Prdm1/Blimp1, an essential regulator of placenta development, were identified in ChIP-seq experiments. Comparisons with previously reported ChIP-seq datasets for primordial germ cell-like cells and E18.5 small intestine, combined with functional annotation analysis revealed that Blimp1 has broadly shared as well as cell type-specific functional activities unique to the trophoblast lineage. Importantly, Blimp1 not only silences TSC gene expression but also prevents aberrant activation of divergent developmental programmes. Overall the present study provides new insights into the chromatin landscape and Blimp1-dependent regulatory networks governing trophoblast gene expression.Trophoblast stem cells (TSCs) give rise to specialized cell types within the placenta. However, the regulatory mechanisms that guide trophoblast cell fate decisions during placenta development remain ill defined. Here we exploited ATAC-seq and transcriptional profiling strategies to describe dynamic changes in gene expression and chromatin accessibility during TSC differentiation. We detect significantly increased chromatin accessibility at key genes upregulated as TSCs exit from the stem cell state. However, downregulated gene expression is not simply due to the loss of chromatin accessibility in proximal regions. Additionally, transcriptional targets recognized by the zinc finger transcriptional repressor  Most primates and rodents form a hemochorial placenta, where maternal blood directly contacts foetal trophoblasts derived from the outer layer of the blastocyst, the so-called trophectoderm. Following implantation the trophectoderm gives rise to both the extraembryonic ectoderm (ExE) lying proximal to the epiblast, as well as the ectoplacental cone (EPC) which forms on the distal maternal interface. The ExE contains the progenitors of specialized syncytiotrophoblasts that mediate maternal-foetal exchange in the placental labyrinth region, whereas the EPC gives rise to the outer spongiotrophoblast (SpT) layer - the source of diverse trophoblast giant cells (TGCs) including the spiral artery-associated trophoblast giant cells (SpA-TGCs) that invade the maternal uterine tissue and replace arterial linings to promote increased blood flow to the foetus3.The placenta, a specialized organ comprised of both maternal and foetal tissues is essential to support mammalian embryonic development5. TSCs are lineage-restricted and when reintroduced into blastocysts exclusively populate the labyrinth and SpT compartments of the placenta6. Upon growth factor withdrawal, TSCs differentiate into mature trophoblast subtypes. In contrast, pluripotent embryonic stem cells (ESCs) derived from the blastocyst inner cell mass, differentiate to form tissues of the embryo proper. The regulatory mechanisms that govern gene expression changes during ESC differentiation are well characterized7. Pluripotent ESCs have relatively low levels of heterochromatin, and lineage restriction during differentiation is accompanied by chromatin condensation7. Genome-wide histone ChIP and DNase I hypersensitivity approaches have been used to describe the global chromatin landscape in TSCs10. However, the dynamic epigenetic changes that co-ordinately regulate local and long-distance promoter-enhancer interactions governing trophoblast differentiation remain ill defined.Trophoblast stem cells (TSCs) isolated from the trophectoderm or the early post-implantation (E6.5) ExE retain self-renewal capacity in the presence of FGF4 and TGFβ111, Blimp1 (encoded by the Prdm1 gene) governs cell-fate decisions in the embryo and adult organism. Blimp1 is required for primordial germ cell specification13 and plays essential roles in the epidermis15, mammary gland development16, and post-natal reprogramming of intestinal enterocytes18. Prdm1/Blimp1 null embryos die at around E10.5 due to placental insufficiency13. Prdm1 is broadly expressed throughout the EPC and SpT layer19. The lethality is associated with loss of the Blimp1-dependent invasive SpA-TGC lineage19. During in vitro differentiation Blimp1 expression is detectable in day 2 diploid trophoblasts, and later in mature TGCs20. The Prdm1 loss-of-function phenotype is recapitulated in vitro, since Prdm1 null TSCs display restricted differentiation abilities and fail to upregulate SpA-TGC markers20.Originally discovered as a master regulator of plasma cell differentiation21 to describe global chromatin accessibility changes associated with loss of the stem cell state and the emergence of the Blimp1+ trophoblast cell lineage. Additionally, our ChIP-seq and transcriptional profiling experiments identified numerous candidate target genes directly repressed by Blimp1 during trophoblast differentiation. Combined with functional annotation analysis and comparisons with published microarray datasets, collectively the present genome-wide analyses reveal key features of the chromatin landscape controlling trophoblast gene expression profiles and advance our understanding of the signalling pathways that regulate development of the trophoblast cell lineage.To learn more about transcriptional and epigenetic mechanisms controlling maturation of various trophoblast cell types, here we exploited ATAC-seq technology22. To map cis-regulatory elements (CREs) in TSCs we performed ATAC-seq experiments, identifying 57,019 distinct regions of accessible chromatin , while those shared with ESCs are more likely to be promoters   Fig. . In contVs) Fig.  but wereVs) Fig. . Though Vs) Fig. . Interesncy Fig. , strongl5   specification through CRE co-occupancysts Fig. . Moreovein) Fig. .34 and E18.5 small intestine (SI)18. The majority of our ChIP-seq peaks are also detectable in either the SI or PGCLC datasets. However, roughly 30% were exclusively present in the d2 trophoblasts sts Fig. . Functiosts Fig. .Figure 6Prdm1 mutant vs. wild type TGCs at d2, d4 and d6 of differentiation20 with our ChIP-seq data identified 125 candidate target genes represented amongst the upregulated transcripts  Fig. . SimilarARs Fig. . Other Bxp1 Fig. . It therIn sum results above characterize structural changes at discrete chromatin regions associated with trophoblast differentiation, and identify novel targets genes that potentially regulate Blimp1-dependent trophoblast lineage specification.40. However, relatively little is known about epigenetic mechanisms governing TSC differentiation and specification of mature trophoblast subtypes.The epigenetic landscape and transcriptional networks that govern self-renewal and maintenance of the stem cell state have been extensively studied in TSCs and ESCs41. It is tempting to speculate that formation of the apical domain during the establishment of the trophectoderm fate may occur via accessible chromatin domains common to 8-cell embryos and TSCs that have been identified here.The present ATAC-seq experiments examine genome-wide changes in chromatin accessibility during trophoblast differentiation. We identified thousands of DARs present at early stages of TSC differentiation, as well as regions that distinguish TSCs from ESCs. Remarkably, a subset of these is detectable as early as the 8-cell stage embryo. Recent experiments demonstrate that apical-basal cell polarity leading to segregation of the trophectoderm and inner cell mass becomes apparent at the 8-cell stage32. Interestingly, a subset of Blimp1 targets are present at CREs previously occupied by Esrrb, including Ankmy2, Foxp1 and others . Moreover, we found that Esrrb ChIP-seq peaks in TSCs are significantly associated with genes downregulated during TSC differentiation. Thus gene expression changes during TSC differentiation can be explained in large part due to the concomitant loss of Esrrbers Fig. . Blimp1 42. Considerable evidence suggests that species-specific ERVs provide crucial regulatory signals driving rapid evolution through re-wiring transcriptional networks in TSCs9. Here we found that TSC DARs are enriched for the RLTR13 family of ERVs, previously identified by profiling of active histone marks in TSCs and containing binding sites for key TSC regulators Cdx2, Eomes and Elf59. Strikingly, however, d2 DARs seem to lack annotated repeat regions. Rather d2 DARs are highly enriched for distinct transcription factor binding motifs such as for Tfap2c, and Ets and GATA sites potentially bound by Elf5 and Gata2 respectively, known to be involved in trophoblast differentiation43.Both the structural and functional characteristics of the placenta display considerable diversity across mammalian speciesPrdm1 mutant trophoblasts we found both shared and partially overlapping, as well as cell type-specific targets. For example, Irak2 and Mmp2 appear to be common targets in all three cell types, while Vsig10 appears to be unique to trophoblasts. As for transfected P19 embryonal carcinoma cells33, here we observe occupancy by Blimp1 and Tfap2c at common target sites in trophoblasts, though it is not clear whether they bind independently or cooperatively in this case. Tfap2c functions as a transcriptional repressor governing trophoblast differentiation within the SpT layer29, but a cooperative Blimp1 and Tfap2c functional relationship has not previously been reported during placenta development. Interestingly, coexpression was recently confirmed in trophoblasts at the single-cell level20. Future studies will explore whether Blimp1 and Tfap2c function as transcriptional partners to cooperatively regulate target gene expression in discrete spongiotrophoblast cell lineages.When we compared Blimp1 ChIP-seq datasets from trophoblasts, PGCLCs and SI to genes derepressed in in vivo using ChIP approaches is confounded by the diversity of Blimp1+ cell populations20. Gene expression changes may also be masked by organ-wide ensemble averaging. The present trophoblast-specific in vitro microarray combined with ChIP-seq analysis, however, demonstrates a significant overlap with genes upregulated in Blimp1 mutant placenta at E9.5 , analogous to mouse SpA-TGCs45 form atypical podosomes that regulate extracellular matrix degradation and cell migration46. Disabled 2 interacting protein (Dab2ip) inhibits cell invasion in a variety of contexts48, and upregulation in human trophoblasts in vitro is associated with reduced invasive and migratory abilities49. Blimp1 repression of Dab2ip may be required to promote trophoblast invasion.Identification of direct Blimp1 targets 9.5 Fig. . Many of50. Many of the 125 Blimp1 targets identified here, however, are either poorly characterized or unstudied in the