We show that TPO profoundly alters the transcriptome of HSPCs, with key hematopoietic regulators being transcriptionally repressed within 30 min of TPO. By examining cis-regulatory dynamics and chromatin architectures, we demonstrate that these changes are accompanied by rapid and extensive epigenome remodeling of cis-regulatory landscapes that is spatially coordinated within topologically associating domains (TADs). Moreover, TPO-responsive enhancers are spatially clustered and engage in preferential homotypic intra- and inter-TAD interactions that are largely refractory to TPO signaling. By further examining the link between cis-regulatory dynamics and chromatin looping, we show that rapid modulation of cis-regulatory activity is largely independent of chromatin looping dynamics. Finally, we show that, although activated and repressed cis-regulatory elements share remarkably similar DNA sequence compositions, transcription factor binding patterns accurately predict rapid cis-regulatory responses to TPO.Thrombopoietin (TPO) is a critical cytokine regulating hematopoietic stem cell maintenance and differentiation into the megakaryocytic lineage. However, the transcriptional and chromatin dynamics elicited by TPO signaling are poorly understood. Here, we study the immediate early transcriptional and  Hematopoiesis—the formation of blood cellular components—is an exquisitely characterized process in which the signaling consequences of extracellular stimuli, such as cytokines and growth factors, must be coherently integrated with chromatin structures to regulate cell-type–specific transcriptional programs . Specificis-regulatory elements such as promoters and enhancers , which recruit chromatin modifiers to establish active or repressive chromatin environments at regulatory elements within a cis-regulatory repertoire  CTCF loops, also referred to as insulated neighborhoods (Chromosome conformation capture (3C)-based methods, particularly high-throughput 3C (Hi-C), have enabled targeted or genome-wide mapping of chromatin architectures . These tborhoods  or loop borhoods . Althougborhoods .cis-regulatory interactions with high resolution and coverage. To overcome this limitation, we recently developed Promoter Capture Hi-C (PCHi-C), a sequence capture approach that selectively enriches Hi-C libraries for interactions involving more than 22,000 annotated mouse promoters, thus allowing global mapping of promoter interactions at restriction fragment level resolution  is a critical regulator of megakaryopoiesis. TPO regulates megakaryocyte and platelet production by activating its receptor, MPL , inducin-renewal . HoweverTo capture the immediate early transcriptional response to TPO, we performed subcellular RNA-seq  before aSupplemental Fig. S1A,B). As expected, this is largely driven by the high intronic content of chromatin-associated RNA-seq libraries, which are enriched for unprocessed and incompletely spliced transcripts . In addition, analysis of small nuclear and small cytoplasmic RNA gene loci revealed compartment-specific transcript localization, confirming the high enrichment of our RNA fractions .We isolated and profiled biochemically fractionated chromatin-associated, nucleoplasmic, and cytoplasmic transcripts in two biological replicates. Principal component analysis and hierarchical clustering indicate high reproducibility between replicates, with the subcellular compartment explaining most of the observed variation in gene expression   and exhibited significantly higher fold changes . This suggests that RNA stability and post-transcriptional regulation contribute substantially to cytoplasmic gene expression changes even in response to transient signaling, underscoring the importance of profiling chromatin-associated or nascent transcripts when studying primary transcriptional responses. Unless otherwise stated, the remainder of our expression analyses focused on chromatin-associated transcripts.We then analyzed the immediate early transcriptional consequences of TPO signaling at the chromatin level. We found that TPO transcriptionally up-regulated 1325 genes and repressed 639 genes A. ImportCish, Pim1, Cited2, and Egr1) and the transcription factor MYC  bone marrow cells , consistent with a slower induction kinetic  and Gene Ontology (GO) enrichment analyses. Transcriptionally up-regulated events were strongly enriched for housekeeping genes involved in RNA and protein metabolism, whose expression is largely driven by a MYC transcriptional program, and for genes that respond to cytokine signaling in the immune system D,E. Howe kinetic . In cont kinetic D–F. A di kinetic . The rap kinetic F suggest kinetic .cis-regulatory elements before and after 30 min TPO stimulation of HPC-7 cells. To this end, we profiled H3K27ac genome-wide by chromatin immunoprecipitation sequencing (ChIP- seq) in two biological replicates and used a sliding window-based approach to detect genomic regions exhibiting significantly altered H3K27ac levels at 1% false discovery rate (FDR)  ; Methodsn = 2669) or decreased  H3K27ac levels within 30 min of TPO treatment . However, the two sets differed in the magnitude of their response to TPO, with activated DARs exhibiting significantly stronger changes in H3K27ac levels . We ruled out the possibility that these broad acetylation changes could stem from technical biases, because H3K27ac promoter signals at the 1000 most highly expressed genes unaffected by TPO were highly correlated (r = 0.94–0.99) and exhibited no systematic difference between replicates and conditions . In addition, changes in the abundance of enhancer RNA (eRNA) transcripts at differentially acetylated intergenic enhancers correlated with changes in H3K27ac levels, further validating our DAR calls . Moreover, to rule out the possibility that differential acetylation events reflect global changes in H3 occupancy, we measured H3K27ac and total H3 levels at a set of differentially acetylated loci by ChIP-qPCR . This analysis indicates that H3K27ac changes at DARs represent bona fide acetylation dynamics.Our analysis identified 10,222 differentially acetylated regions (DARs) exhibiting significantly increased  analysis by linking DARs to their putative target genes . As expecis-regulatory elements is required for transcriptional repression of key hematopoietic regulators . This result is in line with previous studies of extracellular signaling that examined higher-order chromatin architectures 1 h after stimulation in other cellular systems  for differentially transcribed genes. As expected, genes in repressed TADs showed an overall lower expression than genes in activated TADs D. Notablcis-regulatory responses to TPO by correlating the number of differentially transcribed genes with the number of DARs located within the same differentially acetylated TAD. As expected, we found a marked correlation between increased cis-regulatory activity and transcriptional up-regulation , thus explaining the relatively weak association between cis-regulatory and transcriptional repression. Importantly, basal expression levels of these genes were significantly lower than for genes located within TADs hosting at least one differentially transcribed gene . The lower expression levels reduce our ability to detect statistically significant further reductions in transcript levels. However, our results raise the possibility that repressed DARs within these loci might represent the first detectable step toward stable gene silencing of lineage-inappropriate genes.Finally, we examined the relationship between transcriptional and gulation F. In coned genes F. To furcis-regulatory units  . This meSupplemental Fig. S3F).Although by definition intra-TAD interactions occur more frequently than interactions spanning TAD boundaries, topological domains represent a modest twofold enrichment in interaction frequency . TherefoTogether, these results indicate that TPO-responsive enhancers engage in preferential long-range intra- and inter-TAD interactions resulting in their clustering in the three-dimensional nuclear space, and TPO-induced changes in enhancer activity are at least partly uncoupled from this spatial clustering of enhancer elements.Transcriptional enhancers not only interact with each other through long-range interactions but also within dense enhancer hotspots known as super-enhancers (SEs) or stretch-enhancers , which hSupplemental Fig. S5A,B). We next examined the consequences of this perturbation on the transcription of putative target genes as a function of their genomic distance to SEs. Differential acetylation of SEs was more frequently linked to transcriptional perturbation of SE-proximal genes located within genomic distances <1 Mb , with 27% of activated SEs and 22% of repressed SEs localizing within the same CTCF loop as their closest differentially transcribed gene. Nevertheless, the majority of genes located in close proximity to TPO-responsive SEs exhibited little or no detectable transcriptional changes within 30 min of TPO   , for a ttituents D, allowiand Etv6 . Promoted Cxcr4) B,C were d Cxcr4) F, suggesr > 0.99)  A. This w > 0.99) A. These We then focused on the spatial chromatin architecture at differentially transcribed gene loci, taking advantage of the increased resolution offered by PCHi-C over Hi-C data. We found that most promoters of differentially transcribed genes engaged more than 10 promoter-interacting regions (PIRs)  B; althoucis-regulatory units (Methods). For each promoter, we defined a cis-regulatory unit as the union of all its H3K27ac-marked PIRs, and we set a threshold of at least five such regions being required to ensure robust estimates of H3K27ac fold changes and correlation coefficients. This resulted in a set of 907 differentially transcribed loci . Our data revealed that only 5.4% of differentially transcribed gene loci being tested  exhibited a statistically significant correlation between acetylation and looping dynamics in response to transient TPO signaling (Myc and Spred1) .This result was further supported by a permutation test in which the connectivity of ignaling E. Neithe Spred1) A nor rep Spred1) F. Indeedcis-regulatory activity is largely independent of chromatin looping dynamics.Together, these results demonstrate that the extensive and spatially compartmentalized