Engineered DNA-binding molecules such as transcription activator-like effector (TAL or TALE) proteins and the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) (CRISPR/Cas) system have been used extensively for genome editing in cells of various types and species. The sequence-specific DNA-binding activities of these engineered DNA-binding molecules can also be utilized for other purposes, such as transcriptional activation, transcriptional repression, chromatin modification, visualization of genomic regions, and isolation of chromatin in a locus-specific manner. In this review, we describe applications of these engineered DNA-binding molecules for biological purposes other than genome editing. I-SceI and engineered DNA-binding molecules such as zinc finger (ZF) proteins, transcription activator-like effector (TAL or TALE) proteins, and the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) (CRISPR/Cas) system has enabled easier genome editing in target genomic regions in cells of various types and species [Genome editing has been used extensively in multiple fields of biology to knock in or knock out genes of interest. The recent development of meganucleases such as  species ,2,3. Suc species ,2,3. MorZF proteins are the prototypes of engineered DNA-binding molecules, but technical expertise and experience are required for optimal selection and assembly of their DNA-binding modules to generate functional DNA-binding domains. Consequently, ZF proteins have not been widely used for genome editing and other applications. By contrast, design of DNA-binding domains using TAL proteins or the CRISPR/Cas system is more straightforward, which is one reason why both molecules are in wider use today. In light of the current situation, in this review, we describe various applications of TAL proteins and the CRISPR/Cas system to biological research purposes other than genome editing.The discovery and development of TAL proteins and the CRISPR/Cas system as genome editing tools have been described in detail in previous reviews ,5,6,7,8.Xanthomonas sp. This protein typically recognizes specific DNA sequences, about 20 bases in length, via a DNA-recognition domain consisting of assembly of DNA-binding modules corresponding to each nucleotide. Each DNA-binding module consists of a conserved repetitive region of 33–35 amino acids, of which the 12th and 13th amino acid positions  are decisive in recognition of the cognate nucleotide. Thus, by combining the corresponding DNA-binding modules, one can construct TAL proteins that recognize specific DNA sequences of interest. To efficiently assemble the DNA-binding modules, the Golden Gate assembly system has been widely used [Fok I have been used as TALEN, a locus-specific genome editing tool.TAL is a transcription factor of plant pathogenic bacteria such as ely used . In addiely used . TAL proStreptococcus pyogenes (S. pyogenes)) and the immediately upstream 20 bases complementary to the gRNA. Following DNA recognition, Cas9 cleaves the DNA sequence via its intrinsic nuclease activity. For genome editing and other purposes, the CRISPR/Cas system from S. pyogenes has been used most often. The original gRNA is composed of two RNA molecules: the CRISPR RNA (crRNA), possessing the sequence complementary to the target DNA sequence, and the trans-activating crRNA (tracrRNA). One can target a given genomic site by designing an RNA sequence complementary to a 20-base DNA sequence 5′-adjacent to the PAM. Recently, a single chimeric guide RNA (sgRNA) mimicking the structure of the annealed crRNA/tracrRNA has become more widely used than crRNA/tracrRNA, because the former approach entails a simplified system with only two components. sgRNA expression vectors can be constructed time- and cost-effectively using standard methods of genetic engineering. A catalytically inactive form of Cas9 (dCas9), in which two point mutations (D10A and H840A) are introduced, has been widely used for biological research purposes other than genome editing , was discovered as a component of the bacterial adaptive immune system ,11,12. Tting see .S. pyogenes Cas9 (~4.2 kbp) might be problematic to be packaged into routinely used delivery vehicles such as adeno-associated virus. In this regard, use of smaller Cas9 orthologues  can overcome this disadvantage [CRISPR/Cas has one major limitation, namely, that a PAM must be adjacent to the target sequences. By contrast, because TAL does not have such strict sequence limitations, the target DNA sequences can be chosen more flexibly. However, a recent report showed that the specificity of PAM recognition by CRISPR/Cas can be altered intentionally , potentidvantage .One of the major applications of TAL and CRISPR/Cas other than genome editing is locus-specific artificial regulation of transcription to dissect the physiological importance of gene products or manipulate biological outputs such as cellular differentiation or reprogramming. In this section, we describe various modes of artificial transcriptional regulation using TAL and CRISPR/Cas .NTF3 gene in human 293 cells [NTF3 promoter region, induced production of high levels of NTF3 transcript and protein. Likewise, a TAL protein fused with VP64, which is composed of four tandem copies of VP16, was used as a TALa in another study [SOX2, KLF4, c-MYC and OCT4, by 40-fold in an exogenous reporter gene system in 293FT cells. In addition, the endogenous SOX2 and KLF4 transcripts could also be elevated several-fold using TALa proteins that targeted their promoters directly. Although TALa targeting the epigenetically silenced proximal promoter region did not activate Oct4 transcription in mouse neural stem cells [To establish an artificial transcriptional activator, a TAL protein was linked to the activation domain of VP16, a transcriptional activation protein of herpes virus , and useer study . In thisem cells , a TALa em cells . These ret al. [REX1, OCT4, SOX2, and NANOG. By contrast, when multiple (~10) sites in these genes were targeted simultaneously, strong induction of endogenous gene transcription was detected as a result of synergistic effects on transcriptional activation. In Escherichia coli (E. coli), dCas9 fused with the ω subunit of RNA polymerase, which promotes transcription by recruiting RNA polymerase [green fluorescent protein (GFP) gene and the endogenous lacZ gene [CRISPR/Cas has also been applied to locus-specific transcriptional activation. First, Mali et al.  generatelymerase , was useacZ gene . Thus, CREX1 as efficiently as dCas9-VP64/sgRNA.In the CRISPR/Cas-based locus-specific transcriptional regulation system, transcriptional regulators can be tethered not only to dCas9, but also to sgRNA. For example, RNA stem loop structures that interact with the MS2 bacteriophage coat protein (MS2 or MCP) were linked to the 3′ end of sgRNA to recruit two copies of VP64-fused MS2 (MS2-VP64)  Figure a. The reet al. [et al. [Zalatan et al.  also utiet al. , Konerma [et al.  develope [et al. .CXCR4 and CDKN1B genes [When TALa or CRISPRa simultaneously targets multiple (~10) sites in a gene, it can exert synergistic effects on transcriptional activation, even if each individual targeted site barely induces transcriptional activation ,28,29,301B genes . The ind1B genes . Thus, rIn another synergistic transcriptional activation system, dCas9-VP64 was utilized together with a scaffold RNA system in which sgRNA 2.0 recruits MS2 fused to the NF-κB trans-activating subunit p65 and human heat-shock factor 1 (HSF1), which act as transcriptional activators . This syThus, the SunTag, SAM, and VPR systems are more effective than the single activator fusion system such as dCas9-VP64. Notably, transcriptional activation can be induced robustly only when the basal level of transcription of a target gene is low ,32, suggSOX2 and the miR-302/367 cluster in mammalian cells [c-kit and PU.1 genes was also achieved in mouse bone marrow cells by combinatorial use of two kinds of TAL-KRAB targeting the proximal promoter region of each gene [Knockout and knockdown strategies have been extensively used for loss-of-function experiments to address the physiological roles of products encoded by target genes. As