context of placenta. For example, Vsig10 is a robust candidate target gene but its functional activities have not been previously reported. However, it is striking that the Blimp1 target genes identified here are significantly enriched for genes with vascular and immunological, as well as cellular signalling functions. For example, it seems likely that Blimp1 controls intracellular signalling via repression of Ras guanyl-nucleotide exchange factors such as Arhgef18, as well as the signalling-related kinase Irak2, which promotes inflammatory response through NF-kappaB pathway activation51. Arhgef18 has been implicated in cell invasion, which is highly impaired in Prdm1 mutants53. Additionally, Blimp1 regulates expression of matrix metalloproteinase Mmp2, implicated in trophoblast invasion and preeclampsia, though its precise function in the placenta requires clarification56. Multiple other genes, such as candidate tumour suppressor Vwa5a36, and the regulator of trophoblast invasion Cd4437 may also be key to the Prdm1 mutant phenotype. Amongst the other identified targets of Blimp1 is the RING domain containing isoform of Lnx1, which is reported to ubiquitinate specific isoforms of NUMB, leading to its degradation35. NUMB is a multifunctional protein implicated in human trophoblast cell migration and apoptosis57. Numb mouse mutants die mid-gestation probably due to placental insufficiency, though a detailed characterization of the precise placental defects have not been performed58. It is possible that upregulation of Lnx1 contributes to the Prdm1 null placental phenotype through degradation of Numb. The extent to which the Blimp1 targets identified here contribute to the Prdm1 null mutant phenotype will be explored in future studies.A critical feature of placenta development is specification and migration of the invasive trophoblasts that function in remodelling maternal spiral arteries to increase blood flow to the developing foetus. The genes expressed by this specialized trophoblast subset mediate both vascular mimicry and evasion of the maternal immune system59, while Gcm1 is essential for development of the syncytiotrophoblasts that constitute the labyrinth60. However the transcriptional regulatory hierarchies guiding cell fate choices remain poorly understood. Here we have characterized Blimp1-dependent target genes and differentially accessible chromatin regions during trophoblast differentiation. Collectively our experiments demonstrate that TSC differentiation protocols in combination with high throughput genomic techniques represents a powerful entry point to identify components of gene regulatory networks that govern later aspects of placenta morphogenesis.Diverse trophoblast sub-populations that perform essential functions during placental development have been extensively characterized on the basis of location, morphology, and gene expression profiles. Several key transcription factors governing specification of specialized trophoblast sub-types have been identified. For example, Hand1 is required for the formation of trophoblast giant cells at the periphery of the placentaPrdm1+/+ and Prdm1BEH/BEH TSC lines isolated previously20 were grown under defined conditions61. To promote differentiation trypsinized cultures were plated in the absence of FGF4, TGFβ1 and heparin sulfate.Prdm1+/+ and Prdm1BEH/BEH null mutant TSC lines (NCBI GEO accession number GSE74409) were analysed as previously described20. Further analysis using K-means clustering was performed using Cluster 3.062. Gene set enrichment analysis (GSEA)64 was performed using all genes represented on the microarray pre-ranked on Illumina DiffScore derived from the differential expression analysis and compared with the nearest gene transcription start site ± 100 kb from each Blimp1 ChIP-seq peak. For maximum stringency where multiple gene sets are tested we report both nominal P value and family-wise error rate (FWER) P value.Published Illumina Mouse WG-6 v2 Expression BeadChip microarray data corresponding to four independently derived 66. Comparison with gene sets defined by ATAC-seq analysis was performed on quantile normalized data using GSEA with standard parameters.For gene expression comparison with TSCs published Illumina Mouse WG-6 v2 Expression BeadChips microarray data for ESCs was downloaded from NCBI (GEO accession number GSE46308). After background subtraction using GenomeStudio v2009 (Illumina) raw data were quantile normalized and differentially expressed genes identified using ArrayAnalysis67 using 75,000 cells per replicate. Multiplexed 75 bp paired-end sequence reads generated on a single lane of an Illumina HiSeq. 4000 were mapped to the mm10 mouse genome build using Stampy with default parameters68. Mapped data were visualized using Integrated Genome Viewer v2.3.8869. Regions of open chromatin were identified by MACS2 using default parameters70. Regions detectably accessible in both wild type cell lines were identified by overlapping peak coordinates and used for subsequent analyses  as described71.For comparisons with TSC ATAC-seq data 8-cell embryo and ESC ATAC-seq data mm9 peak regions were downloaded from NCBI GEO accession number GSE66581 and converted to mm10 coordinates using liftOverDe novo motif analysis was performed using MEME-ChIP with default parameters72. Matches to known motifs were identified using TOMTOM73. Frequency of de novo identified Blimp1 binding motif within peaks was performed with FIMO74 using a P value cutoff of 1 × 10−4.76 and overlap with ATAC-seq peaks was performed using custom Perl scripts. For comparison repeat annotations were also overlapped with a set of 10,000 randomly generated genomic regions of equivalent size to the ATAC-seq peaks. Statistical differences were determined using Chi-square with Yates’ correction.Repeat element annotations for the GRCm38/mm10 genome build were downloaded from the UCSC Genome Browser databasePrdm1+/+ and Prdm1BEH/BEH TSC lines each were fixed for 15 minutes at room temperature using 1% formaldehyde in culture medium. Samples were then processed for ChIP-seq as previously described77 using 4 × 107 cells per replicate and 14 µg of mouse IgG1 anti-Blimp1 ascites fluid 78. 51 bp paired-end reads generated by multiplexing ChIP and associated input samples on 2 lanes of an Illumina HiSeq. 2500 were mapped to the mm10 mouse genome build with Bowtie280 in Galaxy81 using default parameters, except –k 2. Peaks were called for each ChIP sample relative to its input using MACS270 with default parameters. Non-specific peaks detectable in Prdm1BEH/BEH mutant cells were subtracted from wild type datasets  were downloaded from the published paper32.Tfap2c d1 trophoblast ChIP-seq data was downloaded from the European Nucleotide Archive (accession PRJNA298763) and analysed as above using a MACS2 q-value cut-off of 1 × 10H3K4me1, H3K4me3, H3K9me4, H3K27me3 and H3K27ac TSC ChIP-seq data was downloaded from NCBI GEO accession GSE 42207 and mapped to the mm10 genome build using Bowtie2 and compared with ATAC-seq coordinated using seqMINER as above.83. Plots indicating significance per peak are given in Supplementary Figure P values per dataset comparison and percentage overlap per significantly overlapping peak are given in Supplementary Figure To determine whether overlaps of sets of ChIP-seq and ATAC-seq peaks were statistically significant we applied a multi-step process. First we characterized the pairwise overlap of all peaks within datasets being compared, relative to 1,000 iterations of random regions of equivalent size using GAT84, linking peaks to the nearest transcription start site (TSS) ± 100 kb. Functional annotation of gene lists derived from microarray analysis was performed using Enrichr86. Non-redundant functional terms were selected based on reported significance score and relevance to the biological system.Functional annotation analysis of ATAC-seq and ChIP-seq peak datasets was performed using GREAT version 3.0.087 using Actb to normalize gene expression. Primer sequences used are shown in Supplementary Table −∆∆CT method with expression represented as mean ±S.E.M. Statistical significance was determined using two-tailed homoscedastic t-test.RNA was extracted using RNeasy mini kits (QIAGEN) according to manufacturers instructions. Quantitative PCR (qPCR) was performed as previously describedChIP-seq and ATAC-seq data have been deposited in NCBI GEO (accession numbers GSE74408 and GSE94694 respectively). Details of published datasets used in this study are provided in Methods.Supplementary InformationTable S1Table S2Table S3Table S4Table S5Table S6Table S7Table S8


Cerrena, and included a discussion with regards to regulation of laccase expression.Laccases are a family of copper-containing oxidases with important applications in bioremediation and other various industrial and biotechnological areas. There have been over two dozen reviews on laccases since 2010 covering various aspects of this group of versatile enzymes, from their occurrence, biochemical properties, and expression to immobilization and applications. This review is not intended to be all-encompassing; instead, we highlighted some of the latest developments in basic and applied laccase research with an emphasis on laccase-mediated bioremediation of pharmaceuticals, especially antibiotics. Pharmaceuticals are a broad class of emerging organic contaminants that are recalcitrant and prevalent. The recent surge in the relevant literature justifies a short review on the topic. Since low laccase yields in natural and genetically modified hosts constitute a bottleneck to industrial-scale applications, we also accentuated a genus of laccase-producing white-rot fungi,  Rhus vernicifera and wild-type Bacillus subtilis CotA laccases, respectively) typically have potentials on the low end of this range, whereas fungal laccases have higher redox potentials (0.47–0.79 V)  are a family of copper-containing oxidases found in a variety of bacteria, fungi, insects, and plants  and Trametes  species   promoter , yeasts , filamentous fungi , and plants , are also used  promoter  promoter  promoter, Lentinus edodes priA (fruiting body gene) promoter or S. commune Sc3 (hydrophobin gene) promoter , ACE1 copper-responsive transcription factor binding sites (ACE1), xenobiotic response elements (XREs), antioxidant response elements (AREs), heat shock response elements (HSEs), CreA binding sites (CreA), and NIT2 binding sites (NIT2)  and external factors  influencing laccase synthesis. However, until