epigenome remodeling induced by transient TPO signaling takes place within poised chromatin architectures and suggest that the rapid cytokine-dependent modulation of cis-regulatory dynamics induced by TPO signaling. To identify TFs that control activated and repressed cis-regulatory elements within DARs, we subjected differentially acetylated DHSs to a de novo motif discovery analysis. We found that both activated and repressed DARs were strongly enriched for ETS motifs, which feature a central 5′-GGAA-3′ core, and for motifs recognized by the RUNX and GATA families of TFs  logistic regression models (k-mers (2 ≤ k ≤ 4), DNA shape features (n = 26) and after (n = 3) 30-min TPO stimulation (generated as part of this study or previously published) , and normalized Hi-C signals as a proxy for interaction frequencies. Model performances were evaluated on a test set consisting of differentially acetylated cis-regulatory elements that were not used for learning model parameters , in vivo TF binding profiles were able to accurately predict changes in cis-regulatory activity (AUC = 0.80) , suggesting that key TFs were already included within our chromatin feature set. Furthermore, these results were validated by an independent learning scheme using random forest classifiers  E. In con = 0.80) E, furthecis-regulatory elements . In contrast, binding of MYB, LYL1, and tyrosine unphosphorylated STAT5 (uSTAT5) in the absence of TPO was predictive for repression of cis-regulatory elements . Notably, DARs bound by stably selected features were significantly associated with distinct signaling pathways and metabolic functions, spanning the whole spectrum of biological processes regulated by TPO  STAT5B that ablates TPO-induced tyrosine phosphorylation of STAT5 and does not act as a dominant negative  supplemented with 10% fetal calf serum, 10% SCF conditioned media (produced by the BHK/MKL cell line), 1% L-Glutamine, 1% penicillin/streptomycin, and 74.8 µM monothioglycerol (Sigma). Cell density was maintained between 5 × 105 and 2 × 106 cells/mL.HPC-7 cells were grown at 37°C and 5% COEight- to twelve-week-old C57BL/6 mice (Charles River) were used in this study. All mouse procedures were approved by the UK Home Office and the University of Cambridge Animal Welfare and Ethical Review Board.6 cells/mL in StemSpan SFEM medium (StemCell Technologies) and incubated for 4 h. Serum-starved cells were then stimulated with recombinant murine thrombopoietin  at 100 ng/mL for 30 min. TPO was diluted in a small volume of fresh StemSpan SFEM medium prior to stimulation.Cells were spun down at 1000 rpm for 5 min and washed once in PBS. The cell pellet was resuspended at a density of 1 × 102 prior to stimulation with TPO as described above.Freshly isolated mouse bone marrow cells were stained with AF700-conjugated lineage cocktail (133313), APC-Cy7-conjugated anti-cKit (105826), Bv605-conjugated anti-Sca1 (108133), and FITC-conjugated anti-CD41 (133903). 7-AAD (Invitrogen) was used to exclude dead cells. All antibodies were purchased from BioLegend unless otherwise indicated. Cells were sorted on fluorescence-activated cell sorting (FACS) Aria (BD Bioscience) directly into StemSpan SFEM medium (StemCell Technologies). The sorted cells were incubated for 1 h at 37°C and 5% COHPC-7 cells were starved as described above. Cells were pretreated with HDAC inhibitors, 1 µM of Trichostatin A, or 5 nM of Romidepsin (Selleckchem) during the final 30 min of serum starvation and stimulated with TPO at 100 ng/mL for 30 min.Retrovirus was produced using Phoenix cells (ATCC). HPC-7 cells were infected with retrovirus expressing STAT5B or mutant (Y699F) STAT5B in the presence 8 µg/mL polybrene (Millipore). After 24 h, GFP-positive cells were FACS sorted and cultured in IMDM media (Invitrogen) supplemented with 10% fetal bovine serum and 10% SCF conditioned media.Supplemental Table S2.Total RNA was isolated using Direct-zol (Zymo Research) and reverse transcribed into cDNA using SuperScript IV First Strand Synthesis System (Invitrogen) following the manufacturer's instructions. Quantitative RT-PCR was carried out using SYBR Green Brilliant II low Rox and a Stratagene Mx3000P machine (Agilent Technologies). For primer sequences, see Supplemental Methods.Subcellular fractionation and RNA preparation were performed essentially as described  with modSupplemental Methods; Chromatin immunoprecipitation (ChIP) assays on serum-starved and TPO-stimulated HPC-7 cells were performed as previously described  under accession number GSE100835.RNA-seq, ChIP-seq, Hi-C, and promoter Capture Hi-C raw and processed data from this study have been submitted to the NCBI Gene Expression Omnibus (GEO;


Pax6 is a key regulator of the entire cascade of ocular lens formation through specific binding to promoters and enhancers of batteries of target genes. The promoters and enhancers communicate with each other through DNA looping mediated by multiple protein–DNA and protein–protein interactions and are marked by specific combinations of histone posttranslational modifications (PTMs). Enhancers are distinguished from bulk chromatin by specific modifications of core histone H3, including H3K4me1 and H3K27ac, while promoters show increased H3K4me3 PTM. Previous studies have shown the presence of Pax6 in as much as 1/8 of lens-specific enhancers but a much smaller fraction of tissue-specific promoters. Although Pax6 is known to interact with EP300/p300 histone acetyltransferase responsible for generation of H3K27ac, a potential link between Pax6 and histone H3K4 methylation remains to be established.Cap2, Farp1, Pax6, Plekha1, Prox1, Tshz2, and Zfp536. Pax6 knockdown was accompanied by reduced H3K4me1 at enhancers and H3K4me3 at promoters, with little or no changes of the H3K4me2 modifications. These changes were prominent in Plekha1, a gene regulated by Pax6 in both lens and retinal pigmented epithelium.Here we show that Pax6 co-purifies with H3K4 methyltransferase activity in lens cell nuclear extracts. Proteomic studies show that Pax6 immunoprecipitates with Set1a, Mll1, and Mll2 enzymes, and their associated proteins, i.e., Wdr5, Rbbp5, Ash2l, and Dpy30. ChIP-seq studies using chromatin prepared from mouse lens and cultured lens cells demonstrate that Pax6-bound regions are mostly enriched with H3K4me2 and H3K4me1 in enhancers and promoters, though H3K4me3 marks only Pax6-containing promoters. The shRNA-mediated knockdown of Pax6 revealed down-regulation of a set of direct target genes, including Our study supports a general model of Pax6-mediated recruitment of histone methyltransferases Mll1 and Mll2 to lens chromatin, especially at distal enhancers. Genome-wide data in lens show that Pax6 binding correlates with H3K4me2, consistent with the idea that H3K4me2 PTMs correlate with the binding of transcription factors. Importantly, partial reduction of Pax6 induces prominent changes in local H3K4me1 and H3K4me3 modification. Together, these data open the field to mechanistic studies of Pax6, Mll1, Mll2, and H3K4me1/2/3 dynamics at distal enhancers and promoters of developmentally controlled genes.The online version of this article (doi:10.1186/s13072-016-0087-z) contains supplementary material, which is available to authorized users. Cellular differentiation is regulated by a combinatorial action of sequence-specific DNA-binding transcription factors and extracellular signaling that results in activation and repression of specific batteries of genes –3. TheseGenome-wide studies of chromatin by ChIP-seq have revealed that there is a relatively small number of core histone PTMs, including H3K4me1, H3K4me3, H3K27ac, and H3K27me3, which can be used as landmarks for navigation through the chromatin landscape. Combinations of these PTMs in genomic regions have been shown to be highly associated with the locations of individual promoters and enhancers , 13. ActIn mammalian cells, H3K4 methylations are catalyzed by a family of six distinct complexes. The Mll/Set1 complexes contain enzymes with an evolutionarily conserved C-terminal catalytical SET domain and an evolutionarily conserved WRAD subcomplex . A few additional regulatory proteins discriminate between Mll and Set1 complexes . For exaSey/Sey mice are anophthalmic  and display a range of abnormalities in other organs, including the brain, olfactory system, and pancreas  S-adenosyl methionine as methyl group donor and recombinant histone octamers as the substrates. We found that Pax6-, but not control IgG-immunoprecipitates, were associated with HMT activity . We used Pax6-specific antibodies and tested the enriched proteins by in vitro HMT assay performed in the presence of labeled  S-adenosyl-l-methionine (SAM), for 1 h at 30 °C. The following procedure is done as we described earlier [Pax6 or control IgG antibody precipitates from nuclear extract as described above were incubated with 1 µg recombinant histones in the presence of [ earlier .3H] SAM , and BC200 buffer. After 1-h incubation at 30 °C, the reaction mixture was spotted on P81 phosphocellulose paper (Millipore) and washed 3× with sodium carbonate, pH 8.5 and 1× with acetone before air-drying. Four milliliters of scintillation cocktail was added to the filter paper, and emissions were counted. CPM were normalized to IgG control.N-terminal H3 peptides (1–20) containing unmodified K4, K4me1, K4me2, and K4me3 residues were obtained from Epicypher . The reactions  were conducted with 1 µg of the peptide, specific immunoprecipitate , [αTN4 cells are SV40 T-antigen-transformed mouse lens epithelial cells  that expn = 245) and others as specific peaks by BEDTools (v2.23.0) [p < 2.2e-16 by Fisher’s exact test).Ten 20-cm dishes of control and Pax6 sh2 αTN4 cells were cross-linked with 1 % formaldehyde at room temperature for 10 min and quenched by 2.5 M glycine. The ChIP was performed using antibodies as we described elsewhere . Sequencv2.23.0) . The peade novo enriched motifs with sequences −100 to +100 bp around Pax6 peak summits in lens and αTN4 cells; the setting was 6–20 bps as motif width and returning the top 10 motifs.MEME (4.10.1)  and MEMEThe list of TSSs (transcription start sites) list was downloaded from the UCSC genome browser  using thTotal RNA was extracted from αTN4 cells on a 6-well plate using TRIzol reagent and reverse-transcribed using the SuperScript III First-Strand Synthesis System (Invitrogen). Relative mRNA levels were normalized against average of Gapdh and B2 m. The library construction, sequencing, and data analysis were performed as we described previously . The folloxP [Pax6loxP/loxP and mutated Pax6loxP/loxP; DctCre mice were dissected at E15.5 and RNA was extracted using QIAshredder and RNeasy kits (Qiagen). Reverse transcription of 1 µg of RNA from each sample was performed using qScript cDNA Synthesis Kit (Quanta). cDNA was amplified using the Power SYBR Green Mix (Applied Biosystems) in a 384-well optical reaction plate using ABI Prism 7000 Sequence Detection System (Applied Biosystems). Plekha1 primers used for qRT-PCR are described above, and relative expression was normalized using Tbp and Hprt transcripts. Immunofluorescence analysis was performed on 10-µm paraffin sections as previously described [The mouse lines employed in this study, Pax6loxP  and DctCloxP , have beescribed  using pr