mentioned briefly, TALEN and CRISPR/Cas have been widely used for knockout experiments. On the other hand, TAL or CRISPR/Cas fused to transcriptional repressors can also be used to induce loss of function b. To devan cells ,34. Doubach gene . After tet al. [E. coli and mammalian cells. In this approach, called CRISPR interference (CRISPRi), the dCas9/sgRNA complex is tethered to the coding region or regulatory sites bound to transcriptional regulators such as transcription factors in order to interfere with transcriptional initiation or elongation by RNA polymerase II on the target gene  than conventional expression of dCas9/sgRNA in mammalian cells. CRISPRi was also feasible in Saccharomyces cerevisiae; in this case, dCas9 was fused to Mxi1, a mammalian transcriptional repressor domain bound to the yeast homolog of the histone deacetylase Sin3 [Qi et al.  applied get gene b. CRISPR or the Nanog gene (~5.0 kbp) was targeted. Accordingly, active epigenetic marks at each region were more highly enriched by TALa. By contrast, the impact of transcriptional repression was comparable or higher when using CRISPRi. CRISPRi, but not TAL-KRAB, interrupted recruitment of transcriptional activators such as KLF4 and NANOG to their binding sites in the Nanog enhancer. Although the target sequences of the CRISPR/Cas-mediated system did not completely overlap with those of the TAL-mediated ones, due to the restriction of the PAM sequence, these results imply that when a single site is targeted with a single transcriptional effector, the TAL-mediated system is more suitable for transcriptional activation whereas the CRISPR-mediated system is more effective for transcriptional repression.Gao et al.  comparedTbox3 gene, which encodes a protein required for maintenance of pluripotency, effectively suppressed the gene expression and prevented maintenance of pluripotency via reduction of the H3K4me2 and H3K27ac marks on the enhancer regions. To develop another locus-specific histone modifier, dCas9 was fused to the catalytic histone acetyltransferase core domain of human E1A-associated protein p300 (dCas9-p300) [Because epigenetic marks on promoter regions or distal regulatory regions are associated with the transcriptional states of the relevant genes, artificial alteration of epigenetic marks can lead to desired alterations of gene transcription. On the basis of this concept, TAL- or CRISPR/Cas-mediated epigenetic modifiers have been developed c. For exs9-p300) . By incrs9-p300) .KLF4, RHOXF2, and HBB and restored expression of their gene products. In cancer biology, TAL-TET1 or TAL fused to DNA methyltransferase DNMT3A (TAL-DNMT3A) was used to manipulate expression of endogenous CRMP4, a suppressor of metastasis [CRMP4 promoter in metastatic PC3 cells, thereby attenuating metastasis. Inversely, TAL-DNMT3A induced CpG methylation of the unmethylated CRMP4 promoter in non-metastatic 22Rv1 cells, causing metastasis to occur.CpG methylation, an epigenetic mark associated with gene silencing, is maintained by DNA methyltransferases in mammalian cells . By conttastasis . TAL-TETThus, locus-specific alteration of epigenetic marks is a powerful tool for investigating the physiological significance of specific epigenetic marks and achieving artificial transcriptional modification of the relevant genes. In some cases, TAL or dCas9 exerted stronger effects on transcription when fused to an epigenetic modification enzyme than when fused to a transcriptional activator/repressor such as VP16 or KRAB ,43, suggIt is reported that dCas9 binds to off-target sites ,48,49,50E. coli [Inducible protein expression or binding systems can be combined with the TAL- or CRISPR/Cas-mediated locus-specific transcriptional activation/repression system to achieve spatiotemporal modification of target transcription. For example, CRISPR/Cas-mediated locus-specific transcriptional activation/repression can be reversibly turned on and off when the expression of dCas9 and/or sgRNA is controlled by anhydrotetracycline-inducible promoters in E. coli  or galacE. coli .Another method for inducible transcriptional activation using TAL proteins is an optogenetic two-hybrid system, light-inducible transcriptional effectors (LITEs) . The LITRobust transcriptional activation by TALa or CRISPRa can be easily achieved for exogenous reporter genes, but is difficult for some endogenous genes that are not transcribed in the cell of interest ,20. ThisOct4 promoter had several-fold higher transcriptional activity than the corresponding TALa recognizing unmodified cytosine [OCT4 in 293T cells [Oct4 transcription were restored to a level similar to that in mouse ES cells [DNA recognition by TAL is susceptible to CpG methylation in the binding site ,59, suggcytosine . Another3T cells . In addiES cells .It remains unknown whether CRISPR/Cas-mediated transcriptional activators are influenced by CpG methylation in their recognition sites, although CRISPR/Cas can cleave CpG-methylated sites . Future et al. [et al. [EGFR, PCDH7, ITGB5, ARHGEF1, BRCA3, GPR35, and TFAP2C, responsible for PLX-4720 resistance. These results demonstrate that the CRISPR/Cas-mediated transcriptional regulation system in combination with an sgRNA library represents a powerful approach for genome-wide gain- or loss-of-function screening in various fields, including medical research.CRISPR/Cas9-mediated transcriptional regulation systems have been applied to genome-wide screening for genes essential for cell growth, differentiation, malignant progression of disorders, or drug resistance ,62. Gilb [et al.  utilizedLacZ gene [VioABEDC) [VioA, VioD and VioC genes increased the fluxes into branched metabolic pathways and subsequent production of the corresponding compounds .One potential goal of TAL- or CRISPR/Cas-mediated locus-specific transcriptional regulation is manipulation of biological outputs such as metabolic fluxes, cellular differentiation, or cell fate reprogramming. In this context, CRISPRi was used to knock down the genes encoding components of the lactose regulatory pathway and thereby perturb expression of the downstream acZ gene . CombineioABEDC)  in yeastioABEDC) . CRISPRiSOX2, KLF4, c-MYC, and OCT4 [OCT4 by CRISPRi led to morphological changes in human ES cells [NEUROG2 or NEUROD1 gene were used together, CRISPRa was able to induce differentiation of iPSCs into iNs [On the other hand, TAL- or CRISPR/Cas-mediated locus-specific transcriptional regulation has been used to activate transcription of pluripotency genes including and OCT4 ,20,39,60and OCT4 ,65. KnocES cells , suggestES cells ,68. Wheninto iNs , demonstin vivo. To this end, fluorescence in situ hybridization (FISH) has been employed as a locus imaging technology [Arabidopsis and major satellite regions in NIH3T3 cells, respectively. Next, a locus-specific visualization technology using TAL proteins was developed, named TAL-mediated genome visualization (TGV) [Visualization of specific genomic regions is required for observation of spatiotemporal dynamics of chromosomal organization and segregation chnology . Althougchnology ,71,72; achnology . By targon (TGV)   . Taking on (TGV) .More recently, a CRISPR/Cas-based visualization system was developed  Figure . In thisThus, TAL- or CRISPR/Cas-based visualization systems are ideal tools for locus-specific live imaging. However, simultaneous targeting of multiple sites in a coding region for the purpose of locus visualization may interfere with transcription via CRISPRi effects . In suchet al. [in vitro when the PAM is presented in trans as a separate DNA oligonucleotide  containing the PAM sequence. Recently, O’Connell et al.  showed tcleotide . In this  that interact with genomic regions of interest in vivo. To this end, we developed two locus-specific chromatin immunoprecipitation (locus-specific ChIP) technologies, insertional ChIP (iChIP) [Last, we describe applications of TAL proteins or the CRISPR/Cas system to locus-tagging strategies and subsequent isolation of the tagged loci from cells. Genome functions such as transcription are regulated spatiotemporally in a locus-specific manner. To understand the molecular mechanisms of such genome functions, it would be ideal