now, the bulk of the work has only attempted to decipher regulation of laccase expression by an isolated single factor or a few factors, which is undoubtedly a simplification of the complex network controlling laccase expression. The mechanism underlying the regulatory network of laccase expression awaits elucidation.The efficiency of substrate oxidation by a laccase depends on the difference between the redox potentials of the substrate and the T1 Cu. Due to the lower redox potentials of laccases (≤0.8 V) compared to ligninolytic peroxidases (>1 V) Wong, , laccase·+) and then to the di-cation (ABTS2+) with redox potentials of 472 and 885 mV, respectively. Unlike ABTS, an N-OH type mediator (such as HBT and violuric acid) forms the N-oxy radical upon laccase oxidation and subsequent deprotonation; the radical in turn abstracts the benzylic hydrogen atom from the substrate. Similarly, phenolic mediators also follow a radical hydrogen abstraction mechanism, but with the intermediate being a phenoxy radical  are becoming ubiquitous in the environment and are recognized as emerging trace organic contaminants  and from actinobacteria Streptomyces ipomoeae  in tetracycline degradation have been evaluated in depth with respect to membrane preparation, efficiency, kinetics, and economics , a toxic and recalcitrant intermediate of sulfamethoxazole, is formed upon A. faecalis treatment. Additional laccase, on the other hand, eliminates HO-SMX along with the toxicity   (Ostadhadi-Dehkordi et al., Among these PPCPs, diclofenac, carbamazepine, and triclosan are the most investigated Table ; triclosIn addition to removal of pharmaceutical compounds with laccases, laccase-producing fungi, bacteria and actinomycetes are also evaluated. Laccase is at least partially responsible for pollutant degradation (Marco-Urrea et al., Simultaneous laccase degradation of multiple trace organic contaminants demonstrates that phenolic compounds are generally more easily degraded than non-phenolic compounds (Nguyen et al., Laccase mediators HBT and SA are most often used in degradation of PPCPs. Degradation improvement upon mediator addition is significant in the enzymatic membrane reactor and limited in batch incubation (Nguyen et al., Since the first discovery of laccase over 100 years ago, much has been elucidated about the occurrence, biochemistry, sequences, production, and application potentials of this diverse class of enzymes. We have briefly reviewed some recent developments in laccase research with a focus on production and applications in pharmaceutical degradation. Despite the exciting promise laccases bring, applied research is still mostly performed on the laboratory scale. Emphasis should be placed on augmenting the yields and efficiency and lowering application costs, which constitute bottlenecks in scalable and sustainable applications of laccases. While laccase mediators are widely used to aid laccase-catalyzed oxidation, the transformation metabolites as well as their toxicity should always be analyzed. It is clear that more work needs to be done to realize the full potential of these versatile enzymes.JY, TN, JL, and XY wrote the manuscript. JY, WL, and XD compiled the tables.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Acute myeloid leukemia (AML), a common hematological cancer of myeloid lineage cells, generally exhibits poor prognosis in the clinic and demands new treatment options. Recently, direct sequencing of samples from human AMLs and pre-leukemic diseases has unveiled their mutational landscapes and significantly advanced the molecular understanding of AML pathogenesis. The newly identified recurrent mutations frequently “hit” genes encoding epigenetic modulators, a wide range of chromatin-modifying enzymes and regulatory factors involved in gene expression regulation, supporting aberration of chromatin structure and epigenetic modification as a main oncogenic mechanism and cancer-initiating event. Increasing body of evidence demonstrates that chromatin modification aberrations underlying the formation of blood cancer can be reversed by pharmacological targeting of the responsible epigenetic modulators, thus providing new mechanism-based treatment strategies. Here, we summarize recent advances in development of small-molecule inhibitors specific to chromatin factors and their potential applications in the treatment of genetically defined AMLs. These compounds selectively inhibit various subclasses of “epigenetic writers” , “epigenetic readers” (such as BRD4 and plant homeodomain finger proteins), and “epigenetic erasers” (such as histone demethylases LSD1/KDM1A and JMJD2C/KDM4C). We also discuss about the molecular mechanisms underpinning therapeutic effect of these epigenetic compounds in AML and favor their potential usage for combinational therapy and treatment of pre-leukemia diseases. Epigenetic modifications, including DNA methylation and a myriad of post-translational modifications of the DNA-packaging histone proteins, represent a fundamental means for regulating gene expression and other DNA-templated processes –4. TheseWilm’s Tumor-1 (WT1)], gain-of-function mutation of oncogenic kinases , and stem cell transcription factors (TFs) , Additional Sex Combs Like 1 and 2 , the Cohesin complex (SMC3-SMC1-RAD21-STAG) genes, and Isocitrate Dehydrogenase 1 and 2 (IDH1 and IDH2). These newly identified somatic mutations of DNA/chromatin modifiers and structural organizers are in agreement with previous karyotyping/FISH-based analyses of AML patients, which already identified recurrent chromosomal translocation or abnormality of genes encoding various members of epigenetic “writers”  , a common malignancy of myeloid-lineage precursor cells in the blood, is characterized by two hallmarks, uncontrolled cell proliferation and impaired differentiation. Previously, progression and characteristics of AML were linked to several key pathways , 11, incas MEIS1 –14], as as MEIS1 –26. Amon1/KMT3F) , “eraser1/KMT3F) , 33, and1/KMT3F) , 34. Imp or CHIP , 35, 36  or CHIP –39, suppIn contrast to significant advances in molecular appreciation of human AML’s mutational landscape and putative “driving” pathways, chemotherapy remains as the frontline treatment for most AML patients, with an exception of all-trans retinoic acid (ATRA) used as targeted therapy of the acute promyelocytic leukemia (APL) subtype. AML patients still suffer from low overall survival and a high rate of recurrence, demanding new treatments to be developed. Recent studies of AML and other tumors have increasingly shown that genetic lesion of epigenetic modulator often induces a subsequent chain reaction leading to aberrations in chromatin modification/remodeling, gene-expression program, and cellular states during tumorigenesis , 40–43. Mixed-Lineage Leukemia gene (MLL/MLL1/KMT2A) encodes one of the KMT2 family of methyltransferase enzymes that contain multiple structural domains, including a C-terminal SET domain catalyzing methylation of histone H3, Lys4 (H3K4)  –46. MLL ML cases , 29. OftL fusion ; howeverL fusion , sustaing target . While tIn vitro biochemical assays showed that MM-401 specifically targets WDR5 interaction to MLL1, and not other MLL/KMT2 family enzymes. Treatment with MM-401 blocked proliferation and induced myeloid differentiation of MLL-rearranged leukemia cells while not significantly affecting normal blood stem/progenitor cells  encodes a methyltransferase that catalyzes mono/di-methylation of histone H3, Lys9 (H3K9me1/2), a histone modification correlated with gene silencing. Knockout of G9A in hematopoietic systems led to decreased proliferation of myeloid progenitors without affecting the function of long-term repopulating hematopoietic stem cells  and cell differentiation (such as Egr1) through inducing gene-repressive H3K27me3/2  mediating transcriptional repression through tri-methylation of H3K27 (H3K27me3) . EZH1, agnancies , whereasgnancies –65. Suchong AMLs . Recent ormation . MDS indgression , indicatgenecity –74. Mechthylases , 76. Theon block . Importaarranged , 70, 72 ted AMLs , knockdoEZH2 in solid cancer and lymphoma, several pharmaceutical companies have embarked on high-throughput screening campaigns leading to discovery of a series of small-molecule compounds  is a histone H3 Lys79 (H3K79)-specific methyltransferase that regulates gene transcriptional elongation, telomeric silencing, and DNA damage response . Biochem in AMLs , 84–86. nd MEIS1 –91.HOXA9 and MEIS1 and selectively killed MLL-rearranged leukemia cells and xenografted tumors (NUP98-NSD1 (DNMT3A (NPM1 (IDH1/2 (MN1 (NUP98-NSD1 induced leukemic transformation through direct targeting and epigenetic modulation of AML-promoting “stemness” genes (HOX gene clusters and MEIS1) (DNMT3A hotspot mutation (DNMT3AR882H) was recently found to focally suppress DNA methylation at cis-regulatory elements of these genes thereby promoting their transcription activation  , a DNMT3tivation . In adding cells . LeukemiProtein arginine methyltransferase 1 (PRMT1) encodes a methyltransferase for histone H4 arginine-3 (H4R3) and associates with gene activation. PRMT1 was shown to interact with AML1-ETO, a gene fusion product defining AML with t translocation, activate the downstream target genes of AML1-ETO, and promote progression of AML1-ETO-asssociated leukemia (MLL-rearrangement (such as MLL-GAS7) or the MOZ-TIF2 translocation  remove acetylation off histones to influent gene expression. HDACi Table  includinMLL-rearranged leukemias, terminal differentiation arrest was partially enforced by LSD1, and LSD1i treatment induced myeloid differentiation and suppressed leukemogenesis in vivo , where LSD1i sensitized the pro-differentiation effect of ATRA, an agent only for PML-RARA-positive APLs  is the first identified histone demethylase with specificity toward H3K4 mono/di-methylation (H3K4me1/2) . Severalo Figure . Mechanio Figure . Also, ts Figure . Here, cs Figure . SeveralKDM4C  encodes an “eraser” carrying the H3K9-demethylating and gene-activating activities. Like PRMT1, KDM4C was also found to interact with various AML oncoproteins including MLL-GAS7 and MOZ-TIF2  occur frequently among patients with pre-leukemia diseases such as MDS and apparently healthy individuals with clonal hematopoiesis or CHIP, an aging-related phenotype associated with increased risk of AML , 25, 26.s Figure . We specMLL-rearranged leukemias with PRC2 loss, either pre-existing or acquired, are resistant to BRDi presumably due to enhanced transcription of oncogenes such as MYC (MLL-rearranged leukemia, possibly due to functional collaboration between DOT1L and BRD4 at the highly transcribed super-enhancer genes (Resistance to drug remains a challenge in achieving durable remissions in cancer and epigenetically targeted drugs are no exception. The molecular understanding of resistance in epigenetic therapy is just at its beginning. For example, h as MYC ; furtherh as MYC , 130. Coh as MYC . In addier genes . Future RL and GW wrote the manuscript and generated the figures/tables.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