Mesoplasma florum 2′-deoxyguanosine (2′dG)-sensing riboswitch using a photocaging-ligation approach, and monitor folding to its native state by real-time NMR in both presence and absence of ligand. We further determine transcription rates for two different bacterial RNA polymerases. Our results reveal that the riboswitch functions only at transcription rates typical for bacterial polymerases (10–50 nt s−1) and that gene expression is modulated by 40–50% only, while subtle differences in folding rates guide population ratios within the structural ensemble to a specific regulatory outcome.Transcriptional riboswitches modulate downstream gene expression by a tight coupling of ligand-dependent RNA folding kinetics with the rate of transcription. RNA folding pathways leading to functional ON and OFF regulation involve the formation of metastable states within well-defined sequence intervals during transcription. The kinetic requirements for the formation and preservation of these metastable states in the context of transcription remain unresolved. Here, we reversibly trap the previously defined regulatory relevant metastable intermediate of the  Riboswitches are RNA-based regulatory elements, which regulate downstream gene expression by binding of small molecular weight ligands. Here the authors demonstrate the molecular mechanism of a transcriptional riboswitch that integrates changes in transcription rates, metabolite concentration, and kinetic on- and off-rates of ligand binding. For riboswitches that regulate gene expression on the level of translation, reversible conformational switching allows RNAs to adopt distinct ligand-dependent functional folds at equilibrium during multiple rounds of translation. As a consequence, RNA synthesis and the cellular response to changes in metabolite concentration are separated in time. The functional relevant switch underlying translational riboswitches modulate ribosome access to the SD sequence6, but also more complex regulation mechanisms have been discovered7.Riboswitches selectively bind metabolites to control the transcription or translation of metabolite-associated genes. In general, riboswitch function is described as a ligand-dependent switch between two mutually exclusive conformations to either permit or repress gene expression9. Under these conditions, the nascent transcript can either adopt a sequence-conserved terminator conformation that disrupts the polymerase complex or form one or several alternative, so-called antiterminator conformations that avoid termination. The metabolite concentration EC50, at which riboswitches operate, often exceeds the KD values for ligand binding by at least one order of magnitude14. This observation indicates that fast folding of functional states during well-defined transcription intervals is important for riboswitch function.In contrast, control of gene expression by premature termination of transcription inevitably requires temporal coupling of RNA synthesis and conformational switching. Riboswitches utilize rho-independent transcriptional termination15. The ligand-dependent conformational rearrangement underlying riboswitch function typically involves four distinct sequence segments P, A, T, and H as depicted in Fig. AT concomitantly occurs during transcription of nucleotides 113 to 137. Previous studies assume ligand binding within the first transcription interval (nts 80–110) to stabilize the PA interaction sufficiently to inhibit an interaction with strand T and to ensure OFF function, but it remains uncertain how the PAT state is selected from only one metastable state in the context of transcription. PAT states adopted within the second transcription interval (in green) become metastable as strand H is transcribed (PAT–H) during synthesis of nts 139 to 144 in both, absence or presence of ligand. Within this short period during transcription, the terminator conformation PA–TH represents the most stable state. It follows that deciphering the change of kinetics of antiterminator formation (PAT) depending on the absence and presence of ligand represents the most important information regarding regulation.In a previous study, we investigated the landscape of the thermodynamically stable states adopted in either the presence or absence of metabolite for mRNA transcripts of increasing chain lengthAccording to the equilibrium model, the effects of ligand binding on the overall fold adopted at a specific transcript length is insignificant. Rather, formation of metastable intermediate states as important navigators of the regulatory outcome suggests a correlation between the life times of these states and transcription rates to be critical for riboswitch function. Here, we unravel these kinetic requirements to achieve and to maintain a ligand-dependent reversal of population ratios of functional OFF and ON states during transcription. On the example of the central metastable PA–T conformation of the 2′dG riboswitch, we demonstrate that ligand binding does not induce major changes in equilibration kinetics of the PA–T state, but slows refolding to allow the polymerase to bypass the metastable transcription barrier for the majority of nascent transcripts.AT) folding, we trapped the PA conformation of the 121 mer transcriptional intermediate by incorporating photolabile protection groups in strand T. These protection groups prevent antiterminator helix PT formation and allow monitoring of folding from PA to PT after photolysis by light-induced real-time NMR as reported previously18. The 121 nt transcriptional intermediate forms a complete antiterminator helix PT at equilibrium. While refolding from PA–T to PAT is initiated at nucleotide 113, segment 113–121 only contains four weak base pairs within helix PT, and, therefore, refolding kinetics from PA–T to PAT are most accurately described at length 121 on average.To monitor antiterminator  was prepared by enzymatic splinted ligation and contained three NPE protection groups at the C4 atoms in the nucleobases of Uracil residues 108, 110, and 112  is detectable. In dGsw121,caged, the conformation of the aptamer domain (PA–T) is maintained as evidenced by characteristic imino proton reporter signals, in particular U17 and U1819. Irradiation at 355 nm releases native dGsw121 and subsequently induces formation not of a single, but of two interconverting antiterminator conformations (PAT(I)) and (PAT(M)), identified by helix PT reporter signals G12, U108, U109, and G25I. In the presence of ligand, the antiterminator conformations PAT folds only to ~80%, in agreement with the static experiments15. In the ligand-free form, chemical shifts of helices P2 and P3 are identical in either the aptamer PA or antiterminator PAT conformations, while those signals can be distinguished in the ligand-bound form due tertiary interactions induced by ligand binding.Photocaged dGswM. florum (25 °C). Rates for individual base pairs were determined  folds at a rate of ~1.3 s−1. The second conformation PAT(I) could be monitored by the characteristic and isolated signal G25I, and folds at 10-fold slower rates of 7*10−2 s−1. In the ligand-bound form, the difference in chemical shift of helices P2/P3 in PA–T and PAT allows monitoring of aptamer dissociation in addition to PT formation. The ligand-bound PA conformation folds to PAT(M) at rates of ~9*10−2 s−1 with a second slower rate in the order of ~1*10−2 s−1, which we assign to re-equilibration to PAT(M) after formation of PAT(I).We obtained kinetic traces by real-time NMR at the physiological temperature of the bacterium . florum 5 °C. Ratacy Fig. . We can acy Fig. , where tAT(M) in the absence of ligand (pathway 2) is on the same timescale (1.3 s−1) as bacterial polymerases require to synthesize the subsequent 24 nucleotides (0.4–3.7 s−1)23. The synchronization of transcription with folding rates that are unimolecular and thus concentration independent and sufficiently long life-time of the metastable state ensures ON state function. In contrast, ligand binding slows down folding from the PA conformation to the PAT(M) conformation to a rate of ~0.1 s−1, and, thus, below transcription rates (pathway 3). Hence, in the presence of ligand, antiterminator formation for transcript length for which it represents the stable fold (113–137 nts) is too slow, and, thus, OFF state function is triggered. Further, equilibration between PAT(M) and PAT(I), occurs on the same timescale as antiterminator formation in the presence of ligand, and, therefore, cannot compete with transcription rates. Therefore, our kinetic experiments reveal that only one of the two antiterminator conformations (PAT(M)) represents the regulatory decisive ON state, and, thus, demonstrate a kinetic selection of only a single functionally relevant conformation in the structural ensemble.The analysis of the kinetics reveals three relevant functional pathways as depicted in Fig. on) remain constant within the relevant transcription interval. To facilitate binding of 2′dAP, the ligand selectivity was shifted from G to A by mutating the Watson–Crick ligand recognition site C74 to U74 (dGswC74U), a common approach applied for purine riboswitches  exceeds the KD of 2′dG binding to dGsw (250 nM), the determined koff for 2′dAP of 0.35 s−1 provides an upper limit for koff of 2′dG.OFF state function depends on