By analysing histone modifications in a morphologically-simple, early branching animal, the sponge Amphimedonqueenslandica, we show that the regulatory landscape used by complex bilaterians was already in place at the dawn of animal multicellularity. This includes distal enhancers, repressive chromatin and transcriptional units marked by H3K4me3 that vary with levels of developmental regulation. Strikingly, Amphimedon enhancers are enriched in metazoan-specific microsyntenic units, suggesting that their genomic location is extremely ancient and likely to place constraints on the evolution of surrounding genes. These results suggest that the regulatory foundation for spatiotemporal gene expression evolved prior to the divergence of sponges and eumetazoans, and was necessary for the evolution of animal multicellularity.Combinatorial patterns of histone modifications regulate developmental and cell type-specific gene expression and underpin animal complexity, but it is unclear DOI:http://dx.doi.org/10.7554/eLife.22194.001 Animals come in many shapes and sizes, and vary in how they move, grow and reproduce. The long-held thought that animal complexity is related to the number of genes that are in the animal’s DNA has now been largely dismissed; simple animals like sponges and cnidarians  have comparable gene numbers to vertebrates, insects, mollusks and other complicated bilaterians . This observation led to the idea that gene regulation (how and when genes are turned off and on) is responsible for the evolution of animal diversity.Genomic DNA packs into cells by winding around proteins called histones. Histones themselves can bear certain chemical marks, which in turn determine if the genes contained in the DNA associated with the histones are going to be turned on or off. In bilaterians and cnidarians these marks substantially contribute to gene regulation. Some of these marks predate the evolution of multicellular animals from single-celled organisms. However, the origin of the marks that associate with the gene regulatory elements that are essential for animals to be multicellular remained unknown. In other words, does the evolution of histone marks underpin animal complexity?Amphimedon queenslandica to address this question. Sponges are one of the morphologically simplest animals, lacking a gut, nerves and muscles. By analyzing histone marks in this sponge, Gaiti et al. found they were remarkably similar to the networks of histone marks seen in more complex animals. This is consistent with this form of gene regulation being present at the dawn of the animal kingdom. Indeed, this mode of gene regulation may have been necessary for multicellular animals to first evolve.Gaiti et al. turned to the marine sponge It now appears that most of the genes and regulatory mechanisms underlying the formation of complex animals, like ourselves, had an unexpected early origin – probably as early as the first steps in the evolution of multicellular animals from single-celled organisms. Further studies of animals that are close relatives of sponges, such as comb jellies, and their single-celled cousins, may further improve our understanding of how these simple single-celled organisms became multicellular animals.DOI:http://dx.doi.org/10.7554/eLife.22194.002 As the last common ancestor of modern animals already possessed an extensive repertoire of regulatory genes, including most transcription factors and signaling pathways used in bilaterian development   typically present in complex animals   , would htazoans) .The development of high-throughput chromatin assays like chromatin immunoprecipitation coupled with massively parallel sequencing (ChIP-seq)  has alloAmphimedon queenslandica (herein Amphimedon) has dynamic developmental gene expression akin to eumetazoans  are considered one of the oldest surviving phyletic lineages of animals, diverging from other metazoans around 700 Mya . Despiteetazoans , we set Amphimedon adults and larvae using antibodies against specific histone H3 PTMs that have been used to define chromatin states in model bilaterians  (We carried out chromatin immunoprecipitation (ChIP) on sexually reproducing aterians   .10.7554/cis-regulatory elements such as enhancers; (ii) trimethylated lysine 4 (H3K4me3), enriched in active promoters; (iii) trimethylated lysine 36 (H3K36me3), found with actively transcribed regions; (iv) trimethylated lysine 27 (H3K27me3), enriched in Polycomb-silenced regions; and (v) acetylated lysine 27 (H3K27ac), which occurs around activated regulatory regions. We also used an antibody against total histone H3   monomethylated lysine 4 (H3K4me1), associated with distal stone H3 . An antistone H3   .Amphimedon genome (a priori defined states (from 5 to 15) . We elected to use a 9-state model for all further analyses as it covered all major gene coding and regulatory components  that we expected to resolve with this selection of histone H3 PTMs. Despite the inherent cellular heterogeneity of our starting material, we were able to resolve specificities towards gene components between these nine chromatin states. They fell into two broad categories: one that correlated with actively transcribed genes that include active promoters (‘TssA’) and enhancers , and 5’ and 3’ boundaries of transcribed genes (‘TxFlnk’); and another category with genes with no or little detectable transcription; these include bivalent or poised regulatory , repressed Polycomb , and quiescent (‘Quies’) states  was enriched around transcription start sites (TSSs) of active genes. ‘TxEnhA’ state  associated with coding exons and introns that correspond to potential cis-regulatory elements and short intergenic regions, which are common in the Amphimedon genome (ChIP-seq reads generated from immunoprecipitated and input (whole-cell extract) DNA were aligned to the n genome , resultin genome . Uniquel) states . The ninn genome . In contn genome   (To investigate the distribution of histone H3 PTMs in d RNAPII . Addition genome ), and (ietazoans  (Fisher’ue<0.05) .10.7554/Amphimedon development, we analysed CEL-seq data , resulting in a set of 3,200 ‘high-variance’ expressed genes  . It is noteworthy that the high-variance genes were, on average, also expressed at higher levels than the low-variance genes . The TSSs of high-variance genes were strongly marked by H3K4me3 and occupied by RNAPII and KEGG pathway analyses. High-variance genes, which also include a significantly higher number of transcription factor gene families  expressed with minor changes throughout development. Conversely, the 1,000 genes with the highest coefficients of variation were defined as ‘regulated’ genes. Notably, the ‘regulated’ genes consisted of a small population of genes that differed from the ‘high-variance’ genes described earlier in having much more restricted expression patterns, mainly expressed at late juvenile and/or adult stage , and the gene with the highest coefficient of variation  . The forosophila , regulatectensis  and preve polyps , we obtae polyps .These results suggest that H3K4me3 might not be instrumental for extremely dynamic developmental expression and enforces our interpretation that it is required for tuning the levels of gene expression, a pattern that appears to be a conserved metazoan feature .Amphimedon, we identified the sponge homologs of Drosophila PRC2 components and found that the Amphimedon genome contains four copies of E(z) homologs, two copies of ESC homologs and one copy for each of the remaining components, SU(z)12 and Nurf55 (PRC2 is responsible for the trimethylation of lysine 27 of histone H3 (H3K27me3), one of the best-characterized repressive histone H3 PTMs . As a std Nurf55 .10.7554/Drosophila where PRC2 proteins repress their target genes by recruitment to Polycomb Response Elements (PREs), which contain binding sites for sequence-specific DNA binding proteins, including GAGA factor and members of the Krüppel-like factor family . We searched for short motifs (6–15 bp) on the basis that the known interaction sites of PREbinding proteins in Drosophila are of approximately this length (~8 bp). Conserved binding motifs similar to the GAGA and Krüppel-like factors, in addition to binding motifs similar to homeodomain-containing developmental regulators , were significantly enriched  in the DNA associated with the H3K27me3 silenced regions in both adult and larva   , which hincRNAs) , avoidini.e., higher enrichment of H3K4me3 over H3K4me1 . 43 (20%) lincRNAs could not be assigned to either group, that is, 0.8 < H3K4me1-to-H3K4me3 < 1.2  and canonical promoter-like lincRNAs  . Thus, te3 < 1.2 .10.7554/+ transcripts based on the chromatin status at their TSSs. Although these two populations resemble those found in human, mouse and Capsaspora lincRNAs (These results indicate that sponge lincRNAs can be separated in two distinct populations of poly(A)lincRNAs , their fAmphimedon in silico, we selected distal H3K4me1 regions of enrichment  that did not overlap TSSs (±200 bp) of protein-coding genes and lncRNAs, but overlapped with regions designated as being in an enhancer chromatin state based on the ChromHMM analysis  , suggesting poly(A)+ enhancer RNAs could be transcribed from these regions (To identify putative enhancer elements in atterns) . A subseatterns) . These patterns) , a biochatterns) . Interes regions   terms associated with transcription factor activity and developmental processes   , and comue<0.01) –5.Amphimedon, which has a highly compact genome with minimal intergenic regions  a developmental regulator and nearby functionally unrelated gene(s), whose introns harbor conserved cis-regulatory elements , which plays conserved roles in animal development (Scaper (S-phase cyclin A-associated protein in the ER)  than the mean intron size (Tfap4 (transcription factor AP-4) (Glis2 (GLIS family zinc finger 2) is deeply conserved. Similar to an observation in vertebrates  . The Ampron size , and preron size . Likewisor AP-4)  and Glistebrates , the spoelements . TogetheAmphimedon displays dynamic developmental gene expression akin to eumetazoans  primarily trimethylates histone H3 on lysine 27 and has been conserved throughout opisthokonts evolution, with its core subunits (E(z), SU(z)12, ESC and Nurf55) being present in animals, choanoflagellates and multicellular fungi, but absent in on yeast  + enhancer RNAs   . The pres notion . Althougaterians , where taterians .Amphimedon lacks CTCF  localizes with cohesin genome-wide and is involved in enhancer-promoter long-range interactions and higher-order chromatin structure , Amphimecks CTCF . This liack CTCF . Alternaack CTCF , and mig protein .cis-regulatory elements being important for the maintenance of metazoan-specific microsyntenic gene blocks over 700 Myr of evolution. The emergence of distal enhancer regulation prior to metazoan cladogenesis could explain the pervasiveness of conserved syntenic regulatory blocks in animal genomes and the absence of these blocks in their unicellular relatives  , a rabbit polyclonal antibody against H3K4me3 (RRID:AB_1977252) , a rabbit polyclonal antibody against H3K27me3 (RRID:AB_310624) , a mouse monoclonal antibody against H3K4me1 (RRID:AB_10806625) , a rabbit polyclonal antibody against H3K27ac (RRID:AB_310550) , a rabbit monoclonal antibody against H3K36me3 (RRID:AB_10615601) , and a rabbit polyclonal antibody against histone H3 (RRID:AB_417398)   . The entessfully   were crosslinked in 2% formaldehyde for 5 min at room temperature (RT). Larvae (~350) were pooled, homogenized and crosslinked as above. A similar procedure was then adopted for both developmental stages. Specifically, crosslinking was quenched with 125 mM glycine for 5 min at RT. Cells were washed twice in 0.22 µm filtered seawater and centrifuged at 500 g for 5 min. Pelleted cells were lysed in SDS Lysis buffer , incubated for