to comprehensively identify the molecules  ,84,85,86 (iChIP) ,89,90,91Locus-specific ChIP consists of a locus-tagging strategy and subsequent affinity purification of the tagged loci. In practice, exogenous DNA-binding molecules such as the bacterial DNA-binding protein LexA and its binding DNA elements are utilized for locus-tagging in iChIP ,84,85,86interferon regulatory factor 1 (IRF-1), a gene whose expression is induced by interferon γ (IFNγ) stimulation [Neat1 were identified as novel telomere-binding RNAs. RNA-FISH confirmed that these identified RNAs were partially co-localized with telomere regions [in vivo. In principle, it should be possible to use enChIP to identify interacting chromosomal regions, as well as proteins and RNAs  Compare different conditions for an engineered DNA-binding molecule ; (2) Compare several different engineered DNA-binding molecules for each target genomic region . These controls may also be useful for aforementioned applications such as artificial transcriptional/epigenetic regulation, live imaging, and RNA analysis.Taken together, the findings to date demonstrate that enChIP using TAL or CRISPR/Cas is a feasible tool for identifying genome-interacting molecules in a locus-specific manner and dissecting the molecular mechanisms underlying genome functions. In the near future, such approaches should make it possible to achieve genome-wide mapping of genome-interacting molecules.In this review, we described various applications of engineered DNA-binding molecules such as TAL and CRISPR/Cas other than genome editing, based either on the fusion of these proteins with restriction endonucleases or their intrinsic nuclease activity. Locus-specific transcriptional regulation enables elucidation of the physiological relevance of products transcribed from the targeted genes, as well as manipulation of biological outputs such as cellular differentiation or reprogramming. Locus-specific visualization and locus-specific ChIP technologies enable dissection of spatiotemporal dynamics of chromosomal organization and/or molecular mechanisms of genome functions. RNA-specific analysis may be useful for dissection or manipulation of the physiological functions of RNAs. One advantage of CRISPR/Cas over other systems is the easier design and construction of sgRNAs. Therefore, although applications of TAL proteins preceded those of CRISPR/Cas, CRISPR/Cas can be expected to emerge as the predominant technology for future applications.


Regulation of gene expression requires both transcription factor (TFs) and epigenetic modifications, and interplays between the two types of factors have been discovered. However study of relationships between chromatin features and TF–TF co-occupancy remains limited. Here, we revealed the relationship by first illustrating distinct profile patterns of chromatin features related to different binding events, including single TF binding and TF–TF co-occupancy of 71 TFs from five human cell lines. We further implemented statistical analyses to demonstrate the relationship by accurately predicting co-occupancy genome-widely using chromatin features including DNase I hypersensitivity, 11 histone modifications (HMs) and GC content. Remarkably, our results showed that the combination of chromatin features enables accurate predictions across the five cells. For individual chromatin features, DNase I enables high and consistent predictions. H3K27ac, H3K4me 2, H3K4me3 and H3K9ac are more reliable predictors than other HMs. Although the combination of 11 HMs achieves accurate predictions, their predictive ability varies considerably when a model obtained from one cell is applied to others, indicating relationship between HMs and TF–TF co-occupancy is cell type dependent. GC content is not a reliable predictor, but the addition of GC content to any other features enhances their predictive ability. Together, our results elucidate a strong relationship between TF–TF co-occupancy and chromatin features. Transcriptional regulation exists at both genetic and epigenetic levels. Binding of transcription factors (TFs) to specific DNA sequences is a pivotal step in the control of gene expression. Studies of sequence-associated TF binding preferences have led to the development of sequence-specific Position Weighted Matrix (PWM)  and posiEpigenetic regulation refers to the alteration of DNA accessibility to TFs coordinately with chemical modifications of chromatin . This prcis-regulatory modules , HMs and TF binding events by taking advantage of the wealth of data from the ENCODE project . Statisthttp://genome.ucsc.edu/ENCODE/downloads.html) (All of the data used in this study were downloaded from the ENCODE project (ds.html) . The ENCds.html) , in bothGenome-wide profiles of HMs, including H3K9ac, H3K27ac, H3K4me3, H3K4me2, H3K4me1, H3K79me2, H3K9me3, H3K27me3, H3K36me3 and H4K20me1, and the histone variant, H2A.z, were generated using the ChIP-Seq technique .Genomic locations of 40 193 genes with all information were extracted from the human genome version hg19, obtained from the RefSeq database . Of note, using different overlapping criteria, such as 40% overlapped ChIP-Seq peaks, or other tools, such as IntervalStats  mapped into the 100-bp region to compute methylation level at this TFBS. For 100-bp regions centered at TFBSs with more than one CpG site, the average of methylation levels over these mapped CpG sites was selected to represent the methylation level.We have examined the HMs, DNase I and GC content in the 100-bp region centered at TFBSs (ChIP-Seq peak summits), by counting and normalizing the number of reads mapping to this region to calculate RPKM values. The TF–TF and TF-only binding sites (e.g. CTCF–YY1 versus CTCF-only) were selected as ‘positive’ and ‘negative’ datasets, respectively.We used two non-linear classifiers and two linear methods to build the chromatin models for studying the correlations between TF–TF co-occupancy and chromatin features Figure . The twoIn each cell line, we randomly selected two-third of positive and negative datasets as training and the rest as testing. The ability of chromatin model to distinguish TF–TF co-occupancy from TF-only binding events was assessed by examination of receiver operator characteristic (ROC) curves, plotting the true-positive rate versus the false-positive rate. To test the stability of these predictions, the above procedure was repeated 10 times and the means of the area under ROC curves (AUC) and prediction accuracy (ACC) values were computed to represent the prediction accuracy.The learned models can be applied to different cell lines for the cross-cell type testing purpose. During this process, the model learned for one pair of TFs was used to other cells for the same TF–TF pair. The prediction accuracies were evaluated by the calculations of AUC and ACC values.TFs account for ∼10% of proteins encoded by human genes  and theiA TF may share its binding regions with its partner, namely TF–TF co-occupancy. For instance, CTCF–YY1 co-occupancy refers to the regions co-occupied by CTCF and YY1. Consequently, we defined the genomic regions bound by CTCF but not YY1 as CTCF-only events, and the regions bound by YY1 but not CTCF as YY1-only events . For each TF–TF pair, there are various numbers of co-occupying and solely binding events . The co-occupying TFs preferably bind at specific genomic regions . Genome-wide analyses of TFBSs revealed that CTCF intends to bind at gene bodies; while its partner, YY1, as an example, prefers to regulate its target genes by binding at proximal promoters. When considering co-localized CTCF and YY1, their co-occupied regions are mainly distributed in the gene promoters . In our analysis, we considered the genome-wide TFBSs.De novo binding motifs analyses of each TF–TF pair  illustrated a similar sequence preference for TFs involved in both co-occupying and solely binding regions , indicating that DNA sequence may not be a determinant for the TF–TF co-occupancy.By aligning the profiles of DNA sequences, DNase I and HMs, we were able to examine the chromatin features for TF solely and TF–TF co-occupying binding events. t-test P < 1e-15; see Supplementary