NIPBL are the most frequent cause of Cornelia de Lange syndrome (CdLS), a developmental disorder encompassing several neurological defects, including intellectual disability and seizures. How NIPBL mutations affect brain development is not understood. Here we identify Nipbl as a functional interaction partner of the neural transcription factor Zfp609 in brain development. Depletion of Zfp609 or Nipbl from cortical neural progenitors in vivo is detrimental to neuronal migration. Zfp609 and Nipbl overlap at genomic binding sites independently of cohesin and regulate genes that control cortical neuron migration. We find that Zfp609 and Nipbl interact with the Integrator complex, which functions in RNA polymerase 2 pause release. Indeed, Zfp609 and Nipbl co-localize at gene promoters containing paused RNA polymerase 2, and Integrator similarly regulates neuronal migration. Our data provide a rationale and mechanistic insights for the role of Nipbl in the neurological defects associated with CdLS.Mutations in  •Nipbl interacts with the transcription factor Zfp609 and the Integrator complex•Nipbl, Zfp609, and Integrator are required for cortical neuron migration•Nipbl, Zfp609, and Integrator co-occupy genomic binding sites independently of cohesin•Nipbl, Zfp609, and Integrator directly regulate neuronal migration genes NIPBL mutations cause Cornelia de Lange syndrome, but Nipbl function in brain development is not well understood. Van den Berg et al. show that Nipbl interacts with Zfp609 and the Integrator complex to transcriptionally regulate cortical neuron migration. The cerebral cortex, responsible for higher cognitive function, is generated from a pool of progenitor cells that will give rise to the neuronal and glial lineages of the adult brain. Unperturbed migration of newly born neurons across the expanding cortex to their final destination in specific cortical layers ensures accurate connectivity and neuronal circuit formation. Cell-intrinsic transcription factors play key roles in orchestrating the underlying molecular processes, as was recently shown for the proneural transcription factors Neurog2 and Ascl1 . DevelopNIPBL) have been identified in 50%–60% of cases and are associated with a more severe clinical presentation, while mutations in cohesin complex subunits SMC1A, SMC3, and RAD21 and in the SMC3-targeting deacetylase HDAC8 account for a further 10% of mostly mildly affected cases (Cornelia de Lange syndrome (CdLS) is one particular example of a developmental disorder highlighted by neurological defects, including seizures and intellectual disability. Other characteristics include facial dysmorphism, growth retardation, and upper limb defects. Heterozygous mutations in the cohesin loading factor Nipped-B-like , the single zinc-finger protein Zfp609. We identified Nipbl as a binding partner of Zfp609, which is specifically expressed in neural progenitors in the developing mouse cortex. Zfp609 and Nipbl interact and co-bind genomic regions with the RNA polymerase 2 (RNA pol2)-associated Integrator complex to directly regulate neuronal migration genes. Accordingly, depletion of Zfp609, Nipbl, or Integrator from cortical progenitors in vivo results in neuronal migration defects. Our findings define a Nipbl transcriptional pathway relevant to CdLS.How Nipbl acts in gene regulatory networks and developmental pathways in brain development is poorly understood. In this study, we set out to identify new regulators of cortical development by studying a mouse ortholog of Drosophila scribbler, a single zinc-finger protein that is highly expressed in the larval CNS, where it is proposed to act as a transcription factor  and intermediate zone (IZ) at embryonic day (E)14.5  and in stem cells near the ventricular surface.Zfp608 and Zfp609 are vertebrate homologs of n factor . Zfp608  (E)14.5 . To deliymocytes . At latescribbler mutations affect axon targeting and larval locomotion  in vivo, we electroporated short hairpin RNA (shRNA) constructs into E14.5 mouse embryonic brains  identity and differentiation, as their limitless expansion enables generation of sufficient material for proteomics studies and other genome-wide approaches. To explore whether they could be used as a tool to study Zfp609 function, we analyzed Zfp609 transcript and protein levels in embryonic stem cell (ESC)-derived NSCs . Consistr Zfp608 H. We genr Zfp608 K and S1Lr Zfp608 H. Immunor Zfp608 I.To gain insight into the molecular environment of Zfp609, we purified Zfp609 from NSCs and identified its interaction partners by mass spectrometry. Nuclear extracts from NSCs expressing doxycycline-inducible V5-tagged Zfp609 were subjected to V5 affinity purification, and Zfp609-containing protein complexes were separated by SDS-PAA gel electrophoresis A. NSCs nGel lanes of Zfp609-V5 and control purifications were analyzed by mass spectrometry, and interaction partners present in two Zfp609 purifications are listed in Zfp609, Nipbl transcripts are enriched in the ventricular zone at E14.5 . Out of these, 83% changed expression in the same direction, suggesting cooperativity between Nipbl and Zfp609 in gene regulation. We subsequently focused our analysis on shared target genes of Nipbl and Zfp609, postulated as bound and regulated by both factors. To associate genes with Nipbl and Zfp609 binding sites, we used GREAT (Genomic Regions Enrichment of Annotations Tool)  , which ans Tool) C. A totaNipbl+/− brains  whose downregulation is known to cause neuronal migration defects. Sema3a is a secreted chemoattractive guidance molecule present in a descending gradient from the CP that acts through the co-receptor neuropilin-1 (Nrp1) and specific Plexin receptors, including PlexinD1 (Plxnd1), to promote radial migration of cortical neurons  analysis on these two categories of Zfp609/Nipbl target genes revealed enrichment for terms related to cell motion and the extensive cytoskeletal remodeling that occurs during this process , in particular among the set of target genes activated by Nipbl and Zfp609 D and 4E.− brains C and S4D− brains . We focu neurons . Disrupt in vivo . Indeed, in NSCs F–4H. We The binding of Zfp609 and Nipbl to active promoter regions suggests they may directly contact the basal transcription machinery to activate transcription. Although we did not consistently detect RNA pol2 subunits in Zfp609 pull-downs, we did find thirteen subunits of the Integrator complex, which associates with the C-terminal domain (CTD) of RNA pol2  Table 1Table 1. The interaction of endogenous Zfp609, Nipbl, and Integrator complex was independently verified by their co-immunoprecipitation by an antibody against Ints1. Immunoblotting showed specific co-purification of Zfp609 and Nipbl, independent of DNA B. WesterWe determined the genomic distribution of Integrator binding by performing ChIP-seq with an antibody that efficiently brought down chromatin-bound Ints11 A. Three Integrator was recently shown to physically associate with negative elongation factor (NELF) and the DRB-sensitivity inducing factor (DSIF) complex and to affect RNA pol2 pause release at coding genes . We therSema3a, Plxnd1, and Gabbr2, were downregulated upon Ints1 depletion , that displays phenotypic overlap with CdLS , characterized by moderate-to-severe intellectual disability and seizures  fused to GST. Normal mouse IgG (sc-2025), normal rabbit IgG (sc-2027), and antibodies against Lamin B1 (sc-6216 and sc-6217), RNA pol2 (sc-899), and GFP (sc-8334) were obtained from Santa Cruz Biotechnology. Antibodies against Smc1 (A300-055A), Nipbl (A301-779A), Int1 (A300-361A), and Int11 (A301-274A) were obtained from Bethyl Laboratories. Additional antibodies included V5 , Actin , Vcp , and GFP .8 cells) and scraped in ice-cold PBS, and nuclear extracts were prepared . Complete EDTA-free protease inhibitors (Roche) were added to all buffers. A total of 40 μL anti-V5 agarose beads (Sigma) were equilibrated in buffer C-100∗ ; blocked in 0.2 mg/mL chicken egg albumin (Sigma), 0.1 mg/mL insulin (Sigma), and 1% fish skin gelatin (Sigma) in C-100∗; and added to 1.5 mL nuclear extract in no stick microtubes (Alpha Laboratories) for 3 hr at 4°C in the presence of 225 U Benzonase (Novagen). Beads were washed five times for 5 min with C-100∗ and boiled in 30 μL SDS loading buffer. Eluted proteins separated by polyacrylamide gel electrophoresis were stained with Colloidal Coomassie (Invitrogen), and entire gel lanes were analyzed by mass spectrometry as previously described (Control and Zfp609-V5-expressing NSCs were expanded to ten confluent 14 cm diameter dishes (2 × 10prepared  and diluescribed . CriteriZfp609 fragments were cloned into pGEX-2TK. GST-fusion proteins and GST were expressed in BL21-CodonPlus (DE3)-RP competent cells, and GST pull-downs were performed as previously described .Short hairpin sequences  were cloFor V5, Nipbl, and Ints11 ChIP, NSCs suspended in PBS were crosslinked sequentially for 45 min with 2 mM disuccinimidyl glutarate (DSG) and for 10 min with 1% formaldehyde. Reactions were quenched with 125 mM glycine, chromatin was prepared, and ChIP performed as described . RNA polMice were housed, bred, and treated according to guidelines approved by the Home Office under the Animal (Scientific Procedures) Act 1986. Experiments were approved by the Crick Animal Welfare and Ethical Review Body and the Home Office (project license 707644).In utero electroporation was performed essentially as described . BrieflyCryosections were post-fixed in 4% paraformaldehyde, treated for 10 min with 100 mM acetylated triethanolamine (pH 8.0), and pre-hybridized in hybridization buffer  for 1 hr at 70°C. The Zfp609 probe template was PCR amplified from NSC cDNA (primer sequences in Conceptualization, D.L.C.v.d.B.; Investigation, D.L.C.v.d.B., R.A., K.O., D.H.W.D., and J.A.D.; Resources, N.U. and B.M.; Writing – Original Draft, D.L.C.v.d.B.; Writing – Review & Editing, D.L.C.v.d.B. and F.G.; Funding Acquisition, D.L.C.v.d.B. and F.G.