at least 10 min on ice, and sonicated for 12 min  in a Bioruptor Sonicator  to generate 200–300 bp fragments. Optimal sonication conditions were previously determined by testing a range of sonication cycles (from 5 to 30); 12 cycles were deemed as optimal. Non-soluble material was removed from the lysate by centrifugation at 12,000 g for 10 min at 4°C. An aliquot of the soluble material was removed for input DNA and stored at −20°C. To reduce the SDS concentration to 0.1%, the remaining soluble material was diluted 10-fold in ChIP dilution buffer . To reduce non-specific background, the diluted soluble material was pre-cleared with Dynabeads protein G beads , and, at the same time, the antibodies were linked to Dynabeads protein G beads  by rotating for one hour at 4°C. At this point, the pre-cleared diluted soluble material was incubated with the antibody-bead mixtures, rotating at 4°C overnight. Immunoprecipitated material was washed three times with Low Salt Wash Buffer , three times with High Salt Wash Buffer , three times with LiCl Wash Buffer , and three times with TE buffer . DNA complexes were eluted 30 min at 65°C with TE-SDS  and decrosslinked overnight at 65°C, along with input DNA, with the addition of 125 mM NaCl. Decrosslinked DNA complexes and input DNA were treated with RNaseA, and subsequently with proteinase K. Finally, immunoprecipitated and input DNA were purified with phenol:chloroform:isoamyl extraction (25:24:1), recovered by precipitation with ethanol in the presence of 300 mM NaOAc pH 5.2 and 2 µl of glycogen carrier (10 mg/ml), and resuspended in UltraPure DNase/RNase-Free Distilled Water (ThermoFisher) for later use. Libraries of immunoprecipitated DNA and input DNA were prepared using the NEBNext ChIP-seq Library Prep Master Mix Set for Illumina  according to the manufacturer’s protocol. The quality and profile of the libraries was analyzed using Agilent High Sensitivity DNA Kit  and quantified using KAPA Library Quantification Kit . Deep sequencing (100 bp paired-end) of the adult libraries – two biological replicates for H3K4me3, H3K4me1, H3K36me3, H3K27me3, RNAPII, input DNA and no biological replicates for H3K27ac and total histone H3 – was performed by the Macrogen Oceania NGS Unit on Illumina HiSeq 2000 instrument . Deep sequencing (40 bp paired-end) of the larva libraries – no biological replicates for H3K4me3, H3K4me1, H3K27me3, H3K27ac, RNAPII, input DNA – was performed by the Central Analytical Research facility (CARF), Brisbane, Queensland, Australia, on Illumina NextSeq 500 instrument .Approximately a cmhttp://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and quality filtered using Trimmomatic v1.0.0  (RRID:SCR_011848) (Amphimedon genome (SCR_005476) (SCR_002105) (SCR_001790). ChIP-seq data sets for each mark were flagged if the scores were below a normalized strand cross-correlation coefficient (NSC) threshold of 1.05, as described in the modENCODE and ENCODE guidelines  developed within the GVL project (Adult raw Illumina sequencing reads were checked using FastQC v0.52 (_011848) . Qualityn genome  using Bo_005476)  with -m _002105) . For allidelines . These a project , 2016 anPearson’s correlation coefficients (Pearson’s r) of genome-wide fold enrichment (FE) signals (see below) was computed for biological replicates and a minimum threshold of 0.5 was required, as per SCR_013291) . Enriched regions were scored on individual replicates (R1 and R2), pooled data  (P) and on subsampled pseudoreplicates  (PR1 and PR2). For each histone PTM, we defined ‘R’ as the set of peaks in P that overlap peaks in R1 and R2, and ‘PR’ as the set of peaks in P that overlap peaks in PR1 and PR2. Next, we defined ‘M’ as the set of peaks that match exactly in R and PR, and ‘T’ as the set of peaks that match exactly in R and PR as well as those that are unique to R or unique to PR. For a statement about reproducibility we required the M-to-T ratio to be at least 0.5  that contain at least one narrow peak passing a P-value of 0.01. For the diffused histone PTMs – H3K36me3 and H3K27me3 – we used the broadPeak representation. RNAPII peaks were detected using the peakzilla software (RRID:SCR_007471) (Histone PTM regions of enrichment relative to corresponding sequenced input DNA controls were determined using MACS2 v2.1.0 (RRID:_013291)  accordin_013291) . Specifieast 0.5 . To obta_007471) , using i_007471) . In line_007471) , all ourAmphimedon genome ; (ii) negative log10 of the Poisson P-value of ChIP-seq counts relative to expected background counts local (ppois); and (iii) subtraction of noise from treatment sample (subtract).For every pair of aligned ChIP and matching input DNA data sets, we also used MACS2 n genome . Input DSCR_011841) (SCR_011848) (SCR_013291) .Larva ChIP-seq data sets were analysed as described above, with the following minor modifications. Adapter contamination prior to read quality filtering was removed using Cutadapt (RRID:_011841) . Reads w_011848) . Histone_013291)  in broadAmphimedon genome . We trained several models in parallel mode with the number of states ranging from 5 states to 15 states and chose a 9-state model as the best model that captures all the key interactions between the chromatin marks and cover all possible genomic locations  that we expected to resolve given the selection of histone PTMs we used . To assign biologically meaningful mnemonics to the nine states, ChromHMM was used to compute the overlap and neighborhood enrichments of each state relative to various types of functional annotations . For the neighborhood positional enrichment plots, the normalization is done across all columns . The functional annotations used were as follows: (1) CpG islands obtained using Hidden Markov Models as described in In both stages, chromatin states across the genome were defined using ChromHMM v1.10 , which in genome . Each biotations . State ehttps://www.bioconductor.org/packages/release/bioc/html/GeneOverlap.html) was used to test and visualise their association with lists of various gene expression groups  , but overlapped with regions designated as being in an enhancer chromatin state  based on the ChromHMM analysis. The activated enhancer elements were predicted intersecting enhancer elements with H3K27ac significant peaks, requiring a 50% minimal overlap fraction. BEDTools v2.23.0 (RRID:_006646)  was usedDe novo motif enrichment analyses were performed using MEME-ChIP against JASPAR CORE and UniPROBE Mouse databases  (RRID:SCR_001783) (_001783) . Each moSCR_005736)   against the non-redundant (nr) NCBI protein database. All proteins were also searched for protein motifs and signal peptides using InterProScan 5 (Gene Ontology (GO) functional enrichment analyses were performed using the Cytoscape plugin BiNGO (RRID:_005736)  with cuspeptides  were ann_001010)   (Transcription Start Site (TSS) input DNA-normalised coverage profiles and heatmaps were calculated using ngs.plot v2.61 (RRID:_011795)  and deep_011795) . As abov_011795) . Express_011795) .Amphimedon   .ChIP-quantitative PCRs (ChIP-qPCRs) were performed using the LightCycler 480 platform . ChIP  and Input DNA libraries were diluted in water, combined with LightCycler 480 SYBR green I master mix  and 0.2 µM primers, then cycled with the following profile: 95°C for 10 min, 40 cycles of 95°C for 10 s, 60°C for 10 s, 72°C for 20 s. Primer sequences are available in 210, representing 10% of input chromatin) was subtracted from the Cq value of the input sample to generate the adjusted input Cq. Two different intergenic regions not bound by our histone PTMs of interest were used as negative controls. Double delta (dd) Cq analysis was computed (see Quantification cycle (Cq) values were extrapolated from manufacturers software (version 1.5.1.6.1 SP2) using High Confidence settings. A melt curve and no template controls (ntc) were also run to ensure single amplicons were responsible for the fluorescent signal. The numerical value 3.32 Fold Change = 2Amphimedon genome using Bowtie (RRID:SCR_005476) , as these developmental time points only have one replicate per time point. To reduce noise, the protein-coding genes and long non-coding RNAs with an overall expression of less than 100 CEL-seq raw counts throughout the whole developmental time course were discarded. The CEL-seq raw gene counts were then normalized using variance stabilizing transformation in DEseq2 1.6.3 (RRID:SCR_000154)  non-overlapping protein-coding genes with an intergenic distance >1 kb that were found in scaffolds larger than 10 kb were considered ‘low-variance’ genes . To define low, medium and high, the 3,200 high-variance genes and 3,999 low-variance genes were sorted based on CELseq data values and separated into three bins of an equal number of genes.CEL-seq raw reads were processed and mapped back to the _005476) . We then_000154)  and the Amphimedon genome using Bowtie (RRID:SCR_005476) , as these developmental time points only have one replicate per time point. To reduce noise, only the protein-coding genes with an expression of at least four CEL-seq normalised counts in at least two developmental time points were retained. To define the transcriptional stability of protein-coding genes, the coefficient of variation of gene expression was calculated for each protein-coding gene , as reported by stable’ genes and the top 1,000 genes with the highest variation in expression as strongly developmentally ‘regulated’ genes.CEL-seq raw reads were processed and mapped back to the _005476) . Read cohttp://www.cnidariangenomes.org/) using kallisto     searches_001010)  with a t_011811) . The ali_011811)  with -waAmphimedon ChIP-seq data sets have been deposited to the NCBI Gene Expression Omnibus (GEO) (RRID:SCR_007303) (Amphimedon genome assembly ampQue1 was used throughout the study. CEL-seq data sets can be obtained from NCBI GEO (GSE54364) (Amphimedon RNA-seq data sets can be downloaded at NCBI's SRA (RRID:SCR_004891) with accession SRP044247 (Nematostella vectensis RNA-seq data sets can be downloaded at NCBI's SRA with accession SRP018739 (N. vectensis ChIP-seq data sets can be obtained from NCBI GEO (GSE46488) (A. queenslandica: Aqu2.1 models (http://amphimedon.qcloud.qcif.edu.au/)  (http://amphimedon.qcloud.qcif.edu.au/lncRNAs/)  (N. vectensis: NveGenes2.0 models (http://www.cnidariangenomes.org/)._007303)  under acSE54364) . AmphimeRP044247 . NematosRP018739 . N. vectSE46488) . We used5, 2017) , lncRNAs5, 2017) ; N. vect 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.cis-regulatory complexity" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by Robb Krumlauf as the Senior Editor and Reviewing Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Veronica Hinman (Reviewer #1); Ulrich Technau (Reviewer #3).Thank you for submitting your article "Landscape of histone modifications in a sponge reveals the origin of animal The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Nematostella and on Capsaspora because in the latter case there was no evidence for enhancer/promoter landscapes. Therefore, the study deals with an important question, is well-designed, and has potential for both high relevant study and impact. However, there are two major weaknesses that dampen enthusiasm: a) an important part of the paper is the epigenetic analysis but the quality of H3K4me1 data is poor and b) and the marks of the lincRNAs is weak. These issues must be addressed in a revision before the paper can be considered further. We normally like to see revisions made within two months but we appreciate that the issues raised may require a longer time period and we are willing to allow you the additional time if needed. This may mean that the resubmission is given a new manuscript number but we will make every effort to ensure that it is handled by the same initial reviewers.The consensus opinion of the reviewers is that