ligand binding kinetics and subsequent RNA folding. We further determined the kinetics for transcripts of different chain length by stopped-flow spectroscopy with a surrogate fluorescence ligand 2-aminopurine 2′-deoxyriboside (2′dAP) to investigate whether the ligand association rates  will be populated in a range of 70–100% by the time the polymerase has reached nucleotide 113. Equilibration can take place during transcription of nts 90–110, transcribing at rates of 0.5-4.5 sAT between transcript lengths 113 and 137 at different transcription rates (0–100 nt s−1)  to (i) experimentally validate that the riboswitch functions only in a defined window of transcription rates and to (ii) assess whether the co-transcriptional folding pathway of the riboswitch is sensitive to the transcription properties of the polymerases from different organisms. We determined the dependence of transcription rates on temperature and NTP concentrations. We further determined the regulation efficiency by quantifying the amount of 235 nt fragments elongated past the terminator sequence.We performed single-round in vitro transcription assays in the absence and presence of ligand using both, t1/2 value of PS1 from 3 s to 50 s in the absence of ligand for E. coli RNAP. The second PS1 is located around nucleotide 116 at the 3′-end of segment T. This second PS1 pauses the transcription complex longer than the first PS1 (24 s at 37 °C). The third PS1 is located at the translation start site. This PS1 is located beyond the regulatory decision point and is, thus, not relevant for the co-transcriptional folding pathway of the riboswitch.For both polymerases, three pause sites (PS1) were determined in time-resolved transcription reactions. The first PS1 is encountered when the aptamer is fully transcribed Fig. . A decreE. coli RNAP, the ligand-dependent ON state fraction is primarily affected by changes in temperature, while variations in the NTP concentration in the range of 0.05–2.3 mM do not affect the transcription rate sufficiently to cause changes in the co-transcriptional folding pathway   the fraction of ON state is 61 ± 9%. In line with riboswitch function, addition of ligand promotes accumulation of nascent transcripts within pathway 3 and decreases the fractions of ON state obtained to 35–43% for transcription reactions performed at 37 °C and 25 °C. For multi-round transcriptions, the effect of ligand addition on the fraction of ON state obtained is significantly more pronounced with 40–50% change in population ratio opposed to ~20% for single-round transcriptions   as determined by our simulation at 4 °C. In agreement with our simulations, slower transcription rates at 4 °C expand the time span available for PA–T to PAT(M) folding and lead to an increase in structures populating the ON state at the regulatory decision point in both absence and presence of ligand. In stark contrast, the B. subtilis polymerase requires a reduction in NTP concentration to induce transcription termination upon ligand addition. Due to the high percentage of full-length transcripts this effect is likely unrelated to ligand binding or ON state folding, but is linked to the inability of terminator formation due to the rapid elongation by the polymerase. The calculated transcription rates from the in vitro assays are not absolute values for single RNAPs, but instead are composite rates, as they indicate the point in time at which half of the RNAPs completed transcription of an RNA fragment of a specific length. This approximate rate can then be used in the simulations to examine the relationships of the different parameters in the regulation mechanism.For 26, ligand association rates for purine aptamer domains of ~104–105 M−1 s−128 can effectively direct ligand-binding to either compete with or lag behind bacterial polymerases synthesizing the relevant ligand-binding sequence at rates of 0.5–4.5 s−1. Remarkably, PS1 induces transcriptional stalling at the latest possible transcript length with highest ligand binding affinity according to previous results29. To determine shifts in population ratios as transcription proceeds further, we present a ligation-based approach that provides access to reversible trapping of metastable states that form within the activation (pathway 1) and repression pathway (pathway 3) of ON function (pathway 2). In the biological context of co-transcriptional folding, single nucleotides are sequentially released to an ensemble of RNA intermediates differing in length that are transcribed at locally varying transcription rates. Real-time monitoring of such folding is experimentally challenging and cannot be accomplished by established methods. In the approach applied here, folding is monitored by immediately releasing a part of the riboswitch expression platform to interact with the aptamer domain. Despite these experimental limitations, we have selected a representative transcription intermediate that most accurately reflects refolding rates of the metastable state on average to obtain high-quality quantitative data. We linked kinetics derived from light-induced release of the stable ON state to unbiased mapping of conformational space15 to assess the kinetics of ligand-dependent conformational switch. We further experimentally validated the impact of transcription rates and polymerase dependent transcription properties on the regulation efficiency of the riboswitch using E. coli and B. subtilis RNAP.To act as transcriptional regulatory elements, the sequence of riboswitches has to be optimized to fulfill a number of kinetics requirements. Our experimental approach delineates the competition between refolding rates of cis-regulatory RNA and processive transcript elongation. Within the regulatory decisive 2′dG concentration range of 0.1–100 µM for dGsw30. Here, we further substantiate this proposal by revealing the difference in co-transcriptional riboswitch folding transcribed by two non-native polymerases. Furthermore, biologically demanding experiments with M. florum polymerase would be required to compare these results to native 2′dG riboswitch folding. General fluctuations in transcription rates resulting from changes in cellular conditions31 may be self-regulated by the negative feedback loop of the riboswitch itself. The kinetic and simulation framework developed here provides an important contribution to the conceptual basis to understand transcriptional riboswitches.Our results demonstrate the requirement for a strict synchronization of transcription intervals of metastable states, corresponding transcription rates, and refolding rates, where transcription elongation must not only be matched to refolding and ligand binding kinetics but also to folding delays induced by thermodynamic stabilizations to preserve metastable states within distinct sequence intervals. A significant offset in temperature from physiological conditions leads to quantitative folding to the ON state from both metastable ligand-bound and ligand-free aptamer domains. Sequence-specific variations in transcription rates impact both ON state folding from pathways 1 and 3 equally. Therefore, it is critical that pausing within the expression platform is adapted to the sequence-specific transcription speed of the native polymerase. In a previous study we could already show that the detailed investigation of transcriptional riboswitches requires the native RNA polymerase85 and mutated dGswC74U constructs with varying length for stopped-flow spectroscopy were prepared by in vitro transcription from PCR products by adjusting the reverse primers accordingly as reported previously32. For dGswC74U constructs, two artificial G’s were incorporated at the 5′-end in the forward primers to enable rapid RNA purification by buffer exchange. dGsw85 was transcribed with a 5′-hammerhead ribozyme, exchanged into water, and directly used for ligation reactions.dGsw86–121 was prepared via solid phase synthesis in a 17 µmol scale. Ultramild phosphoramidites, chemical phosphorylation reagent I, and (S)-NPE-U phosphoramidite  for deprotection primarily generates a byproduct with a mass 53 Da greater than the desired product (Supplementary Table 86–121 fragment could be isolated using 3 mL t-BuNH2:water (1:3) for 6 hours at 60 °C. The mixture was separated in 10 reaction tubes and the solvent evaporated. Each tube was treated as follows: the 2′-deprotection was performed using 6.5 mL of a mixture of NMP:Et3N:Et3N·3 HF (3:1.5:2) for 3 hours at 60 °C. After addition of 30 mL n-BuOH the mixture was stored at −80 °C for 2 hours and then centrifuged at −8 °C for 1 hour. The supernatant was decanted and the residue purified via anion exchange HPLC (Amersham Bioscience with an UV-900 detector and P-900 pumps) using a Tricorn SOURCE 15Q AEX 10/150 column . The collected oligoribonucleotide was desalted using a Tricorn SOURCE 15 RPC 10/150 column . The identity of the oligoribonucleotide was confirmed by ESI-MS: mass calculated: 11784.5 Da, found: 11785.7 Da.Photocaged dGsw121 was prepared by enzymatic splinted ligation using T4 RNA ligase 2. Fragments dGsw85 (15N-G/U) and photocaged dGsw86–121 were preannealed to a 31 nt splint (splint:31AATAATTAAAGAGTGGTTTTTCACTTATAGT) prior to ligation at 25 °C for 15 min. Ligation reactions were performed with 12.5 µM dGsw85, 6.25 µM caged dGsw86–121, 10 µM splint31, 48 µg mL−1 T4 RNA ligase 2 (homemade), 10 mM MgCl2, 2 mM ATP, 25 mM DTT in 50 mM Tris-Cl, pH 7.4. dGsw85 was applied in 2-fold excess to optimize the turnover for photocaged dGsw86–121 (80%). Ligation reactions were incubated at 37 °C for 2 hours followed by overnight incubation at 25 °C, followed by incubation with TURBO DNAse (0.02 U µL−1) (Thermo Fisher Scientific) for 8 hours. Ligation reactions were directly applied to HPLC using a Kromasil RP18 10 × 250 column without further concentration and eluted at room temperature . Purified photocaged dGsw121 was lyophilized and exchanged into NMR buffer . Final yield of photocaged dGsw86–121 was 0.6 µmol (54 %).Photocaged dGsw33 in a pseudo-2D experiment using the jump return echo water suppression scheme34. Laser irradiation within the NMR tube was triggered via a TLL connection to a laser set up . Kinetics of ligand-free samples were recorded after 500 ms and kinetics of ligand-bound RNAs after 1 s of laser irradiation. The signal intensity was maximized by Ernst angle excitation35. Kinetics traces of ligand-free samples were averaged over 10 individual experiments and of ligand-bound samples over two individual experiments on 100 µM caged dGsw121 samples. 