Materials; Figure Analyses of GC content , which dictates nucleosome depletion at mammalian promoters with GC-richness benefiting TF binding , showed Analyses of chromatin features also revealed distinct profiling patterns for different binding events . The HM patterns at TFBSs were characterized by 11 types of histone methylation and acetylation , which are associated with transcriptional activation, suppression or both ,36,45. Dt-test P < 0.05; see Supplementary Materials; Figure We did the same analysis for other TF–TF pairs. Similar enrichment patterns were observed when comparing chromatin feature profiles between TF–TF co-occupancy and TF-only events (student's 2-transformed RPKMs) of chromatin features within the 100-bp window centered at TF peak summits  and YY1–CTCF (distinguished from YY1-only) binding events, in the GM12878 cell line Figure . High prWhen using individual or a subset of chromatin features as predictors, the prediction accuracies obtained from DNase I and combined 11 HMs are closed to that using all features as a whole Figure ,53 and HGC content is another valuable predictor. GC content patterns are not significantly different, especially in the YY1–CTCF and YY1-only comparisons, therefore GC content itself does not enable accurate predictions. However, addition of GC content to any other features enhances their prediction capability . The linear model with NB and LDA gave accurate predictions from individual chromatin features, such as DNase I, which were similar to the results obtained from non-linear models. However, the prediction accuracy from combined chromatin features, especially using 11 histone marks as a predictor, was low , indicating a non-linear relationship of HMs with TF–TF co-occupancy. These results were consistent with the relationship between epigenetic modifications and individual TF binding . Since tThe SVM classifiers were trained and tested for other types of TFs for prediction of CTCF-TF/TF-CTCF co-occupancy. The results showed that, chromatin features are informative of binding events, with mean AUC values 0.90 and 0.72, 0.89 and 0.73, 0.83 and 0.73, 0.88 and 0.75, and 0.90 and 0.74 in the A549, H1-hESC, HepG-2 and K562 cells, for both CTCF–YY1 and TF-CTCF co-occupancy, respectively , and the co-localizations of another six TFs, including ATF3, GABP, NRSF, POL2, USF1 and YY1. The latter test involved 15 binding pairs, as shown in Supplementary Table S8. Enrichment analyses of GC content, DNase I and 11 HMs showed distinct patterns between binding events, illustrated by the given examples of SP1-BCL11A and SP1-TAF1 .We consequently constructed and tested the computational models for each TF–TF pair in each cell type. High prediction accuracies were generally obtained. For instance, all chromatin features as a whole were able to accurately predict the GABP-USF1 co-occupancy with AUC values >0.86 in the GM12878 and K562 cell lines . For the prediction of SP1-TF combinational binding events, the average accuracies were ∼0.80, 0.77, 0.80 and 0.80 in the GM12878, H1-hESC, HepG-2 and K562 cells, respectively . For the 15 TF–TF co-occupancy with ATF3, GABP, NRSF, POL2, USF1 and YY1, chromatin features enable highly accurate predictions with AUC values >0.8 in all of five cell types .The prediction abilities with a subset of or individual chromatin features for above TF–TF co-occupancy were similar to those for CTCF-TF co-occupancy. In general, DNase I and the combination of 11 HMs are able to achieve high prediction accuracy and addition of GC content enhances their performance . H3K27ac, H3K4me 2, H3K4me3 and H39Kac perform better than other HMs . This is consistent with our previous findings about the contribution of single HM to binding affinity of individual TFs .The models were trained on each cell line and then applied to the other four cell lines to test their prediction abilities. The results indicated that the cross-cell line applications of classifiers do not reduce their performances in predictions. For example, when we applied the models trained in the GM12878, H1-hESC, HepG-2 and K562 cell lines to the A549 cell for CTCF–YY1 predictions, the average prediction accuracies were 0.86, 0.77, 0.88 and 0.89, respectively, compared to 0.91 using the model trained by the A549 cell itself . Consistent with our results from CTCF–YY1 studies, crossing-cell line applications achieved satisfactory accuracies . The biggest changes were seen when models from other four cell lines were applied to H1-hESC and We observed that the same features had different prediction powers across cell lines . Of note, for some TF–TF pairs, such as SP1-ATF2, DNase I did not enable highly accurate predictions. In summary, all results suggest that the correlations between TF–TF occupancy and DNase I/GC content are more conserved than HMs across cell lines.The accurate regulation of gene expression involves in a complicated interplay between TF, histone modifying enzymes and other factors. The relative importance of epigenetic modification and TF bindings in the regulation of gene expression is still under debate. Statistics analysis has revealed that these two factors regulate gene transcription in a highly coordinate manner , and arecobindR software , which t-test P < 1e-17 in the five cell lines; Supplementary Figure S23), indicating their differentially functional effects. By profiling co-occupancy of CTCF–YY1 and other TF–TF pairs with chromatin features, we demonstrated the important roles of chromatin modifications in gene regulation and the strong associations between genetic and epigenetic regulations.The co-occupied TFs may have different regulation functions from solely bound TF. For example, co-localizations of CTCF and YY1 can enhance transcriptional activity of genes that they are co-occupied . AnalysiOur analysis further illustrated the generalization of this correlation across cell types, which led to the possible application of a prediction model trained from one cell line using combination of chromatin features to other cells for accurate predictions of TF–TF co-occupancy. When applied individual chromatin features in our models, the associations of DNase I with TF–TF co-occupancy were very conserved, and the cross-cell type applications of models with DNase I did not result in dramatically changes of prediction accuracy. This observation suggested that, although DNase I shows distinct profiling patterns in different cell types , these pP-values < 10e-8; Supplementary Figure S24A), consistent with the previous findings (TF–TF co-occupancy may have an effect on transcriptional output. Comparisons of the expression levels for RefSeq genes showed that the genes overlapped YY1-only binding events are significantly more highly expressed, followed by genes overlapped with CTCF–YY1 binding events, in contrast to genes overlapped CTCF-only binding events (Wilcoxon rank-sum test, findings . This obet al. showed that, even for the similarly expressed genes, the distribution of DNase I may differ among different chromosomes (We further tested whether transcriptional output has relationship with both TF–TF co-occupancy and chromatin features, such as DNase I. Comparisons showed that, although either CTCF–YY1 co-occupancy or DNase I is associated with transcriptional activity ,19,68, tomosomes .DNA methylation is another type of epigenetic modification involved in the regulation of gene expression, cell growth and disease development ,72. EarlTFs prefer working together to regulate gene expression by targeting the same genomic regions, namely TF hotspots ,22,28,77In summary, we have presented a statistical and computational approach to investigate the complicated interplays between genetic and epigenetic regulations of gene expression. Although our analysis cannot demonstrate the relative importance or causative role of TFs or chromatin features in transcriptional regulation, we have elucidated a strong relationship between TF–TF co-occupancy and various chromatin features through a large-scale statistical and computational experiments, which will help us in understanding the mechanisms of combinational regulatory landscape.Supplementary Data are available at NAR Online.