Here, we determined the crystal structures of Apo- and DNADRE-bound DUX4HD2 and revealed a clamp-like transactivation mechanism via the double homeobox domain. Biophysical characterization showed that mutations in the interacting interfaces significantly impaired the DNA binding affinity of DUX4 homeobox. These mutations, when introduced into DUX4/IGH, abrogated its transactivation activity in Reh cells. More importantly, the structure-based mutants significantly impaired the inhibitory effects of DUX4/IGH upon B-cell differentiation in mouse progenitor cells. All these results help to define a key DUX4/IGH-DRE recognition/step in B-ALL.Oncogenic fusions are major drivers in leukemogenesis and may serve as potent targets for treatment. DUX4/IGHs have been shown to trigger the abnormal expression of ERG DUX4/IGH, derived from the insertion of chromosome fragments containing DUX4 gene into the IGH locus, has been reported in ~7% of B-ALL patients [Oncogenic fusions are important causes/targets in leukemia and related treatment , 2. Acutpatients , 6, 7.DUX4 gene. At physiological condition, DUX4 is usually silenced [alt [alt, derived from an abnormal transcription starting from the intron 6 of ERG, could induce lymphoid leukemia in Arf−/− mice [alt biogenesis is recognized as a reporter and a potent secondary hit in DUX4-driven leukemogenesis. However, due to the lack of structural information, it is not clear how DUX4/IGH recognizes, binds, and activates the transcription of its target genes via the double homeobox (HD1–HD2) domain.Double homeobox 4 (DUX4) is the core component of human subtelomeric macrostalelite D4Z4 . It has silenced . In pathsilenced , the D4Zsilenced . Furtherced [alt  and is iced [alt –14. ERGaApo-DUX4HD2 and DNA-bound complex. The atomic structures, together with functional characterization, reveal a two-step clamp-like binding mechanism between double homeobox and TAATCTAAT DNA site, the latter of which is frequently observed in DUX4 target genes, and hence termed DUX4-Responsive-Element (DRE). Consistent with previous observation that a single HD domain is not sufficient for productive protein–DNA interaction, our data showed that both HD1 and HD2 are essential in DRE recognition. Single mutation in the major-groove-binding interfaces of HD1 and HD2 can significantly impair DUX4-DRE binding and DUX4-driven transactivation. When investigated in the context of DUX4/IGH and B-cell differentiation, structure-based mutations also abolish DUX4/IGH’s transcriptional activities and their aggressive/inhibitory roles upon B-cell differentiation. All these results have led to the novel proposal that the DUX4-DRE recognition and two-step clamp-like binding mechanism are critical for DUX4/IGH-driven transactivation and B-ALL leukemogenesis.In this report, we determined the crystal structures of Apo-DUX4HD2 and DUX4HD2-DNADRE have been deposited into the Protein Database Bank. The PDB codes are 5Z2S and 5Z2T, respectively.Information concerning bacterial and eukaryotic cells, DNAs, and other materials used in this study is described in Supplementary Data. The experimental details of structure determination, functional/genetic characterizations including biolayer interferometry assay, transactivation assay, luciferase assay, B-cell differentiation assay, RNA-seq analysis etc. are also described in Supplementary Data. The statistics of the data collection and structure refinement are shown in Supplementary Table PCDH17, STAP1, AGAP1, ERGalt, etc.  was used. The recombinant DUX4HD2 and DUX4HD1–HD2 were shown to bind to a DRE sequence at KD values of 7.2 and 1.4 μM, respectively  could be more important, a series of single mutations were designed. The DUX4/IGH-driven transactivation activities  of wild type and mutants were monitored in Reh cells. The single mutation in QNR motifs  was sufficient to inhibit the DNADRE recognition and abolish subsequent ERGalt transactivation  of DUX4, whose molecular boundary is not yet clearly defined, might contribute to the transactivation activities of WT DUX4 and DUX4/IGHs. WT DUX4, overexpression of which often triggers apoptosis, is the main reason of FSHD disease . In markion Fig. , suggest S4 Fig. . Similar S4 Fig. , suggestAGAP1, CHST2, CLEC12A, PCDH17, PTPRM, STAP1, etc., which all contain DREs, were found upregulated in patients harboring DUX4 fusions. This is further supported by the observation that knock-down (KD) of DUX4/IGH through shRNA in NALM6 cells [AGAP1, but none of other target genes, led to a partial arrest of B-cell differentiation in murine progenitor cells  did not show obvious apoptotic effects  homeobox subclass because their HD domains share the most sequence identity , it contains two HD2 molecules and a DNA duplex of TAATCTAAT (DRE) sequence. Based on homology modeling, a HD1 molecule can be mapped onto DRE site. Supportively, the C-termini of HD1 and the N-termini of HD2 are both correctly positioned, giving ample space to envisage the missing linker in between. More importantly, the QNRHD1 motif is predicted to insert into the DNA major groove engaging the direct hydrogen bonds with TAAT. In parallel, the RRRR motif of HD1 wraps around the DNA minor groove  could ~100% inhibit transactivation, suggesting both HD1 and HD2 are essential. In marked contrast, single mutations of RRRRHD1–RRKRHD2 were less dramatic, implying that (1) poly-Arg motif is less critical when compared to the QNR motif; (2) the existence of cooperative binding between HD1 and HD2. This is further supported by the observation that double mutations of RRRRHD1–RRKRHD2  resulted a complete loss of transactivation even in the presence of QNRHD1–QNRHD2 motifs . In this step, the HD1 subdomain might take the leading role to make the first kiss with target DNA. The non-specific association between RRRRHD1–RRKRHD2 with DNA in turn enables the double homeobox to recognize the DRE site via QNRHD1–QNRHD2. The double kiss between TAATs and QNR motifs might be critical to “clamp” DUX4-DRE into an intimate association that enables productive transactivation of its target genes Supplementary Table 1(DOC 34 kb)Supplementary Table 2(DOCX 13 kb)Supplementary Figure 1(PDF 847 kb)Supplementary Figure 2(PDF 1197 kb)Supplementary Figure 3(PDF 80 kb)Supplementary Figure 4(PDF 487 kb)Supplementary Figure 5(PDF 71 kb)Supplementary Figure 6(PDF 228 kb)


We show that only the combined matching of transcription rates and ligand binding enables transcription intermediates to undergo ligand-dependent conformational refolding.In bacteria, the regulation of gene expression by cis-acting transcriptional riboswitches located in the 5'-untranslated regions of messenger RNA requires the temporal synchronization of RNA synthesis and ligand binding-dependent conformational refolding. Ligand binding to the aptamer domain of the riboswitch induces premature termination of the mRNA synthesis of ligand-associated genes due to the coupled formation of 3'-structural elements acting as terminators. To date, there has been no high resolution structural description of the concerted process of synthesis and ligand-induced restructuring of the regulatory RNA element. Here, we show that for the guanine-sensing DOI:http://dx.doi.org/10.7554/eLife.21297.001 Riboswitches are cis-regulatory RNA elements controlling cellular processes, in particular transcription and translation . They exRiboswitches regulating transcription control gene expression by connecting ligand-dependent and ligand-independent RNA folding processes with the concomitant synthesis of the RNA by the RNA polymerase.It has been increasingly recognized that the folding of RNA is intimately linked to transcription kinetics, which are directly dependent on transcriptional pausing. The synthesis of the RNA is not performed at a constant speed . InsteadbtuB riboswitch where the use of a mutant RNA polymerase with deficient pause behavior caused the riboswitch to adopt a non-native structure and to be deficient in ligand binding  negatively regulates the transcription of the xpt-gene by the premature termination of RNA synthesis  or the aptamer domain and the subsequent sequence region of the antiterminator (GSWPAT). Both GSW fragments bind the ligand with similar affinities as the full-length riboswitch suggesting formation of the unperturbed aptamer domain  of this transcription intermediate (GSWPAT) in the absence of ligand.For GSWGSWP[AT]  that difGSW[PA]T . Thus, itrans) and H (Htrans) to GSWPA. Using isotope-filter NMR experiments, selectively 15N-labelled GSWPA (containing the P and A sequences), and 14N-labelled Ttrans and Htrans we investigated the sequential refolding of the mRNA transcription intermediates with nucleotide resolution  10−1 s−1, and induced a slower tertiary structure rearrangement (k2 = [1.8 ± 0.2) 10−2 s−1] within the aptamer, consistent with previous experiments   10−4 s−1 10−3 s−1; trans occurred simultaneously (k1 = (2 ± 1) 10−2 s−1 reporting on the ligand-induced refolding of the aptamer domain and k2 = (1.2 ± 0.4) 10−3 s−1 characterising the rate-limiting dissociation of helix AT.In the absence of ligand, GSWrmation) . Under taneously . Under tFrom these kinetic data , we concB. subtilis (12-sensing riboswitches (6-stretch (U107-U112) is suspiciously located between the aptamer domain and the terminator helix  and between the A and T stretches of the antiterminator   and B. subtilis (BsRNAP) in a time-resolved single-round transcription assay (PATH (164 nt), PS2 (141 nt), PS1  to 1.3 ± 0.6 nt s−1 and 47.0 ± 53.3 nt s−1 for the synthesis of the shorter transcription intermediates at 37°C  of PS1 correlated with the loss of regulation in vivo , and theBacillus subtilis, it was shown that FMN binding and subsequent folding into the terminator conformation requires pausing of the bacterial polymerase (B. subtilis and E. coli utilize different pausing sequences (e.g. RNA95 and RNA90) and therefore differently affect conformational change in the synthesized RNA. Thus, the effective biotechnological application of transcription-regulating riboswitches in heterologous organisms may require a match between polymerase pausing sequences and the conformational space of the riboswitch. Our work significantly extends our understanding of the previously introduced concept of kinetic control of riboswitches, and reveals the importance of matching discontinuous transcription rates to folding rates of transient conformations in order to facilitate RNA-based regulation of gene expression.Single molecule force extension experiments showed that the structurally similar aptamer domain of the adenine-sensing riboswitch folds co-transcriptional in a hierarchical manner and that the closure of the PA is the last step in accommodation of the ligand in the holo-form . These flymerase . UnexpecPATH, GSWPAT, GSWPA, dGSW and FSW were synthesised by in vitro transcription using T7 polymerase and purified as described , 7-deazaG147 modified S3trans was obtained from IBA .RNA constructs GSWescribed . Antisen2O (static or kinetic experiments) on Bruker spectrometers  and analysed with Topspin . Hypoxanthine was used as the better soluble ligand. Real-time NMR experiments were performed at 283 K utilising a rapid sample mixing device  ratio, 4 equivalents of hypoxanthine were added. Ligand binding and conformational rearrangements were monitored on the resolved imino proton resonances reporting on the corresponding interactions. Association of GSWPA and Ttrans was performed in a 1:2 ratio in 50 mM potassium chloride, 25 mM potassium phosphate, pH 6.2.Unless stated otherwise, all NMR experiments were performed in NMR buffer  containing 10% or 5% Dg device . SelectiITC measurements were performed with a Microcal VP ITC  at 283 K. A 217 µM solution of hypoxanthine as ligand was titrated to a 15 µM solution of RNA using 20–42 injections. Buffer conditions were 2 mM magnesium chloride, 50 mM potassium chloride, 25 mM potassium phosphate, pH 6.2. Data were analysed with the Origin ITC software  assuming a single binding site.kij for the inter-state transition i to j. For the synthesis steps in the evaluated