1H,15N-BEST-TROSY experiments were recorded with modifications according to Brutscher et al37. NMR experiments were performed on a Bruker 800 MHz spectrometer equipped with a 5-mm z-axis TCI-HCN cryogenic probe. For processing of data, Topspin 3.5 (Bruker Biospin) was used.Time-resolved NMR experiments were performed as reported previouslyC74U (2 µM) with 2-aminopurine 2′-deoxyriboside (2 µM–512 µM) in a 1:1 volume ratio at 25 °C. Both ligand and RNA were provided in NMR buffer with a Mg2+ concentration of 3 mM. 2′dAP was excited at 304.5 nm using 2 mm slit widths and fluorescence was monitored at >340 nm. Kinetic traces were averaged over 10–15 single measurements. Fluorescence decays were fitted with single and bi-exponential decays with three and five parameters, respectively  and dissociation (koff) constants were determined from individual apparent rate constants according to equation (Stopped-flow measurements were performed on a π*-180 CDF-spectrometer (Applied Photophysics). Folding was monitored by rapid mixing of dGsw15, starting from the aptamer domain fold at transcript length of 113 nt, followed by simultaneous elongation to a transcript length of 137 nt at transcription rates of 10–100 nt s−1 and folding to the antiterminator conformations PAT(M) and PAT(I) with rate constants determined by real-time NMR.Simulations of co-transcriptional folding were performed by kinetic Markov simulations. Simulations were performed as described previouslyE. coli and B. subtilis RNAP mediated transcription was composed of the lambdaPR promoter followed by the dGsw-coding sequence (GCAAATCGCGCTGTTAGCGGGCccttgactattttacctctggcggtgataatggttgcATGAATATAAAAGAAACTTATACAGGGTAGCATAATGGGCTACTGACCCCGCCTTCAAACCTATTTGGAGACTATAAGTGAAAAACCACTCTTTAATTATTAAAGTTTCTTTTTATGTCCAAAAGACAAGAAGAAACTTTTTTATTTAGTTGAATTTATAATAAGAGAAAAAGAAAGGATATTATATGGCAAAAATAAAAAACCAATATTACAACGAGTCTGTTTCGCCAATTG). The time-resolved transcriptions were performed as described previously30 at 37 °C, 25 °C, and 4 °C with NTP concentrations of 0.05 mM, 0.1 mM, 1 mM, and 2.3 mM. Transcription rates were determined as described previously38 and PS1 were analyzed as described by Landick 199639.The DNA template used for The data that support the findings within study are available from the corresponding author upon request.Supplementary Information


Xanthomonas bacteria inject transcription activator-like effector proteins (TALEs) into host cells to specifically induce transcription of plant genes and enhance susceptibility. Although the DNA-binding mode is well-understood it is still ambiguous how TALEs initiate transcription and whether additional promoter elements are needed to support this. To systematically dissect prerequisites for transcriptional initiation the activity of one TALE was compared on different synthetic Bs4 promoter fragments. In addition, a large collection of artificial TALEs spanning the OsSWEET14 promoter was compared. We show that the presence of a TALE alone is not sufficient to initiate transcription suggesting the requirement of additional supporting promoter elements. At the OsSWEET14 promoter TALEs can initiate transcription from various positions, in a synergistic manner of multiple TALEs binding in parallel to the promoter, and even by binding in reverse orientation. TALEs are known to shift the transcriptional start site, but our data show that this shift depends on the individual position of a TALE within a promoter context. Our results implicate that TALEs function like classical enhancer-binding proteins and initiate transcription in both orientations which has consequences for in planta target gene prediction and design of artificial activators.Plant-pathogenic  Xanthomonas bacteria cause severe losses of crop production worldwide [Plant pathogenic orldwide . Their vorldwide . Such prorldwide . Transcrorldwide . After eorldwide . TALE-inorldwide .DNA association of TALEs is initiated by their N-terminal region whereas the central part of TALEs provides specific binding to matching sequences ,5,6. ThiBs3 promoter to a position further upstream resulted in a concomitant upstream shift of the TSS [xa5 allele exhibits an increased resistance to Xoo infection due to a loss of efficiency of TALE-mediated transcriptional initiation [xa5 represents a recessive point mutation of the transcription factor IIA small γ subunit. Apparently, this mutation interferes with the interaction of TALEs with TFIIA and likely accompanying basal transcription-complex components [Computational analysis of genomic target sites of natural TALEs showed a preferential occurrence in apparent core promoter regions of -300 to +200 bp around the transcriptional start site . Previo the TSS . These o the TSS . One hositiation –23. xa5 mponents –24. Furtmponents –28.in planta are poorly understood. If the presence of a target sequence alone is sufficient to initiate transcription at any genomic locus or if the target locus has to satisfy additional requirements is still puzzling. Here, we systematically analyze TALE-mediated gene activation potential in different promoter contexts, from different positions or orientations within a promoter, and in a possible cooperative fashion. To dissect whether an effect is dependent on the TALE activation domain or the TALE DNA-binding domain, we replaced the TALE activation domain with a heterologous activation domain and the TALE DNA-binding domain with the Cas9-DNA-binding domain. Our findings suggest that TALEs can not dictate transcriptional activation on their own, but rather rely on further promoter elements. Furthermore, TALEs can support transcription by binding in either forward or reverse orientation, an observation which has not been reported, before. We show that this mode of activation is biologically relevant, because reverse-binding TALEs are able to support virulence of Xoo during an infection. This study is significant for understanding TALE-mediated gene activation, and will have implications for target site prediction in planta.In contrast to the already intensively studied DNA-binding mode, the circumstances for successful TALE-mediated transcriptional initiation Escherichia coli Top10 , Agrobacterium tumefaciens GV3101 pMP90 [Xanthomonas oryzae pv. oryzae (Xoo) strains BAI3 [talC [E. coli cells were cultivated at 37°C in LB, Xoo strains at 28°C in PSA and A. tumefaciens GV3101 at 28°C in YEB medium.The bacterial strains used in this study were 01 pMP90  and Xantins BAI3  and BAI3Oryza sativa ssp. japonica cv. Nipponbare was grown under glasshouse conditions at 28°C (day) and 25°C (night) at 70% relative humidity (RH). Leaves of 4-week-old plants were infiltrated with a needleless syringe and a bacterial suspension of an optical density (OD) at 600 nm of 0.5 as previously described [Nicotiana benthamiana plants were grown under 16 h of light, 40–60% RH, at 23°C (day) 19°C (night) in the growth chamber. Leaves of 4- to 6-week-old plants were inoculated with A. tumefaciens strains using a needleless syringe.escribed . Symptomet al. (2011). Up to six individual repeats were subcloned in an assembly vector, resulting in a hexa-repeat module. Three of these hexa-repeat-modules were then fused to the N- and C-terminal parts of Hax3 and inserted in an expression vector for Agrobacterium or Xanthomonas, respectively. This results in a N-terminally fused GFP for the Agrobacterium vector and in a C-terminally fused FLAG Epitope in the Xanthomonas vector. The dCas9 variant fused to the C-terminal region of Hax3 was used as dCas9 activator and generated as described before [TALEs were constructed by using the Golden TAL technology as described by Geißler d before . The actet al., 2009. Briefly, the PCR-amplified fragments of the promoters  were cloned into pENTR/D-TOPO  and fused to the uidA reporter gene by LR recombination into pGWB3 [A. tumefaciens strains delivering TALE constructs and GUS reporter constructs were resuspended in infiltration medium, resulting in an OD600 of 0.8, mixed in equal amounts and inoculated into N. benthamiana leaves. Two dpi, leaf discs were sampled and GUS activities were quantified using 4-methylumbelliferyl-β-D-glucuronide (MUG). Total protein concentrations were determined using Bradford assays. Data were compiled from triplicate samples originating from different plants. Error bars represent the standard deviation.β-Glucuronidase assays from plant samples were performed as described by Boch to pGWB3 . To anal2 or with the different Xoo strains using an OD600 of 0.5. At 1 dpi, 5 cm segments were harvested and rice total RNA was isolated using the Qiagen RNeasy kit. cDNA was generated from 2 μg RNA using the Fermentas first-strand cDNA synthesis kit  and real-time PCR was performed using the iCycler  as described before . The 5´ends were amplified by using the universal forward primer and the gene specific primer GUS 5`RACE  are sufficient to initiate transcription at that location or if surrounding DNA sequences influence gene induction. To address this, we designed reporter constructs in which the Hax3-box, that is bound by Hax3, a natural TALE from mpestris ,36, is pn assays . We subdter gene . This seter gene . Additioanalysis . The reptivation . In cont by Hax3 . These f by Hax3 .pBs4-50, that was highly responsive to Hax3, and pBs4-202, that was not, and exchanged parts of them  the reason why pBs4-50, but not pBs4-202 allows TALE-dependent expression is not due to sequences directly downstream of the Hax3-box that might interact with C-terminal parts of Hax3 and (ii) the presence of the 5´UTR in pBs4-50 is not crucial for robust activity by e.g. influencing mRNA stability. In contrast, all other constructs showed a decreased or no activity  was used as negative control, respectively. The majority of the analyzed TALEs activated the reporter gene irrespective of their position in the OsSWEET14 promoter  target the same region as highly active forward TALEs . All TALiability .In summary, the systematic analysis of differentially positioned forward and reverse binding TALEs resulted in three important findings: (i) Some