Arabidopsis thaliana it is estimated that approximately 10% of all genes encode TFs or TF-like proteins. It is important to identify target genes that are directly regulated by TFs in order to understand the complete picture of a plant’s transcriptome profile. Here, we investigate the role of the LONG HYPOCOTYL5 (HY5) transcription factor that acts as a regulator of photomorphogenesis. We used an in vitro genomic DNA binding assay coupled with immunoprecipitation and next-generation sequencing (gDB-seq) instead of the in vivo chromatin immunoprecipitation (ChIP)-based methods. The results demonstrate that the HY5-binding motif predicted here was similar to the motif reported previously and that in vitro HY5-binding loci largely overlapped with the HY5-targeted candidate genes identified in previous ChIP-chip analysis. By combining these results with microarray analysis, we identified hundreds of HY5-binding genes that were differentially expressed in hy5. We also observed delayed induction of some transcripts of HY5-binding genes in hy5 mutants in response to blue-light exposure after dark treatment. Thus, an in vitro gDNA-binding assay coupled with sequencing is a convenient and powerful method to bridge the gap between identifying TF binding potential and establishing function.Several transcription factors (TFs) coordinate to regulate expression of specific genes at the transcriptional level. In  Arabidopsis thaliana it is estimated that approximately 10% (~3000) of all genes encode TF or TF-like proteins based on the TAIR (The Arabidopsis Information Resource) annotation. TFs often play central roles in developmental phases during the formation of organs and tissues. Moreover, in response to environmental changes, several TFs coordinate to regulate the expressional switch of specific genes and enable the organism to adapt to its modified surroundings. For example, APETALA2 TF guides proper flower and fruit development [Changes in gene expression profiles induced by environmental stimuli or developmental phase shifts are controlled through specific multiple mechanisms at the transcriptional, post-transcriptional and translational levels. Transcription is positively or negatively controlled by several transcription factors (TFs) ,2. TFs wely 10% ~000 of alelopment , while telopment ,6,7,8.in vitro. Protein-gDNAs are immunoprecipitated and then gDNA that was bound to the proteins is hybridized on a tiling array [in vivo binding motifs and genomic positions.Since these TFs may form complicated networks in order to execute their function, it is important to identify the target genes that are regulated by TFs in order to understand the environmental response of the whole plant. Chromatin immunoprecipitation (ChIP) followed by microarray hybridization (ChIP-chip) or next-generation sequencing (ChIP-seq) has generally been used to identify genes targeted by TFs ,10,11,12ng array . This meArabidopsis mutant of the HY5 gene is insensitive to light and shows a long hypocotyl phenotype under different lights including red, far-red and blue lights [in vitro binding assays that HY5 recognizes ACGT-containing sequence motifs [LONG HYPOCOTYL5 (HY5) is a bZIP-type transcription factor that extensively regulates photomorphogenesis through binding to light-inducible or light-repressed genes in plants ,15,16. He lights . Previoue lights . In addie motifs .in vitro DNA binding assays are often produced using a protein synthesis system in E. coli but this system requires different codon usage from eukaryotes and gives no post-translational modifications. Wheat germ extract is a powerful cell-free tool to synthesize large amounts of eukaryotic proteins from in vitro transcribed mRNAs [in vitro gDNA binding assay like the DIP method using HY5 protein produced by the wheat germ extract system as a model TF and performed next-generation sequencing to identify TF-binding regions. We also show the relationship between the binding targets and the blue light response revealed by RNA-seq. Here, we demonstrate a powerful use of an established method to reveal the gDNA binding potential of TFs.TFs for ed mRNAs ,21. Protin vitro transcription and translation in wheat germ extract (WEPRO7240H Expression kit) using the Protemist-DT II, an automatic machine (Cell Free Science), was performed according to manufacturer’s instructions. Protein synthesis was performed in a 6 mL volume. After purification of the proteins the solvent  was replaced with storage buffer  pH 7.4, 10% glycerol, 5 mM EDTA) using Amicon Ultra Centrifugal Filters (Ultracel-10K) (Millipore) to a final volume of 250 µL.The coding sequence of HY5 (AT5G11260) was amplified from a cDNA clone by PCR using PrimeSTAR max polymerase (Takara) and two primers, 5'-ACTCGAGATGCAGGAACAAGCGACTAGCTC-3' and 5'-AGCGGCCGCTCAAGCATAATCTGGTACATCATATGGATAAAGGCTTGCATCAGCATTAGAAC-3', the latter of which includes a sequence for a HA tag, and subcloned into the pCRII-TOPO vector (Life Technologies) creating pCRII-HY5-HA. The HY5-HA fragment arising from restriction cleavage of pCRII-HY5-HA with XhoI and NotI was inserted into equivalent sites of the pEU vector (Cell Free Science) creating pEU-His-HY5-HA. For protein synthesis from pEU-His-HY5-HA, The synthesized proteins were loaded onto a 12% SDS-PAGE gel for electrophoresis and then stained with CBB for visualization. Alternatively, after electrophoresis, proteins were transferred onto an Immobilon-P membrane (Millipore) followed by blocking in buffer , 3% skimmed milk). Anti-HA antibody conjugated with peroxidase (Roche), anti-HY5 antiserum and anti-rabbit antibody with peroxidase  were used for detection of HY5-HA. Proteins were visualized by the ECL Prime Western Blotting Detection System and detected using an ImageQuant LAS 4000 .Genomic DNA (gDNA) was extracted from the whole aerial portion of two-to-four-week-old Arabidopsis Col-0 plants using a DNeasy Plant Midi Kit (Qiagen) and sheared into lengths of approximately 150–200 nt by Covaris S (Covaris).Approximately 10 µg of recombinant HY5-HA protein were mixed with a 60 µL volume of Dynabeads Protein G (VERITAS) and 5 µg of anti-HA antibody (abcam) in 400 µL of solution A  and then incubated with constant rotation for two hours at room temperature. The beads combined with HY5-HA were washed twice using solution A and resuspended in 500 µL of solution A. The sheared gDNA fragments (25 µg) were added into the solution containing the beads with HY5-HA and incubated with constant rotation overnight at 4 °C. The HY5-HA beads bound with gDNA fragments were washed four times in solution A and resuspended in 100 µL of solution B . This was mixed with 5 µL of proteinase K (2 mg/mL) and incubated for two hours at 37 °C. The gDNA fragments were recovered by phenol:chloroform, and chloroform extractions followed by ethanol precipitation. A library for next-generation sequencing was constructed using the TruSeq ChIP Sample Prep Kit (Illumina) and the pair-end sequencing (2 × 150 bp) was performed using a MiSeq sequencer (Illumina) according to the manufacturer’s instructions.q-value < 0.01) of gDB-seq was performed using the MACS2 program [p-value) in MACS2. Using the GADEM program [The reads (approx. 