models, a transcription rate of 20 nt s−1 was assumed. The rates connecting conformational states by refolding reactions were employed as determined here from the real-time NMR measurements. Two sets of simulations were performed, in the first set the transcription rate was adjusted to the experimental value for transcription without any pausing events, while the second set simulated a pausing event between states PA/ligPA and PAT/ligPAT. Both sets of simulations were performed for absence and presence of cognate ligand. For the pausing events the transcription rate was set to the reduced transcription rate determined in the transcription assays. Thereby, the reduction of the transcription rate over the pause site corresponds to a factor of ≈70 in the simulations.Kinetic Markovian simulations were performed in order to evaluate the switching behaviour of the guanine sensing riboswitch under different transcription synthesis velocities. The model assumes N discrete states each representing a distinct length and associated conformation of the RNA chain. The kinetics between the states is described by first order rate equations with a single rate constant B. subtilis MH5636 strain was grown in Bacillus growth medium  at 37°C with shaking at 200 rpm to a OD600 of 1.0–1.2. The cells were harvested by centrifugation with 5000 rpm for 15 min at 4°C. The pellet was frozen at −80°C.The Cells (60 grams) were thawed on ice and resuspended in 120 ml Lysis buffer . After cells were fully resuspended, 0.1 mg/mL lysozyme was added to the cells on ice. The cell suspension was lysed in the cold using a French Press at 1000 PSI. The lysate was then centrifuged for 15 min with 12000 rpm at 4°C. The resulting supernatant was transferred to new centrifuge tubes and again centrifuged for 15 min with 12000 rpm at 4°C.In the cold, the supernatant was transferred to a 250 mL beaker and 8% PEI was added slowly (0.5 ml added every 5 min) with constant stirring to a final concentration of 0.6%. After the last PEI addition, the solution was stirred for an additional 30 min and then centrifuged for 15 min at 12000 rpm at 4°C.2, 5% glycerol, 5 mM ß-mercapthothanol) + 0.4 M NH4Cl. The pellet was properly dispensed in the buffer. The suspension was centrifuged for 15 min with 12000 rpm at 4°C.The supernatant was removed and the PEI pellet was washed with 200 mL TGEZ  and centrifuged at 12000 rpm for 15 min at 4°C. The resulting supernatant was then filtered and loaded onto a His-trap column equilibrated in the same buffer at a flow rate of 1 ml/min. After washing with ten column volumes, the protein was eluted using a gradient of 0.01 M to 0.5 M imidazole in the same buffer over 30 min, then from 0.5 M to 0.75 M over 10 min, and then 1 M imidazole. 2 ml fractions were collected.The fractions were analyzed by 4–12% gradient PAGE and Bradford assay. The RNAP was eluted in fractions 7–23, which corresponded to buffer with 200–850 mM imidazole. The combined fractions were dialyzed overnight in TGEZ +100 mM NaCl +5 mM DTT.The dialyzed solution was filtered and then loaded onto the heparin column equilibrated in the same buffer. After washing with the same buffer for 10 column volumes, the protein was then eluted in the same buffer using a gradient to 1 M NaCl.2, 1 mM MgCl2, and 10 mM DTT) overnight. The total yield of the core enzyme (ε260 = 210025 M−1.cm−1) was 5 mL of 0.83 mg/mL, 2.5 µM solution.Protein-containing fractions were pooled and dialyzed into storage buffer  at 37°C. From this plate, a single transformant was grown at 37°C in 5 ml LB medium with antibiotics. This culture was then used to inoculate 2 liters of LB with antibiotics. The cultures were grown with shaking at 37°C to an OD600 of 0.5. The cultures were then shifted to 16°C and the overexpression of SigA (43 kDa) was induced by addition of 1 mM IPTG. Cultures were maintained at 16°C with shaking overnight. The cells were then harvested by centrifugation at 5000 rpm for 15 min at 4 C. The 8.5 g pellet was frozen at −80°C.Cells were thawed on ice and resuspended in 35 ml SigA Lysis buffer . The resuspended cells were transferred to a prechilled beaker and lysosyme was added (0.130 mg/mL) and the cells were incubated on ice for 15 min. Sodium deoxycholate was then added to 0.05% final and incubated on ice for an additional 15 min. Cells were then subjected to sonication at output 6 with a 20% duty cycle for 30 min. The sonicated lysate was then centrifuged for 15 min with 12000 rpm at 4°C.The SigA-containing cell lysate was filtered and then loaded onto a His-Trap column equilibrated in the same buffer plus 10 mM imidazole. After washing with ten column volumes, the protein was eluted using a gradient to the same buffer containing 1 M imidazole.2, 1 mM MgCl2, and 0.1 mM DTT)) overnight. The resulting protein solution contained 5 mL of 25 mg/mL or 590 µM (ε260 = 24410 M−1.cm−1).The protein-containing fractions were pooled and analyzed by 20% SDS-PAGE. As the fractions contained pure SigA, they were dialyzed into SigA Storage Buffer  or the strong lambda pR promoter, followed by the GSW-coding sequence from the B. subtilis genome . For halted complex formation (HC), 25 nM DNA template and 40 nM RNAP holoenzyme (expression and purification shown in SI) were incubated in transcription buffer 2, and 5 mM ß-mercaptoethanol) with 10 µM ATP and UTP, 7.5 µM GTP, and 150 µM ApA (for the B. subtilis GSW promoter) or ApU dinucleotide (for the lambda pR promoter). For transcriptions using the E.cRNAP, 10 µCi GTP* were used whereas transcriptions using the BsRNAP used 20 µCi GTP*. The HC of EcRNAP seem to be more stable than the ones with BsRNAP. Additional GTP* and therefore increased incorporation of 32P-labeled nucleotides into the nascent RNA, made more of the fewer transcribed RNAs visible on the PAGEs. Ligand  was added to a final concentration of 200 µM. Purified rNTPs were obtained from Promega and dinucleotides were obtained from TriLink BioTechnologies. Synchronized transcription was re-started by adjustment of the ATP, CTP, and UTP concentrations to 150 µM and the GTP concentration to 10 µM, and the addition of heparin to a final concentration of 100 µg/mL. Transcription was stopped at different time points by the addition of an equal volume of 2X STOP buffer . For determination of the transcription rates at high NTP-concentration, the NTP-concentration was raised to 1 mM. After heating to 95°C for 2 min, the reactions were analyzed by 8% PAGE. The gels of the time-resolved transcription assays show several expected RNA signals , and full–length transcript  as well as two additional pausing events at 77 nt (RNA77) and 95 nt (RNA95) for E. coli RNAP mediated transcriptions and 77 nt (RNA77) and 90 nt (RNA90) for B. subtilis RNAP mediated transcription. The positions of the RNA species were as estimated by comparison on high-resolution denaturing polyacrylamide gels of transcripts to RNA sequence ladders generated using 3' deoxyNTPs. To do this, halted ECs were first generated as described above. To generate sequence ladders, transcription was restarted by addition of ATP, CTP, GTP, and UTP at 250 µM each, and one 3′-deoxyNTP in each of 4 separate reactions at 240 µM dGTP, 300 µM dATP, 350 µM dCTP, or 400 µM dUTP, followed by addition of heparin at 100 µg/ml. After incubation for 10 min at 37°C, samples were mixed with an equal volume of 2X STOP buffer before being electrophoresed next to transcription products obtained by regular in vitro transcription as described above. To quantitate the relative amounts of the different RNA species, the signal intensities were plotted against time and analyzed using Lab kapp for FL, Gsw[PA][TH], PS2 and PS1 were derived from the exponential plots as shown in by kapp was derived similar as previously described, by determining the average appearance rate for the first values. Knowing the length of the RNA fragment, the rates in nt s-1 were then calculated from the kapp  was fused directly upstream of the lacZ reporter gene of plasmid pDG268  with lysozyme and Triton-X treatment. 200 μL of o-nitrophenyl-β-D-galactoside (4 mg mL−1) was added, and the sample was incubated at 301 K. The reaction was stopped by addition of 0.5 mL of 1 M Na2CO3 upon appearance of a yellowish colouration. The time for colour change was noted and absorbance at 420 nm and 550 nm was measured. ß-galactosidase activity was calculated in Miller units, i.e. 1000 x [A420 – 1.75 x A550] / [t x V x A600]. Three independent cultures were measured in parallel. The measurements were repeated twice.For reporter gene assays, the 5’-UTR of the d pDG268 . Pause sl medium  with inc In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been favorably evaluated by John Kuriyan (Senior Editor) and four reviewers, one of whom is a member of our Board of Reviewing Editors. The following individual involved in review of your submission has agreed to reveal their identity: Kathleen Hall (Reviewer #4).Thank you for submitting your article "Pausing guides RNA folding to populate transiently stable RNA structures for riboswitch-based transcription regulation" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:The manuscript describes a detailed set of biochemical, biophysical and biological analyses to understand the mechanism used by a guanine riboswitch to control gene expression. The work involves a series of ligand binding studies and folding kinetics that were carried out using truncated riboswitches. This study expands on the original observations that some riboswitches are influenced more by kinetic processes  than by thermodynamic equilibria. The NMR study in this manuscript is well executed, in particular the characterization of secondary structures of sub-state conformations. One of the reviewer’s comments on the beautiful NMR data and the creative use of imino protons to monitor structure formation. Indeed, all of the reviewers were impressed with the NMR work.Essential revisions:1) All of the reviewers were concerned about the fact that the authors did not succeed in establishing how their results go beyond what is currently understood and also that they have not tied their results with those published previously. For example, the authors claim that "Up to now, a high-resolution structural depiction of the concerted process of synthesis and ligand-induced restructuring of the regulatory RNA is missing.". However, there is excellent single-molecule biophysics data from the laboratory of Steven Block for adenine riboswitches (a close relative of guanine riboswitches) . This previous paper should be cited and the authors should compare their findings with this prior work. In particular, what is new from this study? Work from the Micura and Block labs that leads to similar conclusions about the importance of kinetics should also be cited. The authors also state that the RNA "…exclusively populates the functional off-state but cannot switch to the on-state, regardless of the presence or absence of ligand.". However, similar claims have been made for FMN riboswitches  and probably others. This brings up an important point: maybe many riboswitches do not switch between the on- and off-states, but choose a folding pathway based on ligand binding early after the full aptamer has been made. The authors should consider this possibility. As indicated above, the mechanism of the guanine-sensing riboswitch described here is very similar to that of the FMN riboswitch, including the number of pause sites and half time of a pause site. However, in this manuscript, little is discussed regarding the mechanism of the FMN riboswitch and there is no comparison between the two riboswitches. The authors need to provide more detailed discussions about the similarity between the two riboswitches and again why this work goes beyond what is currently understood.2) Related to point 1 is that there is currently no Discussion. Addressing point 1 could be done in a Discussion format although the authors must clarify the novelty in the Introduction as well more clearly.3) The authors use separate oligonucleotides that act as surrogates for the actual base-paired