TALEs  activate transcription in both orientations, suggesting that their DNA target sequences are accessible and allow a flexible interaction with the transcriptional machinery. (ii) The activity of some TALEs  is orientation-dependent, suggesting that the reverse orientation in these cases prevented an efficient recruitment of the transcriptional machinery. (iii) Some TALEs do not efficiently activate gene expression irrespective of their orientation, suggesting that either the TALE-box is not accessible within the promoter context or the relative position is not suitable to recruit the transcriptional machinery. The observed activity of reverse binding TALEs breaks the long-standing assumption that TALEs only activate genes in one-direction .OsSWEET14- reporter were co-transformed into N. benthamiana and GUS activity was measured. In most cases, combinations of TALEs at the promoter increased transcriptional induction  of a plant gene –20. SincOsSWEET14 TSS to positions around 40–60 bp downstream of the respective TALE-box which is in concordance to previous observations  lack a prototype TATA-box [Our reporter gene induction experiments demonstrate that the presence of a TALE binding site alone can not trigger transcription, instead, a specific DNA context is required. This indicates that TALEs function analogous to enhancer-binding proteins that require the basal transcription machinery for their action and function as control agents for gene expression. Previous studies have revealed a binding preference for natural TALEs at -300 to +200 bp around the TSS of a target gene, a region that typically includes core promoter elements ,57,58. OTATA-box ,66. Indeox [Xa23 ,68, OsSWOsSWEET4 , pAGT582AGT582-1 , and theTATA-box ,70. ThisOsSWEET14 [OsSWEET14 promoter  to a position 40–60 bp downstream of their binding position upon gene activation –20, but OsSWEET14 promoter. This shows again that the position within the promoter dictates the overall efficiency of the activator and that the activation domain can functionally be replaced by related ones. Our TALE-VP64 fusions still contained most of the C-terminal domain of native TALEs and the portion that interacts with TFIIAy [OsSWEET14 promoter is so well suited for activators, we propose that our TALE-positioning screen actually identified a region spanning a potential natural enhancer element that regulates the expression of OsSWEET14 in rice [In a breakthrough study one of these interactions could recently be shown . TALEs ih TFIIAy . This do in rice ,84.OsSWEET14 promoter. This indicates that both orientations are not equally well suited to induce gene expression and that TALEs prefer the forward orientation. Possibly, in reverse orientation, the TALE protein itself is blocking the path of the polymerase and an efficient establishment of a novel TSS can only occur downstream of the TALE. This is supported by our observation that both, forward- and reverse-binding TALEs upstream of the TATA-box trigger the same TSS downstream. This further suggests that TALEs bound in either one of the orientations can activate already present, but paused polymerase II complexes [We were highly surprised to realize that TALEs also function when bound in a reverse orientation relative to the open reading frame. This has, so far, not been considered for the identification of TALE targets and the reverse binding mode will be an important implementation to identify novel TALE virulence targets. It further points out that the orientation, relative to the TSS, of the TALE activation domain, which is interacting with host components, does not play a role under certain, but not all, conditions. This observation further suggests that TALEs may initiate transcription bidirectionally. A bidirectional transcription describes a situation in which—originating from one promoter—two divergent transcripts are initiated ,86. Thisomplexes .Comparing the activity of our TALE collection at the wild-type and a promoter-derivative lacking the TATA-box and surrounding sequences revealed that those TALEs that are located closely upstream of the TATA-box function less well, whereas further upstream positioned TALEs and TALEs downstream of the TATA-box are not influenced. This indicates that TALEs can either use existing elements or use novel downstream promoter sequences if they are suitable. In the latter case, the TSS is changed. This observation is consistent with our promoter-swap experiments which revealed that the region downstream of the TALE is crucial for TALE-dependent gene activation. Intriguingly, the activity of the reverse-oriented TALEs in particular is negatively impacted by the removal of the TATA-box. This indicates that transcription initiated by reverse TALEs is especially dependent on the presence of potent other promoter elements. This restriction is likely the reason why so far most natural TALEs bind in forward orientation at their respective target promoters.OsSWEET14 promoter revealed that TALEs can act synergistically in plants. This feature was only shown in human cells before, where TALE derivatives with a truncated C-terminus were fused to the VP64 activation domain [in planta may not only act synergistically with each other but are further supported by promoter elements which are possibly occupied by host transcription factors (e.g. the TATA-box). Whether the synergistic activity of TALEs is based on increased chromatin remodeling or other effects remains to be elucidated.Our combination of several weakly active TALEs targeted to either forward or reverse binding sites in the n domain ,45,96. IOur systematic analysis of variably positioned TALEs shows that they act in principle as enhancer-binding proteins. When placing designer TALE activators in promoters to trigger efficient target gene activation one should consider the presence of existing promoter elements, e.g. the TATA-box, and the expected possible shift of the TSS. The novel reverse binding mode of TALEs now allows for the detection of formerly overlooked virulence targets in host plants.Therefore, we have added the option to search for reverse-binding TALEs in the TALE-target prediction programs TALgetter , and theS1 FigBs4 promoter  or from the Bs4 open reading frame (oBs4). Potential cis-regulatory motifs according to the PlantCARE  prediction were marked with different colors on either the forward or the reverse strand of the fragments. The potential function of those motifs according to PlantCARE was noted behind the name of the motif.Sequence overview of the analysed 75 bp long DNA fragments that originate either from the (PDF)Click here for additional data file.S2 FigOsSWEET14 promoter fragment 1kb upstream of the ATG. The binding sites and binding orientation of artificial TALEs (coloured) and the natural TALEs TalC and AvrXa7 (white) is marked with arrows. Reverse binding TALEs are labeled with "-" in front of the number. The 33 bp deletion in the sweet14-11 promoter is indicated.Overview of the (PDF)Click here for additional data file.S3 Fig(A) Schematic overview of the reporter constructs. To compare the activation potential of each TALE independent of their position or orientation in the OsSWEET14 promoter their 18 bp TALE-boxes were inserted in forward orientation in front of the 75 bp pBs4 minimal promoter and a promoterless uidA reporter gene. (B) Activity of artificial and natural TALEs. Agrobacterium strains delivering the 35S-controlled TALE constructs and reporter constructs, respectively, were co-inoculated into N. benthamiana leaves. The TALE Hax3 and its Hax3-box reporter were used as positive control. The quantitative β-glucuronidase measurement was performed 2 dpi. Error bars were calculated on the basis of three independent replicates . (C) Detection of TALE proteins in N. benthamiana. In parallel to the β-glucuronidase assay, six leaf discs of the inoculated area were harvested to analyze protein levels of the TALEs in N. benthamiana. SDS-PAGE followed by immunoblotting with an anti-GFP antibody directed against the N-terminal GFP that was fused to all analyzed TALEs shows a stable protein production. The expected size of artificial TALEs with 17.5 repeats and Hax3 with 11.5 repeats is indicated in kDa. The expression of GFP served as positive control, the sample with not-inoculated plant material (empty) as negative control.(PDF)Click here for additional data file.S4 Fig(A) Overlapping target sequences of the TALEs TAL2 and TAL-2 within reporter constructs. The TAL2/-2-box encompassing both TALE-boxes and the TAL2/-2mut-box with point mutations (red letters) that interfere only with binding of TAL-2, but not TAL2 were fused to the minimal promoter (75 bp Bs4) and a promoterless uidA reporter gene. (B) The activities of the TALEs were determined by quantitative β-Glucuronidase (GUS) measurement in N. benthamiana. Agrobacterium strains delivering the 35S controlled TALE constructs and the corresponding reporter constructs, respectively, were co-inoculated into N. benthamiana leaves. The TALE Hax3 and its Hax3-box reporter construct were used as control. GUS measurement were performed 2 dpi, error bars were calculated on the basis of three independent replicates .(PDF)Click here for additional data file.S5 FigOsSWEET14 promoter including the location of TALE-boxes (coloured boxes) was aligned to DNase I, MNase or bisulfite sequencing profiles indicating occupied promoter regions. DNase I and MNase profiles for rice leaves were downloaded from PlantDHS  in bigwig format. In addition, DNase I hypersensitive sites (DHS) and nucleosome tracks were downloaded from PlantDHS in gff format. DNase I reads of rice seedlings from  where downloaded from NCBI Sequence Read Archive (SRA), accession SRX038423, and mapped to the rice MSU7/TIGR7 genome using bowtie2  with seed length 15 and at most 1 seed mismatch. MNase-Seq data from  of length 75 bp (SRR1536134) and 36 bp (SRR1536112) where also downloaded from SRA and mapped using bowtie2 with identical parameters. Bisulfite sequencing (BS-Seq) data of rice panicles from  were downloaded from SRA  and nucleotide-wise methylation percentages where determined using Bismarck  with bedGraph output.The 1 kb fragment of the (PDF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S1 References(DOCX)Click here for additional data file.