2 × 12.6 M reads) obtained as fastq files were mapped onto the genome by the Bowtie 2 program  and peak program . Approxi program , we dete program .Determination of distance from TSS to HY5-binding sites B was donhy5 (SALK_096651) [Welch’s t-test (p < 0.05) and 2- or 0.5-fold changes.Seeds were sown on filter paper that was put onto medium  and incubated under white light for three days. Total RNAs were extracted using a RNeasy Plant Mini Kit (Qiagen). Labeling of the RNAs with Cy3 was performed by a One-color Quick Amp Labeling Kit (Agilent). Hybridization to Agilent Arabidopsis 44K microarray was performed using a Gene Expression Hybridization Kit in an oven according to the manufacturer’s instructions (Agilent). Signals were obtained using a DNA Microarray Scanner (Agilent). Three replicates of the microarray analyses were performed in wild type and _096651) . For datWelch’s t-test (p < 0.05), FPKM average > 1 and 2- or 0.5-fold changes.Three-day-old wild-type Col-0 plants grown on filter paper in the dark, as described above, were exposed to blue light and harvested after one-hour exposure. Total RNAs were extracted using Trizol reagent (Life Technologies) and purified by PureLink RNA Mini Kits (Life Technologies). Directional RNA-seq libraries were constructed using TruSeq Small RNA Sample Prep Kits and TruSeq RNA Sample Preparation Kits according to the Directional mRNA-Seq Library Prep. Manual (Illumina) and sequenced using a HiSeq sequencer (Illumina). The reads  were mapped onto the genomic sequence by the Tophat program   and FPKMTotal RNAs were extracted using the RNeasy Plant Mini Kit (Qiagen) and then digested with DNase I (Takara). Reverse transcription from 1 µg of the total RNA was performed using the PrimeScript II first strand cDNA Synthesis Kit (Takara). Quantitative PCRs were performed using PrimeSTAR max polymerase (Takara). Primers used here are listed in in vivo synthesized HY5 and its predicted molecular weight has been reported previously [in vivo form for the following experiments.A cell-free expression system in wheat germ extract was used to produce recombinant HY5 proteins with a His-tag at the N terminal and a HA-tag at the C terminal (His-HY5-HA). Purified recombinant proteins were subjected to gel electrophoresis and visualized by Coomassie Brilliant Blue (CBB) staining A. Produceviously . This diin vitro gDNA immunoprecipitation like DIP-chip [in vitro. After immunoprecipitation using anti-HA antibodies, gDNA fragments bound with His-HY5-HA were recovered and subjected to next-generation sequencing. The read sequences were mapped onto the Arabidopsis genome and then peak binding locations were detected in silico. We detected 2503 HY5-binding peaks located in the Arabidopsis genome and 17 peaks located to the mitochondrial or chloroplast genomes . Of the nuclear genome peaks, 2177 mapped to the regulatory regions of 3195 nuclear genes. These regions are defined as being from 2000 bases upstream to 500 bases downstream of the gene -seq and PBM (protein-binding microarray) methods [in vivo interferences, such as environmental conditions, cell types and interaction with other cellular factors.Some studies have established novel  methods ,31,32,33in vivo ChIP-chip analysis and also with light-regulated genes. About 36.5% (1166) or 14.6% (468) of candidate genes in gDB-seq overlapped with genes in ChIP-chip or light-regulated genes, respectively  in the MACS2 program resulted in the detection of a peak located in the ANNAT1 gene . In this microarray, probes of 3050 genes of the 3195 HY5-binding genes were plotted and accumulation of most of their transcripts in the hy5 mutants was equivalent to those in wild-type plants (hy5 mutants, respectively (HY5 gene had a much larger impact on the whole transcriptome than any direct effect.To identify direct targets regulated by HY5 binding, we performed microarray analysis of three-day-old e plants A. Howeveectively . These ihy5 overlapped with HY5-binding genes (CRY3 and EFO1, possess gDB-seq peaks at the promoter and gene body, respectively (As shown in ng genes . For exaectively C.MAPKKK13, JAC1 and F3H in hy5 mutants compared with wild type (in vivo. Interestingly, it is possible that HY5 binds and affects gene expression not only by binding promoters but also by binding gene bodies, such as the EFO1 locus (To understand the role of HY5 binding on blue-inducible genes, we examined the change in accumulation of the transcripts of nine blue-inducible HY5-binding genes by semi-quantitative RT-PCR analysis. We detected delayed induction of eight transcripts including ild type D. This rO1 locus D.in vivo ChIP-based methods identify physiological TF-binding sites, while in vitro gDB-seq, reveals the candidate genes regulated by a TF. The information from gDB-seq will be useful to elucidate the whole picture of genes that are controlled by TFs.The result above is one example of how gDB-seq analysis will contribute to future research that will clarify the regulatory mode of transcription by TFs during environmental change in plants. Generally, BBX22 expression [in vivo regulation of gene expression by HY5. Therefore, additional studies through other approaches may be required to reveal regulation of genes targeted by HY5 based on gDB-seq information.HY5 regulates blue-light signaling pathway through physical interaction with other bZIP-type TFs, HYH and GBF1 ,35. GBF1pression . The outin vitro methodology for identifying TF-binding sites based on protein synthesis in wheat germ extract, DNA immunoprecipitation and next-generation sequencing. This method was applied to the HY5 transcription factor, a key regulator of photomorphogenesis. Comparison between our gDB-seq and previous ChIP-chip analyses suggests that in vitro gDB-seq covers the in vivo binding sites of HY5 to some extent. In vivo ChIP-chip and ChIP-seq reveal physiological binding sites of TFs, while in vitro gDB-seq identifies a comprehensive set of physical binding sites. Therefore, this method will be useful to understand the overall nature of physical TF binding sites and identify their affinity strengths and conserved motifs.In this research, we established an


Bacillus subtilis. Recently the key players that repress and activate pLS20 conjugation have been identified. Here we studied in detail the molecular mechanism regulating the pLS20 conjugation genes using both in vivo and in vitro approaches. Our results show that conjugation is subject to the control of a complex genetic switch where at least three levels of regulation are integrated. The first of the three layers involves overlapping divergent promoters of different strengths regulating expression of the conjugation genes and the key transcriptional regulator RcoLS20. The second layer involves a triple function of RcoLS20 being a repressor of the main conjugation promoter and an activator and repressor of its own promoter at low and high concentrations, respectively. The third level of regulation concerns formation of a DNA loop mediated by simultaneous binding of tetrameric RcoLS20 to two operators, one of which overlaps with the divergent promoters. The combination of these three layers of regulation in the same switch allows the main conjugation promoter to be tightly repressed during conditions unfavorable to conjugation while maintaining the sensitivity to accurately switch on the conjugation genes when appropriate conditions occur. The implications of the regulatory switch and comparison with other genetic switches involving DNA looping are discussed.Plasmid conjugation plays a significant role in the dissemination of antibiotic resistance and pathogenicity determinants. Understanding how conjugation is regulated is important to gain insights into these features. Little is known about regulation of conjugation systems present on plasmids from Gram-positive bacteria. pLS20 is a native conjugative plasmid from the Gram-positive bacterium  Bacillus subtilis, which is also a gut commensal in animals and humans. Here we describe in detail the molecular mechanism by which the key transcriptional regulator