this study is of great potential interest and could make a significant contribution to the field. These analyses could bridge the gap between published studies on A) Epigenetics: The H3K4me1 and H3K27ac data is of poor quality and there are a variety of issues with the technical aspects of the paper. This includes missing ChIP-qPCR validations, questionable antibody quality and an absence of supplementary figures dealing with data integrity.Some of the reviewers feel it would be important to perform additional replicates of H3K4me1 and H3K27ac ChIPs. If this is possible it could enhance the paper and address the weakness of the existing data. However, we are willing to stop short of this request because of the time involved and instead request that more analysis and data be provided to confirm existing peaks. Minimally, you need to provide more thorough statistical analysis and test the effects of different thresholds on the chromatin states. This will not eliminate the issues associated with data noisiness (only new ChIPs can do that) but will allow the reader to gain a better insight into the quality of the data that was used to reach the conclusions. This involves:1) Extensive qPCR (20 – 25 targets / ChIP) validations (including a number of negative controls).2) Heatmaps (not average profiles) followed by HCL or K-means clustering of their ChIP-seq data over various genomic features , including both replicates side by side.3) Thorough comparisons of the ChIP-seq data .4) BAM files and signal files (BigWig) should be uploaded to GEO and made available for reviewers to independently assess data quality.5) For all the relevant genomic examples (named above), both experimental replicates should be shown.6) This paper, and all conclusions, rely entirely on the use of off-the-shelf antibodies. All of the data analyses derive from experiments with these antibodies. Therefore, I think these need to be "gold standard experiments." It’s reassuring that the proteins are 100% identical in the region that the antibody recognizes, but this does not indicate that these antibodies are specific. A great control would be to perform ChiP-PCR or western blot on control versus some stage treated with a histone methylation/acetylation inhibitors. This would confirm specificity.Nematostella . The difference between the top bin and the bottom bin is striking in Nematostella and much more modest, perhaps even not significant, in the sponge. This however is where a genuine statistical analysis is needed to make the stronger point. If this is a qualitative analysis the authors should be clear about this in the discussion of this data. I also wonder here is this analysis is confounded by inclusion of the many genes for which the one peak of expression is in the adult  These could be genes that are nonetheless very stably expressed in the adult, but the resolution of the time-course assigns "adult" as one time equivalent to very restricted developmental stages. This analysis might be better done by considering only the developmental stages, and might provide a better significance.7) Data for B) lincRNAs: There is a concern about the ability to reach conclusions the lincRNA aspect of the study based on figures presented. This could simply be omitted. The consensus opinion of the reviewers is that this study is of great potential interest and could make a significant contribution to the field. These analyses could bridge the gap between published studies on Nematostella and on Capsaspora because in the latter case there was no evidence for enhancer/promoter landscapes. Therefore, the study deals with an important question, is well-designed, and has potential for both high relevant study and impact. However, there are two major weaknesses that dampen enthusiasm: a) an important part of the paper is the epigenetic analysis but the quality of H3K4me1 data is poor and b) and the marks of the lincRNAs is weak. These issues must be addressed in a revision before the paper can be considered further. We normally like to see revisions made within two months but we appreciate that the issues raised may require a longer time period and we are willing to allow you the additional time if needed. This may mean that the resubmission is given a new manuscript number but we will make every effort to ensure that it is handled by the same initial reviewers.A) Epigenetics: The H3K4me1 and H3K27ac data is of poor quality and there are a variety of issues with the technical aspects of the paper. This includes missing ChIP-qPCR validations, questionable antibody quality and an absence of supplementary figures dealing with data integrity.Some of the reviewers feel it would be important to perform additional replicates of H3K4me1 and H3K27ac ChIPs. If this is possible it could enhance the paper and address the weakness of the existing data. However, we are willing to stop short of this request because of the time involved and instead request that more analysis and data be provided to confirm existing peaks. Minimally, you need to provide more thorough statistical analysis and test the effects of different thresholds on the chromatin states. This will not eliminate the issues associated with data noisiness (only new ChIPs can do that) but will allow the reader to gain a better insight into the quality of the data that was used to reach the conclusions.Thank you for these comments and recommendations. We also agree that additional ChIPs would not be necessary, especially given the larval ChIPs corroborate the adult ChIPs despite being a different stage comprised of different cell types with markedly overall different transcriptional profiles. We agree that without this confirmation additional replicates would be necessary. In the revision, we have made sure that we highlight, wherever possible, the consistencies between larval and adult ChIP results. Nonetheless, as outlined below, we have undertaken a range of new validations and we have extended our statistical analyses, and included new figure plates and statements.This involves:1) Extensive qPCR (20 – 25 targets / ChIP) validations (including a number of negative controls).As per request of the reviewers, we have now performed ChIP-qPCRs validations of our ChIP-seq data sets for H3K27ac (n =20 targets) and H3K4me1 (n =21 targets) to confirm existing peaks. In addition, we have validated a subset of H3K4me3 (n =10 targets) and H3K27me3 (n =10 targets) peaks. For each of these four histone PTMs, ChIP-qPCRs confirmed our ChIP-seq findings for >80% of the targets, compared to negative controls (two different intergenic regions not bound by our histone PTMs of interest). Importantly, in the great majority of the cases, we observed high signal when corrected for background.in-silico predicted enhancer elements, and were able to successfully validate >85% and >95% of them, respectively.Specifically, in the case of H3K4me3, we designed primers to target regions of enrichment at transcription start sites (TSSs) of genes identified using our peak calling strategy. We were able to validate 90% of the targets. For H3K27me3, we designed primers to target transcriptionally silenced regions of enrichment and validated 80% of them. Most importantly, for H3K4me1 and H3K27ac we designed primers to predominantly target regions of enrichment corresponding to our Nematostella) or unicellular holozoans . If requested, we can add these data to the manuscript.At the end of this response letter, we provide the methods on how these experiments were performed. We also have added to this revision, as a ‘Related Manuscript File’, a file that shows the ChIP-qPCR results. We have elected not to include these data in the paper as such confirmation experiments for histone H3 modifications are not typically found in equivalent reports from basal metazoans  Heatmaps (not average profiles) followed by HCL or K-means clustering of their ChIP-seq data over various genomic features , including both replicates side by side.Amphimedonprotein-coding genes, and heatmaps in Amphimedonacross TSSs of lincRNAs, we have now added heatmaps over the newly identified activated enhancer elements in In addition to 3) Thorough comparisons of the ChIP-seq data .We re-evaluated reproducibility of high-throughput experiments by measuring consistency between the biological replicates within our ChIP-seq experiments. First, Pearson’s correlation coefficients (Pearson’s r) of genome-wide fold enrichment (FE) signals was computed for biological replicates and a minimum threshold of 0.5 was required. In addition, to ensure consistency between biological replicates, we further required an Irreproducible Discovery Rate (IDR) of at least 0.5, as described in the modENCODE and ENCODE guidelines . ChIP-seq data sets that met these criteria were then merged across biological replicates. This is now stated in the Materials and methods section:“Pearson’s correlation coefficients (Pearson’s r) of genome-wide fold enrichment (FE) signals (see below) was computed for biological replicates and a minimum threshold of 0.5 was required, as per Ho et al. . In addition, to ensure consistency between biological replicates, we further required an Irreproducible Discovery Rate (IDR) of at least 0.5 (see below), as described in the modENCODE and ENCODE guidelines . ChIP-seq data sets that met these criteria were then merged across biological replicates see .”A complete summary statistics and quality metrics of the ChIP-seq datasets used in this study is shown in In addition, we have now incorporated a supplementary figure addressing data integrity. Specifically, in Together, this evidence provides further support for the quality of the data that we used to reach the main conclusions of our study.4) BAM files and signal files (BigWig) should be uploaded to GEO and made available for reviewers to independently assess data quality.http://www.ncbi.nlm.nih.gov/geo/info/seq.html [the further processed data  should be supplied instead]. As per GEO guidelines, all raw data files (fastq) along with peak files (BED), and signal files (wig) are now available at the following link, allowing review of record GSE79645 while it remains in private status: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=qxyhmmiajrolnqt&acc=GSE79645 .GEO GSE79645 has now been updated as requested. Please note that GEO does not accept alignment files (BAM) as processed data, as indicated at 5) For all the relevant genomic examples (named above), both experimental replicates should be shown.As mentioned above in comment #3, we have now incorporated these into a supplementary figure addressing data integrity. In 6) This paper, and all conclusions, rely entirely on the use of off-the-shelf antibodies. All of the data analyses derive from experiments with these antibodies. Therefore, I think these need to be "gold standard experiments." It’s reassuring that the proteins are 100% identical in the region that the antibody recognizes, but this does not indicate that these antibodies are specific. A great control would be to perform ChiP-PCR or western blot on control versus some stage treated with a histone methylation/acetylation inhibitors. This would confirm specificity.e.g., Schwaiger et al. 2014; Sebé-Pedrós et al. 2016) where sequence conservation has been deemed sufficient to justify the usage of antibodies against the N-terminal tail of histone H3 in these organisms. In fact, the antibodies used in our study recognize the correct epitopes in even more distantly related eukaryotes, where their usage has been successfully reported . Although we agree with the reviewers that the conservation of the entire amino acids sequence of histone H3, along with the relevant histone methyltransferase and acetyltransferase, which we also show, does not per seindicate that the antibodies used for chromatin immunoprecipitation