Type II DNA topoisomerases (TOP2) regulate DNA topology by generating transient double stranded breaks during replication and transcription. Topoisomerase II beta (TOP2B) facilitates rapid gene expression and functions at the later stages of development and differentiation. To gain new insight into the genome biology of TOP2B, we used proteomics (BioID), chromatin immunoprecipitation, and high-throughput chromosome conformation capture (Hi-C) to identify novel proximal TOP2B protein interactions and characterize the genomic landscape of TOP2B binding at base pair resolution.Our human TOP2B proximal protein interaction network included members of the cohesin complex and nucleolar proteins associated with rDNA biology. TOP2B associates with DNase I hypersensitivity sites, allele-specific transcription factor (TF) binding, and evolutionarily conserved TF binding sites on the mouse genome. Approximately half of all CTCF/cohesion-bound regions coincided with TOP2B binding. Base pair resolution ChIP-exo mapping of TOP2B, CTCF, and cohesin sites revealed a striking structural ordering of these proteins along the genome relative to the CTCF motif. These ordered TOP2B-CTCF-cohesin sites flank the boundaries of topologically associating domains (TADs) with TOP2B positioned externally and cohesin internally to the domain loop.TOP2B is positioned to solve topological problems at diverse cis-regulatory elements and its occupancy is a highly ordered and prevalent feature of CTCF/cohesin binding sites that flank TADs.The online version of this article (doi:10.1186/s13059-016-1043-8) contains supplementary material, which is available to authorized users. TOP2A and TOP2B, that originate from an ancestral gene duplication event [Top2a in mice leads to defects in nuclear division and early embryonic lethality [The type II topoisomerase (TOP2) enzymes resolve DNA topology problems in core biological processes such as transcription, replication, recombination, DNA repair, chromatin remodeling, chromosome condensation, and segregation –3. TOP2 on event , 5. TOP2on event . TOP2A ion event , 8 and kethality –11. In cethality .Top2b in mice leads to perinatal lethality mediated by defects in neuronal differentiation [Top2b mouse knockout studies have demonstrated TOP2B’s importance during retinal development [The full knockout of ntiation . Conditielopment  and ovulelopment . Studieselopment –17 and lelopment . In contelopment . DespiteIt has been proposed that TOP2B’s role in development involves the activation or repression of specific developmental genes , 21. HumTOP2B is also actively studied in the context of cancer. For example, TOP2B-mediated cleavage occurs at known chromosomal breakpoints in prostate cancer  and has Identifying the protein–protein and protein–DNA interactions of TOP2B is essential for understanding its roles in development, transcription, and cancer. Here we report a comprehensive proximal protein interaction network for TOP2B that includes several members of the cohesin complex. Using ChIP-seq and ChIP-exo in combination with high-throughput chromosome conformation capture (Hi-C) data, we find that TOP2B interacts with CTCF and the cohesin complex with a distinct spatial organization at the borders of long-range chromosomal domain structures.We first set out to characterize a TOP2B protein–protein interaction network. Topoisomerases are large and relatively insoluble proteins  that pren = 6). Control experiments involved parental cells (no BirA*), a BirA*-FLAG fused to green fluorescent protein (GFP-bait), and a BirA*-FLAG tag fused to a nuclear localization signal (NLS-bait)  see “”. Mass sit) see “. We deteit) see “.Supporting the sensitivity of the BioID method, we recovered several previously known interaction partners of TOP2B: TOP2A forms active heterodimers with TOP2B in HeLa cells ; TOP1 foTo investigate whether the physical interactions of TOP2B are also reflected at the genomic level, we profiled DNA occupancy of TOP2B in primary mouse liver cells using chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq). The mouse liver is an optimal TOP2B-expressing tissue for our in vivo experiments as it provides an abundant and relatively homogenous source of non-dividing cells and is an actively used model for mammalian gene regulation with a wealth of functional genomic datasets available –42 Addi.p <10–16, Fig. q < 10–3, Fig. As expected, we found an enrichment of TOP2B binding at gene promoter regions  data , we founConsistent with its preference for DHS regions, we find that TOP2B occupancy is enriched at nucleosome-free regions delineated by MNase-seq experiments performed in mouse liver . SpecifiNext, we characterized the sequence properties of TOP2B bound regions using de novo motif discovery see “”. The mor = 0.403, p <10–16)   Fig. . We foun16) Fig. . We idenTo investigate whether CTCF and cohesin sites occupied by TOP2B possess unique biochemical and evolutionary features, we first explored the genomic co-occupancy of these proteins. TOP2B was found at approximately half of the CTCF/RAD21 sites   to measup <10–16; Fig. p <10–16; Additional file We then asked whether TOP2B binding at CTCF/RAD21 binding sites corresponds to shared orthologous CTCF sites using CTCF ChIP-seq data previously ascertained for human, macaque, rat, and dog liver tissue . We founp <10–16; Fig. Since rodent-specific transposable B2 SINE (Short Interspersed Element) sequences are a source of lineage-specific CTCF binding sites in rodent genomes , 52, we p <10–16, Fisher’s exact test)  Fig. ; Fig. 5cst) Fig.  was founTo determine the precise spatial organization of triple sites, we performed ChIP-exo experiments  for TOP2In order to obtain insights into the exonuclease protection signal of TOP2B and RAD21 relative to the CTCF motif at single base pair resolution, we plotted an average number of 5′ nucleotides of ChIP-exo reads aligned to each base pair around oriented CTCF motifs Fig. . We analOur ChIP-exo protection signal further confirmed the orientation-specific binding of RAD21 and TOP2B relative to CTCF Fig. . Specificore plus the upstream CTCF motif” peaks showed the previously reported increase in ChIP-exo protection signal at positions –16 (reverse strand) and –25 (forward strand) [core plus the upstream CTCF motif” peaks   (Additio strand) . InteresCTCF and cohesin proteins are key architectural components of the genome that anchor long-range interactions that structure chromosomal domains –64. MultUsing recently published mouse liver Hi-C datasets , we studConsistent with other reports , 56, we To test the possibility that TOP2 proteins are involved in supercoiling at CTCF sites we reanalyzed the supercoiling domain data of Naughton et al. . NaughtoWe present the first effort to characterize TOP2B’s protein–protein interactome. BioID enabled us to characterize a network of TOP2B proximal protein interactions that are consistent with its localization and function. Most notably, we identified members of the cohesin complex as well as CTCF as significant proximal interacting proteins. From a protein domain perspective, our BioID results show several interactions with zinc finger containing proteins, many of which are novel and relevant to known TOP2B biology . For example, MORC ATPases have been shown to be involved in heterochromatin silencing in eukaryotes  and follTOP2B shuttles between the nucleoplasm and nucleolus and this shuttling involves an RNA interaction with its C-terminal domain . Our BioTOP2 poisons can affect RNA polymerase I (Pol I) transcription at rDNA loci and this has been shown to involve TOP2A . In addiIt is a long-held hypothesis that TOP2 is attracted to a pre-existing combination of DNA sequence and/or chromatin structure . A modelTOP2B-bound open chromatin regions have distinct functional properties. For example, while it is known that CTCF and cohesin play major roles in genomic regulation, CTCF/cohesin sites co-bound by TOP2B showed stronger ChIP-seq signal, are more likely to be evolutionarily conserved, and are enriched at chromatin domain boundaries compared to CTCF/cohesin only sites. HNF4A binding sites co-bound with TOP2B are also enriched for conserved orthologous HNF4A binding. Thus TOP2B co-occupancy not only occurs at chromatin regions that exhibit topological stress induced by genome regulatory function (e.g. promoters and enhancers), but also includes regions of the genome that are fundamentally important for chromatin architecture.r = 0.8) [Positioning of TOP2B at promoter, enhancer, and topologically associating domain (TAD) boundaries suggests mechanisms by which tissue-specific DNA damage could be imparted. If TOP2B-induced DSBs are not faithfully re-ligated, adjacent genomic regions are potentially susceptible to genome rearrangements , which cr = 0.8) . While tCTCF/cohesin sites anchor both chromosomal domains  , 89 as wTADs contain supercoiling domains whose borders are also enriched for CTCF binding sites . Based oWe only have a basic understanding of how the ubiquitously expressed TOP2B selectively regulates gene expression in vivo. Detailed information about protein–protein and DNA–protein interactions of TOP2B is important for understanding its role in development, rapid gene expression, and chemotherapeutic responses. We identified cohesin and several other chromatin proteins that are in close proximity to TOP2B in vivo. We demonstrated that TOP2B binding occurs at evolutionarily conserved TF binding sites and topological domain boundaries. The prevalent occupancy of TOP2B at conserved gene regulatory and chromatin architectural regions indicates that TOP2B is intrinsically