G3 Mouse GE 8x60K microarray . After washing, the arrays were scanned using a NimbleGen scanner, and images were analyzed using Agilent Feature Extraction software. The resulting values were processed and analyzed using custom R scripts. Expression data were deposited in the Gene Expression Omnibus (GEO) repository (Libraries were prepared as previously described  with modhttp://www.ncbi.nlm.nih.gov/geo) under accession number GSE69978.Sequence reads were trimmed  to remove adapters  and mapped to the mouse genome (mm10) using Bowtie 2.1.0 with the following arguments: “--local -D 20 -R 3 -N 1 -L 20 -i S,1,0.50.” SAM files were processed using HOMER version 4.3. HOMER tag directories were created using the makeTagDirectory tool with the options “-unique” and “-fragLength 150” and were used to create BedGraphs for visualization. All data output from HOMER analysis was normalized to 10 million mapped reads. BedGraphs were created using HOMER's makeUCSCfile tool with default options. Enriched regions were identified using the findPeaks tool from HOMER with the options “-style histone” and “-minDist 500” with the input sequences as controls. High-confidence enriched regions of at least 500 base pairs enriched in both replicates were identified. The HOMER analyzeRNA tool with the “-rpkm” option was used to quantify regions of interest. To generate heat maps, HOMER's annotatePeaks tool was used (options “-ghist” and “-hist 50”) to generate a matrix of RPKM (reads per kilobase per million mapped reads) values, which was processed using custom R scripts. Illumina sequencing data were deposited in the GEO repository (Details of cell culture conditions, RNA extraction, cDNA synthesis, protein extractions, ChIP, and immunoblotting are provided in the Supplemental Material.