are specific, we believe that the ‘gold standard’ had already been set by rich body of literature that had not attracted any major criticisms over the years.We have followed current approaches adopted by previous studies in non-bilaterian model organisms (7) Data for Amphimedonand Nematostellaand performed a Mann-Whitney U test to show that the difference in H3K4me3 enrichment between stable and regulated genes is statistically significant. This is now mentioned in the main text. Specifically:We agree with the reviewers that a genuine statistical analysis is needed to make the stronger point. We have now compared the log2(H3K4me3/input DNA) distributions between regulated and stable genes in both “Although stable and regulated genes had similar levels of RNAPII and total histone H3 , the stable genes were strongly marked by H3K4me3 and the regulated genes had significantly lower levels of H3K4me3 , regulated genes showed higher levels of H3K27me3  and lower levels of H3K36me3  than did stable genes . Analyzing RNA-seq–based gene expression through the development of the cnidarian Nematostellavectensis and previously published ChIP-seq data sets in Nematostellaadult female polyps , we obtained the same pattern  lincRNAs: There is a concern about the ability to reach conclusions the lincRNA aspect of the study based on figures presented. This could simply be omitted.Amphimedon. Specifically:We have now expanded this analysis by performing additional statistical analysis and showed that the difference in H3K4me1-to-H3K4me3 signal is significantly different between the two predicted subpopulations of lincRNAs in “Similarly to Ilott et al. , we arbitrarily adopted a H3K4me1-to-H3K4me3 ratio of >1.2 and <0.8 to define elincRNAs and plincRNAs, respectively. […] Lastly, 43 (20%) lincRNAs could not be assigned to either group, that is, 0.8<H3K4me1-to- H3K4me3<1.2 .”CapsasporalincRNAs , where similar subpopulations of lincRNAs have been identified based on their H3K4me1- to-H3K4me3 signatures, and we think represent interesting findings that are worth reporting in the manuscript.These results strongly resemble those recently found in


Drosophila PhoRC during embryogenesis and revealed extensive co-occupancy at developmental enhancers. By using an established in vivo assay for Polycomb response element (PRE) activity, they show that a subset of characterized developmental enhancers can function as PREs and silence transcription in a Polycomb-dependent manner, thereby suggesting that reuse of the same elements by the PcG system may help fine-tune gene expression and ensure the timely maintenance of cell identities.Here, Erceg et al. studied the occupancy of the  cis-regulatory functions are thought to operate through distinct dedicated elements. By examining the occupancy of the Drosophila pleiohomeotic repressive complex (PhoRC) during embryogenesis, we revealed extensive co-occupancy at developmental enhancers. Using an established in vivo assay for PRE activity, we demonstrated that a subset of characterized developmental enhancers can function as PREs, silencing transcription in a Polycomb-dependent manner. Conversely, some classic Drosophila PREs can function as developmental enhancers in vivo, activating spatio–temporal expression. This study therefore uncovers elements with dual function: activating transcription in some cells (enhancers) while stably maintaining transcriptional silencing in others (PREs). Given that enhancers initiate spatio–temporal gene expression, reuse of the same elements by the Polycomb group (PcG) system may help fine-tune gene expression and ensure the timely maintenance of cell identities.Developmental gene expression is tightly regulated through enhancer elements, which initiate dynamic spatio–temporal expression, and Polycomb response elements (PREs), which maintain stable gene silencing. These two  Drosophila, where PcG loss-of-function mutations cause derepression of Hox genes in body segments where they are normally not expressed, leading to dramatic changes in segment identity (Polycomb group (PcG) proteins are an evolutionarily conserved chromatin-modifying system that functions to maintain gene silencing during development, having an essential role in lineage commitment and differentiation . The sysidentity . Biochemidentity . PRC2 coidentity , a chromidentity , while Pidentity . H3K27meidentity  such thaidentity . This stidentity .Drosophila, they target only a subset of genes. In Drosophila, PcG proteins are recruited to chromatin via Polycomb response elements (PREs), cis-regulatory elements that silence transcription in a PcG-dependent manner , which appear to act in a dominant manner to silence transcription of any linked gene (Hox loci), most non-Hox target PREs are close to the transcriptional start site (TSS)  to activate specific patterns of spatio–temporal expression , providcis-regulatory elements during embryonic development. To initiate this study, we performed ChIP-seq (chromatin immunoprecipitation [ChIP] combined with high-throughput sequencing) of both components of the PhoRC (Pho and dSfmbt) during a narrow 2-h time window of embryogenesis when major cell lineages are specified within the mesoderm and ectoderm. This identified almost 1000 regions cobound by both proteins, a surprising fraction of which is bound to characterized developmental enhancers. To determine whether Polycomb can mediate silencing through these elements, we investigated whether PhoRC-bound enhancers can act as PREs in vivo. Using two established functional assays for PRE activity, we demonstrated that 50% of enhancers tested can function as PREs in vivo, silencing transcription in a PcG-dependent manner. Conversely, we show that a subset of characterized “classic” PREs can function as developmental enhancers in vivo, activating transcription in specific spatial domains. Therefore, in addition to dedicated enhancers and dedicated PREs, this study identified cis-regulatory elements with dual activity, functioning as developmental enhancers to activate spatio–temporal expression in one cell type and PREs that stably silence transcription in another. Having both functions mediated through the same element may provide more fine-tuning of gene expression and ensure that key enhancers are rapidly and stably silenced during key lineage transitions.To better understand the relationship between PREs and enhancer elements, we performed an in-depth analysis of the functional properties of Supplemental Table S2), representing 79.6% of Pho-bound regions and 40% of dSfmbt-bound regions . These cobound regions, referred to as PhoRC-bound regions, have quantitatively higher levels of both Pho and dSfmbt ChIP signal compared with regions bound by either protein alone , suggesting that they bind with higher affinity as a complex to target sites. There is also a difference in the distribution of regions bound by PhoRC compared with regions bound by either protein alone : The majority (72.3%) of dSfmbt-only peaks is located very close to the promoter (97-base-pair [bp] median distance to the closest TSS), while Pho-only peaks are more loosely distributed around promoters (1890-bp median distance from the closest TSS) , as observed in whole embryos .To dissect the role of PhoRC in the regulation of cell type-specific developmental programs, we obtained a high-resolution map of Pho and dSfmbt occupancy specifically in mesodermal cells using BiTS-ChIP-seq (batch isolation of tissue-specific chromatin for immunoprecipitation [BiTS-ChIP] combined with high-throughput sequencing)  during t embryos . The 994Drosophila PREs, including those at hox loci , and contain the known Pho motif  and identify many more putative PREs within these loci; e.g., abd-A , cad  and Sox21b . The only exceptions are the prod and α-PKC loci , which show no evidence of Polycomb-mediated repression in mesodermal cells at these developmental stages.PhoRC-bound regions overlap 93% (28 out of 30) of all functionally characterized ho motif , demonstAlthough PhoRC often binds in close proximity to gene promoters (47% within 500 bp), more than half of the PhoRC peaks are found at greater distances ; (3) ChISupplemental Table S3). This fraction represents a significant enrichment over matched background regions   and is likely an underestimate, since an additional 16.2% of PhoRC peaks reside within intergenic regions  and may represent as yet unidentified enhancer elements.In addition to binding close to TSSs, 225 of the 994 PhoRC sites reside within developmental enhancers, characterized either from transgenic embryos  or within ChIP-defined putative enhancers , representing 22.6% of all bound regions   D. Moreovo-sided)  in enhano-sided) C, indicao-sided) , a signio-sided) F.The extensive occupancy of PhoRC at enhancers suggests that a proportion of Polycomb's activity is mediated through developmental enhancers and not only via spreading from previously characterized PREs and promoters  indicateAntp and Bithorax hox loci) and is referred to here as “repressed promoters.” The bimodal distribution of H3K27me3 at repressed promoters , with the exception of four regions that have no detectable DHS signal  reporter, which does not contain any inherent enhancers and is therefore ideal to assay enhancers for PRE activity. Ten developmental enhancers with characterized spatio–temporal activity and occupancy of PhoRC were placed in front of a mw promoter in a construct where, upon successful integration of the plasmid into a specific landing site in the Drosophila genome, it reconstituted a functional mw reporter gene  pairing-sensitive silencing (PSS) , which iter gene . mw expro copies C. When ao copies . While mo copies .wg_del-wg and Dad enhancers. Three enhancers have even lighter eye color when homozygous compared with their heterozygous siblings, indicative of reporter gene silencing through PSS   may hav [eve_RP .ph-null allele (ph heterozygous and homozygous mutant backgrounds by in situ hybridization against the mw reporter in embryos at late stages of embryogenesis (when ph zygotic phenotypes are visible). Upon the removal of ph, the enhancer's activity was dramatically expanded in five out of 10 cases (50%), and therefore the maintenance of tissue-specific repression was lost . Additionally, the corresponding endogenous gene activity was also disturbed as observed previously in a ph mutant background . The well-characterized Ubx_BXD-C enhancer , we next determined whether they are sufficient to recruit the Polycomb system to the transgenic enhancer de novo. Embryos were collected from each of the five transgenic reporter lines in addition to a transgenic line with an integrated empty vector and used for ChIP-qPCR against H3K27me3 as an indicator of PRC2 recruitment. H3K27me3 was detected using primers that specifically amplify signal from the integrated transgenic enhancer. The ChIPs on all five transgenic enhancer lines indicate that H3K27me3 is highly enriched on the integrated enhancer transgenes, which is not the case in the transgenic line with the empty vector (For these five enhancer elements with clear PRE activity (y vector D. These mw reporter gene expression) only in specific permissive contexts. This may reflect the inherent properties of each enhancer's sequence, its potential to interact with the local genomic environment such as nearby silencers, or the relative levels of PhoRC occupancy. The latter, for example, may explain our results in the eve locus, where we tested two enhancers for PRE activity: the neuronal eve_RP enhancer , which has high levels of PhoRC occupancy and is PcG-responsive , and the muscle–heart eve_MHE enhancer, which has a smaller PhoRC peak and cannot function as a PRE, at least in this transgenic context . In fact, rather than being silenced by polycomb, the MHE enhancer appears to lose