positioned to function at actively utilized points of genome control.TOP2B gene was generated via Gateway cloning into pDEST 5′ BirA*-FLAG-pcDNA5-FRT-TO. TOP2B (accession #NM_001068) was cloned into pDONR223 entry vector using pooled human cDNA and sequence verified. TOP2B bait protein tagged with a N-terminal BirA*-FLAG tag (n = 6 replicates) was stably expressed in Flp-In T-REx HeLa cells as described [n = 6) and stable cells expressing BirA*-FLAG fused to a green fluorescent protein  or to a nuclear localization sequence  were used as negative controls for the BioID experiments and processed in parallel to the TOP2B bait expressing cells. Stable cell lines were grown to 80 % confluence before expression was induced via 1 μg/mL tetracycline and biotinylation by the addition of 40 μM biotin for 24 h. Subsequently, cells were washed and harvested in ice-cold PBS and frozen at −80 °C until purification.Construct for escribed . ParentaEqual quantities of starting material were used for each BioID experiment. HeLa cell pellets were thawed in 1.5 mL ice-cold modified RIPA buffer (50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1 % NP-40, 1 mM MgCl2, 1 mM EGTA, 0.1 % SDS, and 0.4 % sodium deoxcycholate). Sigma protease inhibitor cocktail  and PMSF (1 mM) were added prior to use. The lysates were sonicated at 4 °C using three 5 s bursts at 35 % amplitude with 3 s pauses. Samples were treated with 250 U of TurboNuclease (BioVision) for 15 min followed by removal of insoluble material by centrifugation at 20,000 g. The supernatant was transferred to a new tube and 30 μL of pre-washed streptavidin-sepharose bead slurry  was added. Biotinylated proteins were captured on the beads for 4 h at 4 °C with rotation. The beads were washed once with 1 mL of 2 % SDS in 25 mM Tris (pH 7.4), once with 1 mL of standard RIPA buffer, once with 1 mL of TNNE (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.1 % NP-40, 1 mM EDTA). Lastly, the beads were washed three times with 1 mL of 50 mM ammonium bicarbonate, pH 8.0 (ABC). Following the final wash, the beads were pelleted and any excess liquid was aspirated off. The proteins captured on the beads were resuspended in ABC, reduced with 5 mM DTT at 50 °C for 30 min, and alkylated using 50 mM iodoacetamide for 20 min at room temperature in the dark. The proteins were digested overnight with gentle rotation at 37 °C with 1 μg of trypsin  in a total volume of 50 μL. In the following morning, an additional 0.5 μg of trypsin was added for an additional incubation of 2–4 h. The beads were pelleted and the peptide supernatant was transferred to a fresh tube. The beads were rinsed twice with 75 μL HPLC-grade water and the wash fraction was combined with the supernatant. The peptide solution was acidified with 50 % formic acid to a final concentration of 5 % and the samples were dried in a centrifugal evaporator. Tryptic peptides were re-suspended in 15 μL 5 % formic acid and stored at −80 °C until analyzed by mass spectrometry. Mass spectrometry and data analysis were carried out as described previously . BrieflyRaw mass spectrometry files were stored, searched, and analyzed using the ProHits laboratory information management system (LIMS) . The WIFhttp://maxquant.org) and the Global Proteome Machine .Briefly, the spectra were searched against a total of 72,230 proteins consisting of the NCBI human and adenovirus complements of the RefSeq database , supplemented with “common contaminants” from the Max Planck Institute  were compressed to three samples, meaning that after SAINTexpress was run on each sample individually, the three highest SAINTexpress scores were averaged for the final scoring and Bayesian FDR assessment. To increase the stringency in the identification of true positives, the 12 controls were also compressed to four; in this case, the compression is performed before running SAINTexpress by selecting the four highest spectral counts for each prey protein for modeling [www.crapome.org) [The results from each search engine were analyzed through TPP   via the modeling . All conome.org)  and assiq <0.05; Additional file Significantly enriched pathways were computed with the g:Profiler software , using oPost-mortem liver material from male C57BL/6 × A/J mice (aged ~6–8 weeks) were kindly provided by Dr. Duncan Odom (Cambridge Research Institute). C57BL/6J mice (aged ~6–8 weeks) post-mortem livers used for ChIP-seq and ChIP-exo were kindly provided by Dr. Jayne Danska. Fresh liver tissue was fixed for 20 min in 1 % formaldehyde as described previously .n = 5), anti-CTCF , anti-RAD21 , anti-H3K36me3 , anti-H3K4me3 , anti-H3K4me2 . The DNA was end-repaired, dA-tailed, ligated to the sequencing adapters, PCR amplified by 16 cycles using multiplexing index primers (NebNext), size selected , quantified with 2100 Bioanalyzer (Agilent), and 50 bp reads were sequenced with the HiSeq2500 (Illumina).ChIP-seq experiments were performed as described previously . The folWe used an Illumina ChIP-exo protocol  adapted Publicly available datasets used in this study include: mouse liver ChIP-seq of multiple liver expressed regulatory factors and histone modifications (Data accession: E-MTAB-941) , mouse lhttp://bio-bwa.sourceforge.net/) [https://sites.google.com/site/anshulkundaje/projects/blacklists). Only uniquely mapped reads were used for further analysis.ChIP-seq sequencing reads were trimmed to 36 bp and aligned to the reference mouse genome assembly  available at UCSC genome browser database using the Burrows-Wheeler Aligner (ge.net/)  with defq = 0.01.Quality of the datasets processed from the raw sequencing reads was assessed following the ENCODE ChIP-seq guidelines . Qualityn = 2) or IgG sc-2027 (n = 1)) was used for each ChIP  . RIME asq = 0.01 and fold change cutoff of 5. The “–keep-dup” option was set to “all” to keep duplicated reads. For histone modifications and RNA polymerase II binding, peaks were called with additional “—broad –broad-cutoff 0.05” options. To compare ChIP-seq and ChIP-exo peaks, the SWEMBL (www.ebi.ac.uk/~swilder/SWEMBL) peak caller was used with parameter “-R 0.005.”The reads of biological replicates and corresponding input samples were merged for peak calling. Read counts and peak numbers used in our analyses are listed in Additional file p values were calculated with R using the one-sided binomial test. The overlap of TOP2B ChIP-seq binding sites with binding sites of other factors was calculated using bedtools intersect [q values were smaller then 10–3.The genomic distribution of TOP2B binding was annotated using the cis-regulatory element annotation system (CEAS) . p valuentersect . The signtersect  with 100Using pairwise Pearson correlation coefficients as a distance measurement, we clustered multiple ChIP-seq experiments using hierarchical clustering and visualized the result as a heatmap with the R bioconductor package DiffBind . Peak reProcessed RNA-seq gene expression values for mouse liver (GSM1015152)  were logTo analyze the binding of TOP2B and other factors at rDNA loci, we constructed a customized mouse genome with the single rDNA repeat sequence included as an extra chromosome. Mouse rDNA sequence and structure were obtained from GenBank accession no. BK000964. Reads were aligned to this customized genome using bwa with default parameters. Only uniquely mappable reads were used for downstream analysis. Reads were extended to 150 bp prior to plotting. After normalizing to the number of mapped reads for each ChIP-seq and input experiment, input reads were subtracted from ChIP-seq reads at each base pair of the rDNA repeat. Plotting was performed using R package “Sushi” . MappabiAligned Dnase I Digital Genomic Footprinting (DGF) data for mouse liver were obtained from the ENCODE database  by the Ren lab as part of the mouse ENCODE release . In order to match the genome assembly used in Ensembl 70, CTCF peaks identified in mm9 were lifted over to mm10 using the liftOver tool and the chain file provided by UCSC Genome Browser Database . Triple http://www.repeatmasker.org) was run on the genome sequences of CTCF peaks. Only peaks having the B2 SINE repeat overlapping their peak summits were included in the analysis.The Repeatmasker method . Peak overlaps were performed and plotted using DiffBind [Sequencing reads for ChIP-exo experiments were aligned without trimming. All reads were used in the analysis. ChIP-seq of the same factors was performed in parallel for comparison. We applied the SWEMBL peak caller algorithm that is sensitive for point-source data  were used to investigate the allele-specific binding preferences of TOP2B at locations bound by specific TFs in mouse liver. Single nucleotide polymorphisms (SNPs) obtained from the Sanger Mouse Genomes Project version 2 were used to acquire a list of SNPs between the A/J and reference (C57BL6/J) genomes . Alignedp <0.05) were annotated based on the mouse strain possessing TF-preferred allele. A one-sided Wilcoxon ranked sum test was used to compare the allele frequency of each factor between allelic biased regions and non-biased regions.Considering only the overlapping regions of the TOP2B and the TF in question, we counted the number of allelic reads mapped to each parental genome to determine an allele frequency. Peaks showing significantly biased allelic read distribution  . Please 2(bTMP cell /bTMP input) – log2(bTMP genomic DNA – bTMP input) [p values were smaller than 10–16.Processed files containing DNA microarray probe intensities were obtained from ArrayExpress (E-GEOD-43450). Data were processed as previously described with normalized bTMP incorporation calculated as logP input) . GENCODEP input)  were use