interactions that occur in a unimolecular riboswitch. It is not clear that these interactions will have kinetic properties that accurately reflect analogous interactions that occur in the natural construct during RNA synthesis. If not, then the author's conclusions (such as those made in the eighth paragraph of the Results) might be flawed.4) Related somewhat to 3: The associations between PA and T and between T and H are bimolecular reactions in the NMR experiments. However, during co-transcriptional folding, these associations are intramolecular reactions. It is not clear in the manuscript whether the authors have considered differences between these two types of reactions and corrected the NMR-determined associations rates for simulation. Further, the authors did not provide clear descriptions in this manuscript how the simulation was performed, how the pausing was implemented, and what rates were used. For example, t0,1~5s and t0,2~61s were labeled for ligand binding at the aptamer in 5) One of the major concerns of the current study is how transcription intermediates and pause sites were identified. It is possible that a transcription intermediate gives an aborted product in multi-round transcription, but the appearance of an aborted product in multi-round transcription can be a result of many other factors. The appropriate approach for identifying a transcription intermediate is to measure accumulation in time-resolved single-round transcription assays, such as what the authors have done to confirm pause site 2 (PS2). In this regard, in contrast to PS2, the identity of pause site 1 (PS1) is not clear, as no transcription intermediate at PS1 was detected in the time-resolved single-round transcription assay. However, in this manuscript, PS1 has been a critical intermediate for designing truncated riboswitch constructs, performing kinetic measurements, and simulating co-transcriptional pathways (see below). If PS1 is a true pause site, the authors need to clearly confirm the presence of PS1 and characterize its pausing properties.E. coli transcription and B. subtilis transcription. For example, there are five major products in the E. coli transcription, but there are least eight major products shown in the B. subtilis transcription. If aborted products were all transcription intermediates, this would suggest there are many more pause sites for the B. subtilis polymerase than for the E. coli polymerase, which is not likely. To properly evaluate transcription systems, the authors need to perform time-resolved single-round transcription assays and compare differences in transcription intermediates.6) Similar to the above concern, it is not appropriate to compare aborted products in multi-round transcription reactions to evaluate transcription intermediates between 7) In the figure for simulation, the authors labeled pausing at PS1 but did not label pausing at PS2. In the manuscript, the authors specifically discussed pausing at PS1 but did not discuss pausing at PS2. The experimental data identified PS2 but did not identify PS1. It is not correct to simply assume the presence of PS1 and ignore PS2. In addition, it is also not clear whether the half time at PS2 was used in the simulation, or whether it was used for PS1.8) Similar to the above concern, since PS1 was not identified in the in vitro assay, while mutating PS1 sequences do affect in vivo gene expression, it is difficult to evaluate these influences as mutation-induced changes in pausing or other factors, such as folding. The authors need to clearly confirm the presence of PS1 and may also need to characterize pausing properties of these mutations.E. coli RNAPs: These enzymes are quite different, and they are not considered to be indistinguishable on the basis of their pause kinetics. However, if the authors have new information on their comparative properties, they need to reference it. The authors are using E. coli RNAP on a B subtilis riboswitch, which is quite ok, but they need to be clear that they are measuring properties of EC RNAP.9) Regarding the Bacillus and eLife. Your revised article has been favorably evaluated by John Kuriyan as the Senior Editor, Lewis Kay as the Reviewing Editor, and two reviewers. Your paper has been sent for re-review to two of the previous experts who now feel that the work is far improved and suitable for publication at eLife.Thank you for submitting your revised article "Pausing guides RNA folding to populate transiently stable RNA structures for riboswitch-based transcription regulation" for consideration by Both reviewers feel that the work represents an important quantitative advance in the description of transcriptional regulation by riboswitches and that the data is at much higher resolution than earlier studies. One of the reviewers comments about the utility of the simulations of 1) The authors wrote in their response that there are no quantitative models to describe transcriptional regulation by riboswitches. This statement is not correct. While the kinetic model established in the current study represents one of the few quantitative models and has its own merits in advancing the understanding of transcriptional regulation by riboswitches, a similar regulatory model with quantitative kinetic parameters has been previously reported for the FMN riboswitch . The authors need to revise their Introduction to be consistent with the literature.2) One major concern of the original manuscript was the application of multi-round transcription assays to evaluate transcription intermediates. In the revised manuscript, the authors have performed time-resolved single-round transcription assays to properly establish transcription intermediates. However, despite having these appropriate measurements, it is somewhat surprising that the authors have kept applying the multi-round transcription data to evaluate transcription intermediates . The aut3) In the revised manuscript , the authors changed the length of one of the transcription intermediates from the original 125 nucleotides to 140 nucleotides. Since this RNA fragment was proposed to represent pause site 2 (PS2), this new length will mark PS2 around nucleotide A150, which is different from PS2 at nucleotide U141 shown in both 4) Similar to the above concern, the labels for PS1 and PS2 are confusing in the new 5) In the new supplementary Table 8 , the aut6) One other concern of the original manuscript was the lack of details for the co-transcriptional folding simulation. While the authors have provided more details in their revised manuscript, a key aspect of the simulation remains not clear. The authors need to describe in the method how pausing was implemented in the simulation. Essential revisions:1) All of the reviewers were concerned about the fact that the authors did not succeed in establishing how their results go beyond what is currently understood and also that they have not tied their results with those published previously. For example, the authors claim that "Up to now, a high-resolution structural depiction of the concerted process of synthesis and ligand-induced restructuring of the regulatory RNA is missing.". However, there is excellent single-molecule biophysics data from the laboratory of Steven Block for adenine riboswitches (a close relative of guanine riboswitches) . This previous paper should be cited and the authors should compare their findings with this prior work. In particular, what is new from this study? Work from the Micura and Block labs that leads to similar conclusions about the importance of kinetics should also be cited. The authors also state that the RNA "…exclusively populates the functional off-state but cannot switch to the on-state, regardless of the presence or absence of ligand.". However, similar claims have been made for FMN riboswitches  and probably others.In this contribution, we apply high resolution structural and kinetic techniques to decipher the full co-transcriptional folding pathway, enabling monitoring the behaviour of the decisive structural units during synthesis for the first time.” Nevertheless, we have included reference to the Block paper and point out the new findings in our study: “In single molecule force extension experiments it could be shown that the structurally similar aptamer domain of the adenine-sensing riboswitch folds co-transcriptional in a hierarchical manner and that the closure of the PA is the last step in accommodation of the ligand in the holo-form . Although this finding supports our derived model that puts the formation of the base-paired PA-stem to be the committed-step in the off-pathway, the single- molecule force extension experiments are not investigating the full length riboswitch, which is important to map out the coexistence of ligand-dependent and ligand-independent functional OFF-states .”We do not agree with the statement that the single-molecule work by Steven Block can be compared in its resolution to NMR structural work where each base-paired nucleotide can be determined. We are not aware that the previous reports would have tried to develop a quantitative model to describe transcriptional regulation, from measurement of ligand binding, RNA refolding from time-resolved NMR and transcription speed. Such studies, even though within reviewing process of this work over the last four years such claims are repeatedly put forward, have not been conducted nor conceptualized before. It is a fundamental difference to put forward a schematic picture or to develop a quantitative kinetic model. Therefore, our work substantially advances the field of transcriptional regulation by riboswitches. From a fundamental point, we find that the regulatory ON state is achieved by a metastable state! This has not been shown before. To highlight this we have extended the Introduction: “This brings up an important point: maybe many riboswitches do not switch between the on- and off-states, but choose a folding pathway based on ligand binding early after the full aptamer has been made. The authors should consider this possibility. As indicated above, the mechanism of the guanine-sensing riboswitch described here is very similar to that of the FMN riboswitch, including the number of pause sites and half time of a pause site. However, in this manuscript, little is discussed regarding the mechanism of the FMN riboswitch and there is no comparison between the two riboswitches. The authors need to provide more detailed discussions about the similarity between the two riboswitches and again why this work goes beyond what is currently understood.In order to address this point and to include a discussion in the light of the new results from transcriptional analysis we have rewritten the entire Discussion section that reads now: “In single molecule force extension experiments it could be shown that the structurally similar aptamer domain of the adenine-sensing riboswitch folds co-transcriptional in a hierarchical manner and that the closure of the PA is the last step in accommodation of the ligand in the holo-form . […] Our work significantly extends our understanding of the previously introduced concept of kinetic control of riboswitches, and reveals the importance of matching discontinuous transcription rates to folding rates of transient conformations in order to facilitate RNA-based regulation of gene expression.”2) Related to point 1 is that there is currently no Discussion. Addressing point 1 could be done in a Discussion format although the authors must clarify the novelty in the Introduction as well more clearly.B. subtilis. Previously, the Schwalbe lab was able to conduct these experiments for E. coli polymerase but not with the more demanding B. subtilis polymerase. From this work, we show different transcription rates and utilization of different pause sites. This has not been done before.In new single round transcription assays, we show accumulation of several transcriptional intermediates, with pausing and stalling events be present. We were able to do so because the Schwalbe group sent a postdoc to the lab of Landing to conduct such single round