activity in the visceral muscle in ph mutants , suggesting that it may even be positively influenced by PcG.Taken together, these three inherent properties  indicate that a subset of characterized developmental enhancers can function as PREs in a PcG-dependent manner. The latter two requirements are fulfilled in five out of 10 (50%) enhancers tested, suggesting that PcG-bound enhancers may be PcG-responsive  . For example, the PcG-dependent PRE P{C4-418bis}  , we usedreporter A. Six wee system . Enhance-418bis}  is suffi-418bis} B, where s}  to stably maintaining a silenced state (PRE).cis-regulatory element, depending on the cellular context. This is not a rare event—almost 25% of PhoRC occupancy is at developmental enhancers. Of the 16 elements that we tested experimentally (either enhancers for PRE activity or PREs for enhancer activity), nine have dual function, being sufficient to activate transcription in a specific spatio–temporal pattern and mediate PcG-dependent silencing in vivo.While enhancers initiate spatio–temporal transcriptional activity, PREs maintain a previously determined transcriptional state of their target genes, thus leading to transcriptional memory . PREs arThese dual elements have interesting implications for transcriptional regulation during embryonic development. First, at the level of PcG protein recruitment, this subset of enhancers is highly enriched in the Pho motif, which distinguishes them from other developmental enhancers. This suggests that the recruitment of Pho to PhoRC enhancers is direct via sequence-specific DNA binding, consistent with an instructive model of recruitment , althougprat2 locus , the vast majority of which is key developmental regulators of cell identity.Third, this may have broader implications for cell fate decisions during rapid developmental transitions. When multipotent cells become specified into different lineages, a specific transcriptional program often needs to be activated in one cell while being repressed in other cells from the same progenitor population . Having Drosophila Pho and dSfmbt proteins, Yin Yang 1 (YY1) and SFMBT, respectively, the conservation of PhoRC as a complex and its involvement in mammalian PcG silencing remain unclear  databases under accession numbers E-MTAB-4585 and ERP000560, and processed data are provided on the Furlong laboratory's Web site (http://furlonglab.embl.de/data).Genome-wide binding profiles for Pho and dSfmbt in mesodermal embryonic nuclei were obtained using BiTS-ChIP  using ch


After more than three decades since the discovery of transcription activation domains (ADs) in gene-specific activators, the mechanism of their function remains enigmatic. The widely accepted model of direct recruitment by ADs of co-activators and basal transcriptional machinery components, however, is not always compatible with the short size yet very high degree of sequence randomness and intrinsic structural disorder of natural and synthetic ADs. In this review, we formulate the basis for an alternative and complementary model, whereby sequence randomness and intrinsic structural disorder of ADs are necessary for transient distorting interactions with promoter nucleosomes, triggering promoter nucleosome translocation and subsequently gene activation. Transcriptional activators are the main triggers of gene activation, determining not only the specific selection of genes transcribed in response to distinct stimuli, but also the level of transcription and its modulation –4. A typThe growing number of human diseases associated with the malfunction of gene expression argues for the development of new drugs affecting specific transcriptional activators or functionally substituting them –11. TechGAL1-HIS3 and GAL1-LacZ reporters revealed that 92 known yeast activators can work in this new context, with 32 showing strong activation potential [One of the remarkable properties of ADs is the general functional interchangeability among gene-specific activators. Testing all 6000 ORFs of the yeast genome for functionality as activators in the context of Gal4 DBD, and otential . This fuotential –22.Based on the ease of interchangeability, it is logical to expect some similarities in structure and/or sequence composition. Contrary to these expectations, comparison of the amino acid sequences of known ADs does not reveal any amino acid consensus or specific structural motif. In fact, the majority of known natural ADs have no distinct structure and are characterized by a high level of intrinsic disorder , 24. DesE. coli genomic DNA fragments fused to the Gal4 DBD [bona fide AD potential and that these sequences are significantly enriched with acidic residues. Later it was shown that hydrophobic amino acids are equally as important [Over the years, there have been several attempts to gain insight into ADs’ composition and other characteristics using a yeast one-hybrid fusion protein system, whereby random sequences were screened for functionality as ADs while connected to a variety of DBDs using a viability test based on a single reporter. The pioneering screen was done by Ma and Ptashne in 1987, testing randomly fragmented Gal4 DBD . The resmportant –30. A simportant  and in amportant . The Lex45 for a stretch of 45 nucleotides. The high variability of AD sequence compositions, in combination with very high frequency of functional AD sequences isolated from random pools [It is paradoxical that despite the easy functional interchangeability among ADs, even those belonging to transcriptional activators from different phyla, they share no clear consensus sequence, and the only compositional similarity between different interchangeable ADs is an excess of hydrophobic and acidic amino acid residues. Even for this loose rule, there are known exceptions, with some ADs containing a positive total charge or at least an absence of acidic residues . Functioom pools –28, 32, The lengths of ADs are another important characteristic. Natural AD length varies over several dozen amino acid intervals. Surprisingly, the above-mentioned screens of randomized sequences revealed that ADs with potency comparable to natural sequences can be as short as 12, 11, nine, or even eight amino acids , 27. In The minimal size of functional ADs is not limited to eight amino acids, as it has been shown that the AD peptides can be substituted with non-natural compounds or other molecular sequences that are much smaller . For insBased on conservation of function between different AD sequences, it is logical to expect a conservation of specific structural motifs, as was clearly shown for DBDs , 6. ContIn sum, despite being easily interchangeable, with conservation of function between activators belonging even to different biological phyla, as well as being easily isolatable in large numbers from random pools of sequences, ADs do not share any consensus sequence and/or structural motif, at least in the absence of their interaction partners. The size of ADs is also highly variable, ranging from several dozen to as few as eight amino acid modules. Even the chemical nature is elusive, varying from different amino acids of L or D chirality working almost equally well, to certain RNA sequences, and to relatively small (MW 290) organic compounds. The paradox of conservation of function without conservation of sequence and/or structure shows the deficiency of our understanding of ADs and the necessity for further investigation, perhaps fundamentally revising research strategies and mechanistic concepts.The commonalities arising from the investigation of a large number of natural and synthetic ADs are limited to the combination of acidity and hydrophobicity, and the intrinsically disordered nature of identified peptides. A large fraction of functional amino acid sequences (up to 1 %) isolated from randomized pools suggests by itself that although some specificity is required, potentially interacting targets in vivo might be numerous, and interactions are likely of low specificity.Since their discovery nearly three decades ago, the mechanism of ADs’ function was proposed to be a direct physical recruitment of transcription-related activities to gene promoters , 26, 48.A subsequent shift of attention in the transcription field to TBP-associated factors (TAFs) resulted in the proposition that the obligatory interacting targets of ADs are TAFs . HoweverAn alternative school of thought , 65 thatExplosive development in the 1990s and early 2000s of the field of chromatin-remodeling and histone-modifying activities, in combination with the demonstration of how critical some of these activities are for the initiation of transcription –81, led HO promoter was subjected to consecutive chromatin immunoprecipitation experiments. The results were indicative of the ordered recruitment of activities. Chromatin remodeling by the SWI/SNF complex was followed by recruitment of SAGA, which in turn facilitated Mediator and Pol II recruitment [In an attempt to systematize and logically connect the recruitment events involved in chromatin remodeling, the yeast ruitment , 100. BuPerhaps the culmination of this line of investigation, demonstrating a wide range of possible mechanisms and synergistic interactions on long- and short-range scales, is a recent attempt to investigate the functionality of 223 chimeric activators created by fusion of individual subunits of known chromatin regulators to zinc finger DNA-binding proteins [In an initial attempt to unite this multiplicity of possible interacting targets in a uniform mechanism, the acidic ADs were proposed to function as “acidic blobs” recruiting numerous protein complexes by simple electrostatic interactions , 102. ThMore recent investigation of GCN4–Mediator interactions , based oConsidering that ADs interact with targets utilizing nonspecific, low-affinity “fuzzy” interactions with very few contact points , itIf the best target exists for a specific activator in the context of a specific promoter , why does the experimental selection of functional ADs from a pool of random sequences or the evolutionary selection not yield a specific AD consensus sequence? Or similarly, why is there a lack of sequence conservation among ADs?What determines the sequence of recruitment events of seemingly distinct enzymatic complexes to the gene promoter, and how are ADs involved in this process?The structural analysis of AD sequences certainly reveals the absence of a clear motif. In fact, ADs comprise one of the best examples of intrinsically disordered protein domains. Why is the structural disorder apparently so important for ADs’ function?Why does the same AD retain functionality and often the degree of activation potential if transferred within evolutionarily very distant contexts of mammalian, plant, and fungi cells , 18–22?Summarizing ADs’ properties, interacting targets, and the mechanism of AD function, it is evident that despite enormous efforts over a long period of time, there is no clear mechanistic understanding of ADs’ function. Although each specific AD performs essentially the same function, namely facilitating chromatin remodeling in broad sense and assembly of the transcription initiation complex, ADs do not have a consensus sequence. That is true even for synthetic ADs selected for functioning within the context of the same activator and the same reporter gene(s) , 31, 32.One can invoke the direct-recruitment model to argue that the high level of randomness of sequence and structure is exactly necessary for the recruitment of multiple targets from a large pool, but this does not help with resolving the first three questions. Challenges regarding understanding the mechanism of ADs’ function have been stated on multiple occasions , 15, 16.In an attempt to help with solving problems formulated above, we propose to consider additional mechanistic