tightly represses the conjugation genes during conditions unfavorable to conjugation without compromising the ability to switch on accurately the conjugation genes when appropriate. We found that conjugation is subject to the control of a unique genetic switch where at least three levels of regulation are integrated. The first level involves overlapping divergent promoters of different strengths. The second layer involves a triple function of the transcriptional regulator. And the third level of regulation concerns formation of a DNA loop mediated by the transcriptional regulator.Plasmids are extrachromosomal, autonomously replicating units that are harbored by many bacteria. Many plasmids encode transfer function allowing them to be transferred into plasmid-free bacteria by a process named conjugation. Since many of them also carry antibiotic resistance genes, plasmid-mediated conjugation is a major mechanism in the dissemination of antibiotic resistance. In depth knowledge on the regulation of conjugation genes is a prerequisite to design measures interfering with the spread of antibiotic resistance. pLS20 is a conjugative plasmid of the soil bacterium  Bacteria exchange genetic material at high rates by different processes, which are collectively named Horizontal Gene Transfer (HGT). HGT can be beneficial for bacteria because the newly acquired DNA may endow them with novel features enabling them to adapt to changing conditions in the environment, i.e. rapid evolution. On the other hand, HGT is notorious for its role in the dissemination of virulence/pathogenicity determinants and antibiotic resistance. The main mechanisms responsible for HGT are transformation mediated by natural competence, transduction and conjugation Some basic features of the conjugation process are conserved among plasmids Bacillus subtilis had not been reported until recently. This is most remarkable taking into account that (i) it is one of the best-studied Gram-positive bacteria; (ii) it has important industrial applications; and (iii) it is closely related to pathogenic and fastidious bacilli B. subtilis strains are gut commensals in animals including humans B. subtilis plasmids may therefore play an important role in HGT in different environments. We chose the B. subtilis plasmid pLS20 for our studies. Originally, this 65 kb plasmid was identified in the Bacillus subtilis natto strain IFO3335 that is used in the fermentation of soybeans to produce “natto”, a dish that is popular in South Asia LS20, that suppresses the development of natural competence of B. subtilisVarious conjugative plasmids have been studied in considerable detail LS20rco, which encodes the master regulator of conjugation responsible for keeping conjugation in the default “OFF” state. Activation of conjugation requires an anti-repressor, RapLS20, that belongs to the family of Rap proteins. Inactivation of the LS20rap gene on pLS20 severely compromises conjugation, and conjugation was enhanced when LS20rap was expressed from an ectopic locus. The activity of RapLS20, in turn, is regulated by a signaling peptide, Phr*LS20. The small LS20phr gene, located immediately downstream of LS20rap, encodes a pre-protein. After being secreted, PhrLS20 can be processed by a second proteolytic cleavage, resulting in generation of the functional pentapeptide, Phr*LS20, corresponding to the five C-terminal residues of PhrLS20. When (re)imported, this peptide inactivates RapLS20. Therefore, activation of conjugation is ultimately regulated by the Phr*LS20 signaling peptide. The Phr*LS20 concentration will be relatively high or low when donor cells are predominantly surrounded by donor or recipient cells, respectively. Hence, conjugation will become activated particularly under conditions in which recipient cells are potentially present. In addition, Phr*LS20 has a crucial role in returning conjugation to the default “OFF” state Recently, we have reported a global view of the regulatory circuitry of the pLS20 conjugation genes. A conjugation operon encompassing more than 40 genes is located next to a divergently oriented single gene, in vitro and in vivo approaches, we show that the genetic switch controlling pLS20 conjugation involves at least three layers of regulation. Together, they tightly repress the main conjugation promoter under conditions that do not favor conjugation, while maintaining the ability to accurately switch on the conjugation genes when appropriate conditions occur. The three layers involve coinciding or overlapping divergent promoters of different strengths, autoregulated expression of RcoLS20, which turns out to be a tri-functional transcriptional regulator, and formation of RcoLS20-mediated DNA looping. The sophisticated regulatory mechanism that combines three layers of control into a single switch is novel for plasmids of Gram-positive bacteria. The implications of the uncovered regulation mechanisms for conjugation are discussed in the context of regulatory systems present on other HGT elements and with other regulatory systems involving DNA looping.Despite identification of the players involved in regulation of the conjugation genes, our knowledge on regulation of the genetic switch responsible for activating conjugation is still very limited. Using a combination of various 27, encoding the main repressor of conjugation genes, RcoLS20, reads leftwards. Flanking genes 28 to 74, which are all transcribed in the opposite direction, probably constitute a large conjugation operon 28 we cloned the ∼600 bp intergenic LS20rco-gene 28 region in the appropriate orientation in front of a promoterless lacZ reporter, and subsequently placed a single copy of this cassette at the B. subtilis chromosomal thrC locus (strain PKS3). Transcriptional fusions to several sub-fragments of this region were also constructed. In addition, all the fragments were cloned in the opposite orientation to analyze the divergent promoter of the LS20rco gene (see below). For simplicity, the cloned fragments are indicated with Roman letters. Fragments cloned in the orientation to analyze the conjugation or the LS20rco promoter are indicated with the extension “c” or “r”, respectively. The entire intergenic region is referred to as Fragment I (or F_I). A schematic representation of the different strains and fusions described in this work is given in According to our standard presentation , the trac-lacZ fusion were blue when grown overnight on Luria-Bertani (LB) agar plates supplemented with the chromogenic substrate 5-bromo-4-chloro-indolyl-β-D-galactopyranoside  activities that were in the range of 300 and 500 Miller Units (MU) during mid-exponential and stationary phase, respectively. These results indicate that cP is a rather strong promoter that does not seem to be regulated by host-encoded factors when grown under these conditions.Colonies of strain PKS3 containing F_Ide Xgal; , demonstcP is the main conjugation promoter it is expected that (i) its activity would generally be lower in the presence of pLS20cat and (ii) there would be a correlation between promoter cP activity and the efficiency of conjugation. The following results show that this is indeed the case. Thus, we introduced pLS20cat into strain PKS3, and colonies of the resulting strain, PKS8, were white after overnight growth on Xgal-containing plates , is responsible for repression of the cP promoter. To this end, we placed the LS20rco gene under the control of the isopropyl β-D-1-thiogalactopyranoside (IPTG)-inducible spankP promoter and introduced this cassette at the amyE locus of strain PKS3 (harboring the F_Ic-lacZ fusion at the thrC locus). Colonies