transcription in 3) The authors use separate oligonucleotides that act as surrogates for the actual base-paired interactions that occur in a unimolecular riboswitch. It is not clear that these interactions will have kinetic properties that accurately reflect analogous interactions that occur in the natural construct during RNA synthesis. If not, then the author's conclusions (such as those made in the eighth paragraph of the Results) might be flawed.4) Related somewhat to 3: The associations between PA and T and between T and H are bimolecular reactions in the NMR experiments. However, during co-transcriptional folding, these associations are intramolecular reactions. It is not clear in the manuscript whether the authors have considered differences between these two types of reactions and corrected the NMR-determined associations rates for simulation. Further, the authors did not provide clear descriptions in this manuscript how the simulation was performed, how the pausing was implemented, and what rates were used. For example, t0,1~5s and t0,2~61s were labeled for ligand binding at the aptamer in Clearly, our experimental setup does not completely reflect the co-transcriptional RNA folding scenario in vivo. Instead, we present the structural basis for folding of the different transcription intermediates, which occur during riboswitch transcription in the cell. We show that the individual base-pair interactions of hybridized oligos represent the RNA structural motifs of the unimolecular RNA of the corresponding size. At the RNA concentrations used for NMR (>0.1 mM), we are confident that we mimick a scenario very close to the unimolecular situation. In a previous study , we could show that for translational riboswitches, the aptamer domain and the expression platform are conformationally decoupled. Therefore, the intramolecular effect in RNA refolding is small. Further: our data from biophysical studies, mainly NMR, are fully consistent with the single-round transcription experiments that we can conduct under full control. This consistency makes us confident that our data will advance the field.The values in the simulation that have to be adapted for concentration are accordingly adapted to match cellular concentrations comparable to Buckstein 2008, as referenced in the text.5) One of the major concerns of the current study is how transcription intermediates and pause sites were identified. It is possible that a transcription intermediate gives an aborted product in multi-round transcription, but the appearance of an aborted product in multi-round transcription can be a result of many other factors. The appropriate approach for identifying a transcription intermediate is to measure accumulation in time-resolved single-round transcription assays, such as what the authors have done to confirm pause site 2 (PS2). In this regard, in contrast to PS2, the identity of pause site 1 (PS1) is not clear, as no transcription intermediate at PS1 was detected in the time-resolved single-round transcription assay. However, in this manuscript, PS1 has been a critical intermediate for designing truncated riboswitch constructs, performing kinetic measurements, and simulating co-transcriptional pathways (see below). If PS1 is a true pause site, the authors need to clearly confirm the presence of PS1 and characterize its pausing properties.E. coli and the B. subtilis RNAPs in collaboration with the Landick lab. These assays gave distinct signals for the run-off transcript (or full length), for premature transcription termination at the terminator (GswPATH), and the pause-sites PS2 and PS1. In addition, we could identify further pause-sites (RNA77 and RNA95 for E. coli and RNA77 and RNA90 for B. subtilis). For both RNAPs, the ratio between full-length and terminator varied in a ligand dependent manner and the intensities of PS1 were higher than for PS2. We therefore conclude that PS1 plays a more crucial role for the co-transcriptional pathways. However, due to its strong pausing effect on the EC, which leads to a strongly halted complex, the determination of the pausing properties was rather difficult.In order to identify the transcription intermediates, we performed time-resolved single-round transcriptions with the 6) Similar to the above concern, it is not appropriate to compare aborted products in multi-round transcription reactions to evaluate transcription intermediates between E. coli transcription and B. subtilis transcription. For example, there are five major products in the E. coli transcription, but there are least eight major products shown in the B. subtilis transcription. If aborted products were all transcription intermediates, this would suggest there are many more pause sites for the B. subtilis polymerase than for the E. coli polymerase, which is not likely. To properly evaluate transcription systems, the authors need to perform time-resolved single-round transcription assays and compare differences in transcription intermediates.E. coli and the B. subtilis RNAPs.We share the concerns of the reviewers and therefore performed time-resolved single-round transcription assays, as mentioned above, which revealed the nature of the transcription intermediates. We could show that PS1 plays an important role in the transcriptions using both, the 7) In the figure for simulation, the authors labeled pausing at PS1 but did not label pausing at PS2. In the manuscript, the authors specifically discussed pausing at PS1 but did not discuss pausing at PS2. The experimental data identified PS2 but did not identify PS1. It is not correct to simply assume the presence of PS1 and ignore PS2. In addition, it is also not clear whether the half time at PS2 was used in the simulation, or whether it was used for PS1.E. coli and the B. subtilis RNAPs.We share the concerns of the reviewers and therefore performed time-resolved single-round transcription assays, as mentioned above, which revealed the nature of the transcription intermediates. We could show that PS1 plays an important role in the transcriptions using both, the 8) Similar to the above concern, since PS1 was not identified in the in vitro assay, while mutating PS1 sequences do affect in vivo gene expression, it is difficult to evaluate these influences as mutation-induced changes in pausing or other factors, such as folding. The authors need to clearly confirm the presence of PS1 and may also need to characterize pausing properties of these mutations.We could identify PS1 in the newly performed in vitro assays and determine its role for the regulation mechanism.9) Regarding the Bacillus and E. coli RNAPs: These enzymes are quite different, and they are not considered to be indistinguishable on the basis of their pause kinetics. However, if the authors have new information on their comparative properties, they need to reference it. The authors are using E. coli RNAP on a B subtilis riboswitch, which is quite ok, but they need to be clear that they are measuring properties of EC RNAP.E. coli and B. subtilis RNA-polymerase, only the PS found for BS and their properties were included in the simulation of the riboswitch folding trajectory.As outlined above we have performed optimized transcription assays (static and time resolved) using […] 1) The authors wrote in their response that there are no quantitative models to describe transcriptional regulation by riboswitches. This statement is not correct. While the kinetic model established in the current study represents one of the few quantitative models and has its own merits in advancing the understanding of transcriptional regulation by riboswitches, a similar regulatory model with quantitative kinetic parameters has been previously reported for the FMN riboswitch . The authors need to revise their Introduction to be consistent with the literature.B. subtilis was analyzed and it was postulated that transcription speed plays a major role in the regulation mechanism of certain riboswitches. However, the KD values were measured by comparing the termination rates under different conditions. Therefore, only the overall apparent rates were determined. In addition, no transcription rates were quantified. In our work, we combine measuring exchange rates and KD values at nucleotide level with quantified transcription and pausing rates. We can therefore determine the conformational with base pair resolution and gain a more detailed view. In addition, we use a simpler transcription system consisting of only the template, the RNAP and the RNA. This overcomes the problem that additional transcription factors could alter the interaction between RNAP and the riboswitch which was also mentioned in Wickiser et al., 2005.We thank the reviewers for this comment. In the previous work, the effect of transcription speed and pausing on the FMN riboswitch from 2) One major concern of the original manuscript was the application of multi-round transcription assays to evaluate transcription intermediates. In the revised manuscript, the authors have performed time-resolved single-round transcription assays to properly establish transcription intermediates. However, despite having these appropriate measurements, it is somewhat surprising that the authors have kept applying the multi-round transcription data to evaluate transcription intermediates . The autE. coli RNAP showed similar results. These results were taken into account when the shortened riboswitch and the trans added RNAs were designed for the time resolved NMR experiments.We thank the reviewers for this comment. When we synthesized the full-length riboswitch sample by T7 mediated in vitro transcription, the analytic PAGE showed several abortion products. NMR data of the full-length riboswitch revealed that it was unable to switch. When we investigated this phenomenon, we realized that the abortion fragments on the gel could correspond to stable transcription intermediates, which caused the T7 RNAP to abort transcription. Comparison to the 3) In the revised manuscript , the authors changed the length of one of the transcription intermediates from the original 125 nucleotides to 140 nucleotides. Since this RNA fragment was proposed to represent pause site 2 (PS2), this new length will mark PS2 around nucleotide A150, which is different from PS2 at nucleotide U141 shown in both We thank the reviewers for this comment. Compared to the wild type sequence, the NMR construct is missing 10 nt and therefore starts with G10.4) Similar to the above concern, the labels for PS1 and PS2 are confusing in the new One transcription intermediate was identified as 110 which migrates between the bands 110 and 123 of the sequencing ladder. In order to clarify the length of this fragment, a 3’-mapping of the transcriptions was performed using 3’-deoxynucleotides . The gel5) In the new supplementary Table 8 , the autE. coli RNAP was also determined in the previous version of this paper. The values were in accordance with literature and could therefore be considered for the simulations.We thank the reviewers for this comment and have improved discussion of the results in the paper. The mentioned values were determined from signal intensities close to the noise which is also indicated by the high error. However, the transcription speed for the 6) One other concern of the original manuscript was the lack of details for the co-transcriptional folding simulation. While the authors have provided more details in their revised manuscript, a key aspect of the simulation remains not clear. The authors need to describe in the method how pausing was implemented in the simulation.We have corrected this and included the details of simulation in the Materials and methods section by including the following paragraph:ligPA and PAT/ligPAT. […] Thereby, the reduction of the transcription rate over the pause site corresponds to a factor of ≈70 in the simulations.”“Two sets of simulations were performed, in the first set the transcription rate was adjusted to the experimental value for transcription without any pausing events, while the second set simulated a pausing event between states PA/