options that involve local interaction of ADs with promoter nucleosomes. This possibility naturally arises from the fact that a majority of ADs are negatively charged and hydrophobic, thus likely interacting with positively charged and hydrophobic targets. Another premise for our model is that general chromatin remodeling at gene promoters is a well-established and often required step leading to an activation of transcription. According to the AD–nucleosome interaction model, the otherwise unlikely and weakly interacting partners—ADs and nucleosomes—are brought into proximity by the DBD of the activator and interact only in the vicinity of the activator-binding site due to this facilitation by DBD. Interactions of ADs are likely possible at multiple points on nucleosomes and, while being of low specificity, are leading to a structural destabilization of promoter nucleosomes, and either to a translocation of nucleosomes from the promoter, or to an exposure for the chromatin remodelers and histone modifiers triggering additional chromatin-remodeling events. ADs, which remain at the nucleosome-free promoter anchored by DBD, later participate in direct recruitment and more importantly stabilization of transcription initiation complex. Thus, ADs may play a dual function of initially triggering nucleosome distortion and translocation, and later attracting and stabilizing transcription initiation complex Fig. .Fig. 1TrThere are significant distinctions in the interaction of ADs (with interaction partners) in the direct-recruitment model and in the nucleosome-distortion model. For the recruitment model, the requirement is an establishment of a functional link by attraction of a specific enzymatic activity(s) to the promoter; thus, the more functionally productive interactions have to have higher affinity to specific targets interacting in a specific structural region of the target as it was demonstrated for TBP, Gal11, Tra1, and others , 105. InLow-affinity “fuzzy” interactions, instead of creating a conundrum of mutually disruptive and competitive recruitment events of co-activators , are actConsidering the requirement of low- or very low-specificity destabilizing interactions in multiple points of nucleosomes, it is clear why selection of functional ADs from random pools , 31, 32 As a result of AD–promoter nucleosome interactions, the recruitment of distinct enzymatic activities to a specific gene promoter can be dictated, at least in some situations, by the chromatin architecture of a specific promoter and by a specific nucleosome distortion induced by a specific AD. An additional source of specificity could be the particular composition of promoter DNA elements, such as in TATA- and TATA-less promoters.The intrinsically disordered structural nature of ADs is a feature compatible with and actually favorable for “fuzzy” interactions with different destabilizing points of nucleosomes leading to the subsequent recruitment of co-activators.Finally, the retention of functionality of ADs during the transfer of the same sequence between very distant biological phyla is also easily explainable by extremely high evolutionary conservation of histones and nucleosome structure. In contrast, the evolutionary conservation of co-activator complexes is significantly lower. Consequently, for the direct-recruitment model, this would dictate a significant drop of the AD activation potential during transfer into an evolutionarily distant species.Invoking AD–nucleosome interaction model provides solutions to all of the questions raised above:The proposition of the nucleosome-distortion model can be supported by the following considerations. First, the abundance of histones in the cell is at least one to two orders of magnitude higher than the abundance of any interacting targets of ADs  describeThere are many ways the nucleosome structure can be disturbed; however, for nucleosome relocation, at least some of the histone–DNA contacts must first be broken. Considering that AD sequences typically can be reduced to eight or nine amino acids , 27 or eThe in vivo possibility of AD–nucleosome interactions was demonstrated in early experiments. By modified yeast two-hybrid screening, it was shown that histone H3 is among possible targets of such transcriptional activators as human CTF1/NF1  and yeasThese interactions are transient and of very low specificity, possible only with facilitation by the activator’s DBD and only to nucleosomes in close proximity of the activator-binding site. This extremely low-specificity requirement directly originates from experimental data showing high randomness of AD sequences (1–2 % of random pools), which are able to substitute the native ADs , 31, 32.AD interactions with nucleosomes likely occur at multiple low-affinity points which compete with each other. Contrary to the proposed model, any high-affinity interactions would lead to nucleosome stabilization and repression of transcription. The idea that ADs interact with nucleosomes at multiple competing points is also compatible with the multiplicity of AD sequences that can function in identical molecular contexts , 31, 32.The distorting interactions of AD with nucleosomes are likely less strong and specific than with co-activators and transcriptional PIC components. Thus, in typical biochemical experiments, they are likely to be ignored, leaving interactions with co-activators and PIC as the dominant ones and thus more obvious for mechanistic interpretations of AD function.The difficulties with explaining the mechanisms of AD function , 15, 16,These early experiments demonstrating the possibility of AD–nucleosome interactions, in comparison with the demonstration of direct recruitment of co-activators, are certainly not numerous, and critics may ask why. The answer might lie in the nature of the AD–nucleosomal contacts postulated by the AD nucleosome-distortion model. Considering this model, the challenges of demonstration of AD–nucleosome interactions become obvious:The function of ADs has been a fundamental enigma of the transcription regulation field for more than 30 years. One of the reasons we face this problem arguably is due to the traditional approach, which considers vital protein–protein interactions in terms of the key-and-lock concept or an approximation of it, looking for specific or semi-specific interactions between AD and possible target(s), and utilizing classical biochemistry methods designed to detect these specific interactions and discard noisy background. Our review and analysis is an attempt to get away from this stereotype, and to switch to a reality of intrinsically disordered regions functioning at an extremely low-specificity level. The model of transcription activation based on promoter nucleosome distortion by AD requires these extremely low-affinity noise-level interactions and is an addition to the classical direct-recruitment model Fig. . Our pro


Deletion and base substitution analyses of the kinB promoter (PkinB) region using lacZ fusions indicated that either a 5-nt deletion  or the substitution of G at nt -45 with A (G-45A) relieved kinB repression. Thus, we found a pair of SinR-binding consensus sequences  in an inverted orientation (SinR-1) between nt -57/-42, which is most likely a SinR-binding site for kinB repression. This relief from SinR repression likely requires SinI, an antagonist of SinR. Surprisingly, we found that SinR is essential for positive stringent transcription control of PkinB. Electrophoretic mobility shift assay (EMSA) analysis indicated that SinR bound not only to SinR-1 but also to SinR-2 (nt -29/-8) consisting of another pair of SinR consensus sequences in a tandem repeat arrangement; the two sequences partially overlap the ‘-35’ and ‘-10’ regions of PkinB. Introduction of base substitutions (T-27C C-26T) in the upstream consensus sequence of SinR-2 affected positive stringent transcription control of PkinB, suggesting that SinR binding to SinR-2 likely causes this positive control. EMSA also implied that RNA polymerase and SinR are possibly bound together to SinR-2 to form a transcription initiation complex for kinB transcription. Thus, it was suggested in this work that derepression of kinB from SinR repression by SinI induced by Spo0A∼P and occurrence of SinR-dependent positive stringent transcription control of kinB might induce effective sporulation cooperatively, implying an intimate interplay by stringent response, sporulation, and biofilm formation.It is known that transcription of  Bacillus subtilis, entry into the sporulation pathway is governed by a member of the response regulator family of transcription factors known as Spo0A (abrB transcription (σH (spo0H) gene encoding σH. kinA is transcribed by RNA polymerase (RNAP) possessing σH  under positive stringent transcription control is the adenine species; ilvB is the first gene of the ilv-leu operon for branched-chain amino acid synthesis  gene in multiple copies  and proteins to form an extracellular matrix for building the biofilm  analysis indicated that SinR bound not only to SinR-1 but also to SinR-2 consisting of another pair of SinR consensus sequences in a tandem repeat arrangement (nt -29/-8) that partially overlap the ‘-35’ and ‘-10’ regions, respectively, which is likely involved in positive stringent transcription control of PkinB.In this work, we identified a pair of SinR consensus sequences in an inverted orientation (SinR-1) between nt -57/-42 (+1 is transcription initiation nt) as a SinR-binding site for B. subtilis strains used in this work are listed in Table 1. To construct transcriptional promoter-lacZ fusion strains of kinB, PkinB regions comprising nt -75/+10, -75/+10 [with base substitution of A at nt +1 with G (A+1G)], -65/+10, -85/+10, -95/+10, -75/+10 [with 5-nt deletion (Δ5) (-61/-57)], -75/+10 [with 10-nt deletion (Δ10) (-64/-55)], and -75/+10 (with base substitution of G at -45 with A (G-45A) and Δ5] were amplified using the primer pairs of F75c/R10c1, F75c/R93, F90/R10c1, F92/R10c1, F82/R10c1, F95/R10c2, F96/R10c2, and F17/R17 (Supplementary Table Escherichia coli strain DH5α to ampicillin-resistance (50 μg/ml) on Luria-Bertani (LB) medium plates (kinB regions with and without a base substitution and (or) deletion were linearized with PstI, and then used for double-crossover transformation of strain 168 to chloramphenicol-resistance (5 μg/ml) on tryptose blood agar base (Difco) with 10 mM glucose (TBABG) plates, which produced strains FU1191 PkinB (-75/+10), FU1193 PkinB (-75/+10 A+1G), FU1190 PkinB (-65/+10), FU1192 PkinB (-85/+10), FU1182 PkinB (-95/+10), FU1195 PkinB (-75/+10 Δ5), FU1196 PkinB (-75/+10 Δ10), and FU1217 PkinB (-75/+10 G-45A Δ5), respectively.The m plates . The corkinB (-75/+10 G-45A), FU1241 PkinB (-55/+10 A-17G), FU1242 PkinB (-55/+10 G-16A), FU1243 PkinB (-55/+10 T-15C), FU1244 PkinB (-55/+10 G-14A), FU1245 PkinB (-55/+10 T-20C T-19C), FU1246 PkinB (-55/+10 T-18C A-17G), FU1247 PkinB (-55/+10 G-16A T-15C), FU1248 PkinB (-55/+10 C-26T T-25C), and FU1249 PkinB (-55/+10 T-27C C-26T), the upstream and downstream parts of the PkinB region (nt -75/+10) and the PkinB region (nt -55/+10) were separately amplified with the respective two primer pairs F16a/R16b and F16c/R10c1, F55c/R41b and F41c/R10c3, F55c/R42b and F42c/R10c3, F55c/R43b and F43c/R10c3, F55c/R44b and F44c/R10c3, F55c/R45b and F45c/R10c3, F55c/R46b and F46c/R10c3, F55c/R47b and F47c/R10c3, F55c/R48b and F48c/R10c3, and F55c/R49b and F49c/R10c3 for FU1216, FU1241, FU1242, FU1243, FU1244, FU1245, FU1246, FU1247, FU1248, and FU1249 (Supplementary Table kinB (-55/+10) as a template for FU1241 to FU1249. Next, the respective two PCR products were mixed, and extension reactions were carried out without any primer. PCR with the resultant fragment as a template and a primer pair  was constructed as follows. The regions upstream and downstream of the sinR gene were firstly amplified by PCR using DNA of strain 168 as a template, and primer pairs F04a/F04b and F04e/F04f, respectively. The erm cassette was amplified by PCR using DNA of plasmid pMUTIN2  on TBABG plates to produce strain FU1204. Strains FU1206, FU1210, FU1218, FU1219, and FU1224, which carry ΔsinR::erm and each of the lacZ fusions, were obtained by transformation of FU1115, FU1191, FU1216, FU1217, and FU1195 with DNA of strain FU1204 to erythromycin-resistance, respectively.Strain FU1204 ( pMUTIN2  as a temΔsinI::spc) and FU1226 (ΔslrR::tc) were obtained by transformation of strain 168 with DNAs of strain NCIB3610 carrying ΔsinI::spc ] with DNAs of strain FU1225 or FU1226.Strains FU1225 (inI::spc  and straslrR::tc  to resislacZ-fusion strains were grown at 30°C overnight on TBABG plates containing the appropriate antibiotic(s); chloramphenicol (5 μg/ml), erythromycin (0.3 μg/ml), spectinomycin (60 μg/ml), and (or) tetracycline (10 μg/ml). The cells were inoculated with an optical density at 600 nm (OD600) of 0.1 in 50 ml of a nutrient sporulation medium (NSMP)  When the cells reached an OD600 of 0.5, 15 ml each culture was distributed into two flasks, and decoyinine was added to one flask to give a final concentration of 500 μg/ml (18 mM). Before and after decoyinine addition, 1-ml aliquots of the culture were withdrawn at 30-min intervals, and the β-Gal activity was measured.The m (NSMP) , and them (NSMP) . The celm (NSMP) . (In theΔsinR), were measured to obtain the sporulation percentages (S/V x 100) at T0 and T20 (0 and 20 h after entry into the stationary cell phase during sporulation in NSMP). The sporulation percentages for S6 cultures at 0 and 10 h after decoyinine addition (T0 and T10) were also measured.The titers of viable cells (V) and spores (S) that were heat-resistant (75°C for 20 min), for the cultures of strains 168 and FU1204  derivative producing SinR  containing 1 μg of poly(dI-dC) . After the addition of 2 μl of loading buffer , the samples were applied onto a polyacrylamide gel, and electrophoresis was performed in 0.1× TBE buffer at 4°C. The method used for the detection of biotin-labeled DNA was described previously  of the phosphorelay system that phosphorylates Spo0A was identified, and its transcription was examined  . It is co-transcribed with kapB encoding a lipoprotein involved in autophosphorylation of KinB and phosphorylation of Spo0F ; the larger fragment including the patB gene upstream of kinB could not be cloned to plasmid pCRE-test2, presumably because patB is harmful in its multiple copy state in E. coli. No significant difference in lacZ expression by the largest lacZ-fusion strain was observed in the wild-type, ΔcodY, and ΔabrB genetic backgrounds, on cultivation in NSMP or S6 medium with and without decoyinine (data not shown), suggesting that the CodY- and AbrB-binding sites that affect PkinB are unlikely to be located in the PkinB region (nt -95/+120). This finding implied that kinB expression might not be directly regulated by AbrB and CodY.The examined . The kin (nt +1)  . To detekinB during sporulation in nutrient NSMP medium and upon decoyinine addition in minimal S6 medium is dependent on the adenine species at the transcription initiation nt, we constructed lacZ fusion strains with the PkinB region (nt -75/+10) carrying adenine and guanine at the transcription initiation nt, and β-Gal synthesis was monitored during sporulation of the constructed strains, PkinB (-75/+10) and PkinB (-75/+10 A+1G), together with the previously constructed strains, PkinB (-55/+10) and PkinB (-55/+10 A+1G) . The positive stringent transcription control was clearly observed in strains PkinB (-75/+10) and PkinB (-55/+10) for both sporulation in NSMP medium and decoyinine-induced sporulation in S6 medium, that is, some enhancement around T0.5 for sporulation in NSMP, and roughly a 1.5-fold increase after decoyinine addition, respectively . But, this positive control was not observed for strains PkinB (-75/+10 A+1G) and PkinB (-55/+10 A+1G). These results clearly confirmed that positive stringent transcription of PkinB depends on the adenine species at the transcription initiation nt (+1). Furthermore, the basal level of β-Gal synthesis was somewhat repressed in strains PkinB (-75/+10) and PkinB (-75/+10 A+1G) in comparison with that in strains PkinB (-55/+10) and PkinB (-55/+10 A+1G) for both sporulation in NSMP medium and decoyinine–induced sporulation in S6 medium , implying that the PkinB region (nt -75/-55) might possess a binding site or part of one for a transcription repressor.To confirm that positive stringent transcription control of P10 A+1G)  , we constructed a successive series of lacZ-fused PkinB truncation derivatives . When β-Gal synthesis in these truncation derivatives was monitored during sporulation in NSMP , the PkinB (-55/+10)-lacZ derivative exhibited a higher level of β-Gal synthesis than the other truncation derivatives , which showed similar levels. When it was monitored upon decoyinine addition to the S6 cultures , the basal level of β-Gal synthesis by the PkinB (-55/+10)-lacZ derivative before decoyinine addition was higher in comparison with those by the other derivatives. However, the positive stringent transcription control of PkinB was observed to be nearly the same level, approx.1.5-fold increase, for all the truncation derivatives, suggesting that the relief from kinB repression is not involved in this positive stringent transcription control. These overall results suggest that a binding site of a repressor or part of it is likely located in the PkinB region (nt -65/-55), which is responsible for kinB repression but not involved in positive stringent transcription control of PkinB.To localize a repressor-binding site in the PkinB (-75/+10) region to confirm that the repressor-binding site is located between nt -65/-55. When β-Gal synthesis by the derivatives carrying each of the inner deletions  for sporulation in NSMP  and in decoyinine-induced sporulation . Moreover, the higher levels of β-Gal synthesis by these derivatives were in the order of PkinB (-75/+10 Δ5), PkinB (-75/+10 Δ10), PkinB (-55/+10), and PkinB (-75/+10). The differences could be attributed to newly created sequence variation of nt -65/-55 in these deletion derivatives, which might affect the binding of the assumed kinB repressor. However, nearly the same level of positive stringent transcription control was observed for these derivatives . The overall deletion analysis  indicated that the inner deletion of Δ5  disrupted a binding site or part of it for the assumed repressor for kinB transcription, which is not involved in positive stringent transcription control of PkinB.Next, we introduced inner deletions [Δ10  and Δ5 ] into the PsinR gene was isolated as a sporulation inhibition gene in multiple copies (ΔsinR) in NSMP were < 5 × 10-5% at T0, and 80 and 100% at T20, respectively. The sporulation percentages for strains 168 and FU1204 in decoyinine-induced sporulation were 0.4% and 1.5% at T0 (at decoyinine addition time) and 40% and 98% at T10.  Hence, the ΔsinR deletion tended to promote the sporulation, especially on cultivation in S6 medium with decoyinine.The e copies . At firslacZ-fusion with the PkinB region , PkinB (-75/+10 Δ5), PkinB (-75/+10 G-45A), and PkinB (-75/+10 Δ5 G-45A), in the wild-type (sinR+) and ΔsinR genetic backgrounds. In the sinR+ strains cultivated in NSMP medium, the introduction of the inner deletion of Δ5 or the base substitution (G-45A) greatly and equally relieved the severe repression of lacZ expression observed in strain [PkinB (-75/+10)] without the deletion or substitution . Moreover, the introduction of both Δ5 and G-45A gave further relief from the repression. In the ΔsinR strains cultivated in NSMP, the severe repression of the strain without Δ5 and G-45A as well as the residual repression observed in the Δ5 or G-45A strain were well relieved on the introduction of ΔsinR .A previous study involving a B region  suggestesinR+ background strains in S6 medium, Δ5 or G-45A equally well relieved the severe repression in strain PkinB (-75/+10) carrying no deletion or base substitution . Also, it was completely relieved in strain PkinB (-75/+10) carrying both Δ5 and G-45A. In ΔsinR strains cultivated in S6 , the levels of lacZ expression before decoyinine addition were nearly the same in strains PkinB (-75/+10) with and without Δ5 and (or) G-45A, indicating that the repression observed in strain PkinB (-75/+10) was well relieved on the introduction of ΔsinR. Surprisingly, positive stringent transcription control of PkinB, which is inducible through the addition of decoyinine, did not occur in any ΔsinR strain with and without Δ5 and (or) G-45A at all . However, significant repression of lacZ expression was remained even in the genetic background of ΔsinR, as observed in Figure 4D as well as in Figure 4B. These results indicated that SinR is involved in positive stringent transcription control of PkinB.For decoyinine-induced sporulation of the ΔsinR strains for sporulation in NSMP . We could not explain this sporulation phase-dependent variation of β-Gal synthesis without SinR regulation, because SinR repression of kinB transcription and positive stringent transcription control of PkinB did not occur in the ΔsinR strains during sporulation under the cultivation conditions .A mild plateau of β-Gal synthesis around T0 and a clear decrease in it after T1 were observed in all kinB sequence around the Δ5 deletion and the G-45A substitution allowed us to identify a pair of SinR consensus sequences [C1-1 (nt -57/-51) and iC1-2 (-48/-42)] in an inverted repeat orientation (SinR-1 site), each consensus unit containing part of the Δ5 deletion or the G-45A substitution [Figures 4 (Top)]. Therefore, SinR-1 is most likely a SinR-binding site for kinB repression.The consensus DNA binding sequence for SinR comprises a sequence (GTTCTYT) that can be found in inverted and tandem repeat arrangement/orientation and in a monomer state at SinR operator sites . ExaminakinB. Examination of the sequences around the ‘-35’ and ‘-10’ regions revealed another pair of SinR consensus sequences [C2-1 (nt -29/-23) and C2-2 (-14/-8)] in a tandem repeat arrangement (SinR-2 site)[Figures 4 (Top)]. The C2-1 and C2-2 sequences partially overlap the ‘-10’ and ‘-35’ regions of PkinB, which might be possibly a SinR-binding site for positive stringent transcription control of PkinB. We attempted to isolate mutants of strain FU1115 [PkinB (-55/+10)-lacZ] that are defective in positive stringent transcription control of PkinB. We arbitrarily introduced nine base substitutions to the SinR-2 sequence (nt -29/-8) in the PkinB (-55/+10) region , and examined if the strength of each mutant PkinB is comparable to that of the wild-type, and if β–Gal synthesis under the control of the mutant PkinB is positively regulated after decoyinine addition. Thus, we found that only one mutant, FU1249 PkinB (-55/+10 C-26T T-27C) carrying the substitution in the C2-1 consensus sequence of SinR-2, synthesized β-Gal almost at the same level as wild-type strain FU1115, and exhibited partially impaired positive stringent transcription control in comparison with strain FU1115 . Although the other eight substitutions affected the PkinB strength, they did not affect positive stringent transcription control significantly , and (T-18C A-17G) mutations decreased it by several-fold, but did not affect positive stringent transcription control. The G-16A and (C-26T T-25C) mutations considerably enhanced the PkinB activity, but they did not affect positive stringent transcription control significantly. These results suggested that the C2-1 consensus sequence of SinR-2, where the C-26T T-27C substitution only affecting positive stringent transcription control of PkinB is located, is likely involved in its positive stringent transcription control.As described above, SinR is essential for positive stringent transcription control of PΔsinR relieved the repression of kinB transcription involving SinR-1, as described above . It also abolished positive stringent transcription control of PkinB. Thus, we examined the effects of SlrR (kinB repression and positive stringent transcription control of PkinB. We constructed lacZ-fusion strains PkinB (-75/+10) carrying ΔslrR or ΔsinI, and PkinB (-55/+10) carrying ΔsinR, ΔslrR or ΔsinI. As shown in Figure 6A, β-Gal synthesis by strain [PkinB (-75/+10) ΔsinR] was largely relieved from the repression in the wild-type strain PkinB (-75/+10) in sporulation in NSMP medium. ΔslrR did not relieve the repression, but ΔsinI further strengthened it. Thus, kinB repression by SinR seemed hard to be relieved in the absence of SinI. Moreover, ΔsinR also relieved mild repression by SinR which resulted from partial deletion of the C1-1 sequence in PkinB (-55/+10) . Neither ΔslrR nor ΔsinI affected this mild repression. of SlrR , a paral of SlrR , an antakinB (-75/+10) ΔsinR] and [PkinB (-55/+10) ΔsinR] exhibited neither the repression nor positive stringent transcription control of PkinB on decoyinine-induced sporulation in S6 . Strain [PkinB (-75/+10) ΔslrR] exhibited no significant difference in either the repression or positive stringent transcription control in comparison to wild-type strain PkinB (-75/+10) . ΔsinI did not affect positive stringent transcription control of PkinB. But, lacZ expression in strain [PkinB (-75/+10) ΔsinI] was most severely repressed, that is, this strain exhibited the lowest level of β-Gal synthesis before decoyinine addition . On the other hand, the lacZ fusion strains [PkinB (-55/+10) ΔsinI] and [PkinB (-55/+10) ΔslrR] exhibited almost the same level of the repression and positive stringent transcription control of PkinB as the wild-type strain PkinB (-55/+10) (∼1.5-fold increase) . The overall results indicated that SinI deficiency causes stronger SinR-dependent repression and reduces derepression, but SlrR is not involved in the repression, and also indicated that SinR is involved in positive stringent transcription control of PkinB, but SinI and SlrR are not.Strains [PlacZ fusion analysis using ΔsinR as well as Δ5, G-45A, and C-26T T-27C, SinR was found to be responsible not only for kinB repression involving SinR-1 consisting of C1-1 and iC1-2 , but also for positive stringent transcription control of PkinB probably involving SinR-2 consisting of C2-1 and C2-2 . On EMSA analysis using the probes carrying Δ5 and G-45A, and C-26T T-27C, we found that these mutations actually affected in vitro SinR binding to SinR-1 and to SinR-2, respectively, as follows .On Figure 7A-1, the wild-type PkinB (-75/+10) probe gave the two closely located bands on EMSA, which likely resulted from SinR binding to SinR-1 and SinR-2. The upper band is invisible at 12.5 nM SinR, and visible at 25 nM with the PkinB (-75/+10) probe carrying the G-45A substitution, likely resulting from approximately 2-fold less binding affinity to SinR-1 . This band disappeared with the probe carrying the Δ5 deletion or Δ5 and G-45A , suggesting that SinR cannot bind to SinR-1 if part of the C1-1 sequence is deleted by Δ5.As shown in Figure 4C shows that in the wild-type cells with PkinB (-75/+10) during cultivation in S6 medium, as well as in those with PkinB (-75/+10) possessing Δ5 (and G-45A), approximately 1.5-fold positive stringent transcription control of PkinB steadily occurred, regardless of the level of kinB repression before decoyinine addition. The same level of positive stringent transcription control was also observed in the cells of a series of truncation and deletion derivatives of the PkinB region (nt -95/+10 to -55/+10) that exhibited different levels of kinB repression . These results suggested that SinR simultaneously binds to both SinR-1 and SinR-2 to form a larger complex than that on SinR binding to SinR-1 or SinR-2. Nevertheless, a more slowly migrating band other than the two closely located bands did not exist . Thus, the closely located upper and lower bands were considered to probably result from simultaneous SinR binding to SinR-1 and -2, and from SinR binding to SinR-1 or SinR-2, respectively.As described above, Figure 5). EMSA with the probe of PkinB (-55/+10) deleting part of C1-1 of SinR-1 gave only a shifted band most likely resulting from SinR binding to SinR-2 . SinR binding affinity to SinR-2 with the probe carrying the C-26T T-27C substitution  was significantly less than that of the wild-type PkinB (-55/+10) .The C-26T T-27C substitution located in C2-1 partially affected the positive stringent transcription control , and illustrates the covering of the PkinB region by various probes for EMSA. The SinR binding ability to SinR-1 and (or) SinR-2 of the probes  is given in the right columns. SinR bound to SinR-1 and SinR-2 of the PkinB (-75/+10) probe, but it did not bind to SinR-1 of its mutant derivatives . SinR bound to SinR-2 of the PkinB (-55/+10) probe, but it only partially bound to its mutant derivative . SinR bound to SinR-1 of PkinB (-124/–38) (Rm0) and SinR-2 of PkinB-39/+104) (Fm0)  probe but not to the PkinB (-75/-17 Δ5 G-45A) probe  probe but not to the PkinB (-20/+104) probe  allowed us to conclude that both C1-1 and iC1-2 of SinR-1 are necessary for SinR binding to SinR-1, whereas both C2-1 and C2-2 are necessary for SinR binding to SinR-2.kinB (-75/+10) and PkinB (-75/-7) carrying Δ5 G-45A gave an apparent equilibrium dissociation constant (Kd) of approximately 10 nM for SinR binding to SinR-2 , PkinB (-31/+104), and PkinB (-39/+104) (Fm0) probes gave Kd of more than 100 nM for SinR binding to SinR-2  and the wild-type one (Rm0) were used, as illustrated on the left side of Figure 9. The EMSA results as to Rm0 and Rm1 to Rm6 are shown in the upper panel of Supplementary Figure Figure 9. The EMSA data as to Fm0 and Fm1 to Fm6 are shown in the upper panel of Supplementary Figure For EMSA of SinR binding to SinR-2, the mutant probes  and the wild-type one (Fm0) were used, as illustrated on the right side of Figure 9) indicated that SinR-binding to SinR-2 only partially requires C2-1 but it well requires C2-2. The EMSA results with deleted probes as described above  and its complete elimination . However, the role of AGT just upstream of C2-2 in SinR binding to SinR-2 is unknown. These EMSA results suggested that both C2-1 and C2-2 are likely necessary for SinR binding to SinR-2, although C2-1 might not be so strictly required in comparison with C2-2. Thus, the SinR binding site (SinR-2) likely comprises the two SinR consensus of C2-1 and C2-2 sequences in a tandem arrangement, which partially overlap the ‘-35’ and ‘-10’ regions of PkinB, respectively.EMSA analysis with the mutant probes , so it was expected that SinR might bind to SinR-2 to form a transcription initiation complex of SinR, RNAP, and PkinB to exert its positive stringent transcription control. As shown in Figure 10, the electrophoretic band of a complex of the PkinB probe and RNAP appeared to shift to a slightly slower position when SinR was further added. This implies that a positively regulated stringent promoter such as PkinB might form a transcription initiation complex with SinR and RNAP for its positive stringent transcription control.We found that SinR-2 consists of C2-1 and C2-2 in tandem arrangement, which is likely involved in positive stringent transcription control of PE. coli, the target of ppGpp is RNAP, stringent genes being regulated positively and negatively, depending on their specific promoter sequences. In contrast, the ppGpp target is GMP kinase in B. subtilis, the in vivo GTP concentration being reduced  disclosed that positive stringent transcription control has a larger contribution to kinB expression in decoyinine-induced sporulation in minimal S6 medium than in sporulation in nutrient NSMP medium. Both kinB repression by SinR and SinR-dependent positive stringent transcription control of PkinB simultaneously occur, as inferred from that approx. 1.5-fold positive stringent transcription control of PkinB was constantly and steadily observed after decoyinine addition to the S6 culture, regardless of the level of kinB repression before decoynine addition .Decoyinine induces sporulation of  sources . It is k sources , 1982. Rrulation , which msinR strain exhibits the sporulation-deficient phenotype when present in multiple copies . SinR was also involved in positive stringent transcription control of PkinB . SinI is an antagonist of SinR . SrlR, a protein homologous to SinR .The e copies . The Δsi by SinR  . Thus, in the wild-type SinR+ cells, the limited level of derepression of kinB from SinR repression by SinI induced by Spo0A∼P and significant induction of SinR-dependent positive stringent transcription control of PkinB upon stringent response cooperatively induce effective sporulation. It is inferred from the results  that the level of kinB expression on sporulation of the wild-type strain is likely lower than that on sporulation of the ΔsinR strain even if positive stringent transcription control is blocked by ΔsinR. This might be the reason why the ΔsinR strain sporulated a little bit better than the wild-type strain.It should be noted that the relief from kinB region revealed two SinR binding sites (SinR-1 and SinR-2), i. e. a pair of SinR consensus sequences (C1-1 and iC1-2) in an inverted orientation, and another pair of SinR ones (C2-1 and C2-2) in a tandem arrangement, respectively . Such SinR-binding motifs consisting of a pair of SinR consensus sequences in an inverted orientation and a tandem arrangement are often observed in the promoter regions of the operons involved in biofilm formation such as espA-O  and EMSA using various deleted and mutated probes  revealed that both C1-1 and iC1-2 are necessary for kinB repression and SinR binding to SinR-1. Moreover, the base substitution in C2-1, which was involved in positive stringent transcription control of PkinB , also affected SinR binding to SinR-2 . EMSA using various deleted and mutated probes  suggested that both C2-1and C2-2 are necessary for SinR binding to SinR-2.Examination of the sequence of the Ps espA-O  and tapAipW-tasA . In vivosinR deletion (ΔsinR) abolished positive stringent transcription control of PkinB . lacZ-fusion analysis of the other stringently-controlled promoters (unpublished observation by S. Nii and Y. Fujita) indicated that ΔsinR also abolished the positive stringent transcription control of PilvB, PpycA, and PkinA. Interestingly, ΔsinR did not affect the negative stringent transcription control of PptsG and PpdhA. This observation suggested that positive stringent transcription control involves SinR, but negative stringent transcription control does not involve it. EMSA analyses  showed that the PkinB region actually possesses an SinR-binding site (SinR-2), i. e. a pair of C2-1 and C2-2 sequences partially overlapping the ‘-35’ and ‘-10’ regions, respectively, which is likely involved in positive stringent transcription control of PkinB . Furthermore, EMSA indicated that a complex of RNAP, SinR, and PkinB for transcription initiation is likely formed, implying that SinR might be involved in transcription initiation of positively controlled stringent genes . Detailed investigation of the molecular mechanism involving SinR underlying positive stringent transcription control is in progress.The in vivo study of kinB regulation by SinR in B. subtilis from April 2015 to March 2017 in Fukuyama University. YF moved from Fukuyama University to Tokai University April 2017. YF and MO (Tokai University) performed in vitro study of this work from April 2016 to July 2017.YF, SN, and KH performed 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.