of this strain, PKS5, were blue when grown on Xgal-supplemented LB agar plates, but white when the plates contained IPTG , the βG activities obtained with strains harboring lacZ fused to Fragment IIIc, IVc, Vc or VIc were very similar to those obtained when lacZ was fused to Fragment Ic. These results show that promoter cP is located at an unusually large distance of at least 350 bp upstream of gene 28.We next set out to delineate the position of the lacZ fused to Fragment VIIc (GR68) or VIIIc (GR70) revealed that promoter activity was sustained only by Fragment VIIc (c contains (at least part of) the Pc promoter. This 63 bp region contains the sequence TTtACA-gttaaaaaaatgtc-TATctT-3′5′- ttaaaaatttcactgaaatac-, which constitutes a putative σA-dependent promoter endorsing several features characteristic for a strong promoter. First, the hexamer sequences 5′-TTtACA-3′ and 5′-TATctT-3′ are very similar to the consensus −35 (5′-TTGACA-3′) and −10 (5′-TATAAT-3′) sequences recognized by σA. Second, an optimal spacer length of 17 bp separates the putative −35 and −10 boxes. Third, the spacer contains the extended −10 motif . Fourth, AT-rich tracts are located directly upstream of the predicted −35 box which are likely binding sites for the C-terminal domain of the RNA polymerase α-subunit. Additional evidence that this sequence constitutes the cP promoter was obtained by primer extension analysis to determine the transcription start site. The detected extension product is shown in cP core promoter sequences mentioned above (see 28 and downstream genes (shown in green), start close to the divergently oriented LS20rco gene (shown in red).Analyses of strains containing ent VIIc , showingbove see . The poscP, we constructed lacZ fusion strains to characterize the divergently oriented rP promoter responsible for expression of RcoLS20. Surprisingly, no promoter activity was observed when lacZ was fused to the 570 bp Fragment Ir , also did not provide detectable levels of promoter activity. We then introduced pLS20cat into these strains to study whether it encoded a protein that might be required to activate promoter rP. Colonies of the resulting pLS20cat-harboring strains GR39 (F_Ir-lacZ) and GR66 (F_IAr-lacZ) turned pale blue when grown on Xgal-containing plates . Therefore, a lack of rP promoter activity in the reporter assay can be due to the absence of (part of) the rP promoter or the RcoLS20 binding sites required for activation of rP. Since activator proteins generally bind upstream of promoters, we tested constructs having deletions at the 3′ end of Fragment Ir (i.e. flanking the LS20rco gene). Promoter rP activity was detected when lacZ was fused to Fragment VIIr (strain GR82), but not when it was fused to Fragment VIIIr (strain GR116)  located on the 63 bp 5′ region of Fragment VII on which the divergently oriented cP promoter is also located   and 3A. xtension . The detoter see . This imrP is a weak promoter whose activity requires RcoLS20 (see above). It is therefore unlikely that the −35 and −10 boxes will be very similar to the consensus sequences. The following two sequences that may constitute a σA-dependent promoter are located upstream of the determined rP transcription start site: (i) [5′-aaGAtA- 17 bp -TgTAAa-3′] and (ii) [5′-aTaACA-18bp-aAgtAT-3′] (mismatches with respect to consensus −35 (5′-TTGACA-3′) and −10 boxes (5′-TATAAT-3′) given in lower case, see rP promoter. Interestingly, this would imply that the positions of the −10 and −35 boxes correspond exactly to the −35 and −10 boxes, respectively, of the divergently oriented cP promoter.rP and cP promoters overlap. RNA transcripts mapped against the entire intergenic region except for a small region that is located near the start of the LS20rco gene. The divergent promoters rP and cP, responsible for the left- (red) and rightward (green) oriented transcripts, respectively, must both be located in the small nontranscribed region which corresponds to the position of the rP/cP promoters according to their transcriptional start sites determined by primer extension.The results of the RNAseq experiments presented in cP and rP promoters overlap, if not coincide.In summary, results obtained by a combination of different approaches demonstrate that divergent LS20 belongs to the Xre-family of transcriptional regulators and is predicted to contain a Helix-Turn-Helix (HTH) DNA binding motif in its N-terminal region LS20 will exert its transcriptional regulatory effects on rP and cP by binding to DNA sequences. We employed the following in vivo approach to gain insights into the location of the RcoLS20 binding sites. Either pLS20cat or the spankP-LS20rco cassette was introduced into the various lacZ fusion strains  displayed high cP promoter activity, but no promoter activity was detected when RcoLS20 was provided in trans (strains PKS5 and PKS8). Efficient RcoLS20-mediated repression of the cP promoter was lost however when lacZ was fused to Fragment IIIc (strains GR16 and PKS32). This strongly indicates that an RcoLS20 operator site is located on the 368 bp Fragment IIc and that this operator, which would be located at least 85 bp downstream of the cP promoter, is crucial for efficient repression of the cP promoter. Fragment IIc contains an inverted repeated sequence (5′-ATCAAAATCAtgctgcaactTGGTTTTGAT-3′). To test whether this region constitutes an RcoLS20 operator site we constructed lacZ fusions to Fragments IVc or Vc, and also engineered derivatives of these two strains containing pLS20cat or spankP-LS20rco. The 5′ ends of these Fragments are located up- or downstream of the inverted repeat . Together these in vivo results strongly indicate that an ∼160 bp region, located 85 bp downstream of cP, contains an RcoLS20 operator site that is required for efficient repression of this promoter. We name this operator site OI.A schematic summary of the results obtained for promoter peat see . The Pc cP was not repressed by RcoLS20 when the lacZ fusion was based on Fragment IIIc (strain GR16) and cells were grown in the presence of 10 µM IPTG. Interestingly though, promoter cP in strain GR16 was efficiently repressed when the concentration of IPTG was increased to 1 mM  but not when it was fused to Fragment IIIr . To facilitate purification, we constructed an E. coli strain that expresses an RcoLS20-His(6) tagged fusion protein. The (6)his-tag was placed at the C-terminus because RcoLS20 contains a predicted Helix-Turn-Helix DNA binding motif close to its N-terminus. The following result demonstrates that the RcoLS20-His(6) protein is functional in vivo. We constructed B. subtilis strain GR90 in which the expression of LS20-his(6)rco gene is placed under the control of the inducible spankP promoter, and which also contains the F_Ic-lacZ reporter fusion. The activity of promoter cP in strain GR90 was repressed in an IPTG-dependent manner similar to that observed for strain PKS5 containing an inducible copy of native LS20rco (not shown).To study the position of the Rcoin vivo transcriptional fusion results presented above indicated the presence of two operators. One of them, operator OII, located near promoters cP/rP, and another one, operator OI, present in an ∼160 bp region about 85 bp downstream of cP. In addition, this analysis indicated that the ∼200 bp region immediately upstream of gene 28 does not contain RcoLS20 binding sites. Accordingly, we began analyzing binding of RcoLS20 to Fragments X (200 bp region upstream gene 28), III (expected to contain operator OII) and XII (expected to contain operator OI)  and Fragment XIIA (125 bp) both produced up to two shifts, and RcoLS20 did not bind to the 46 bp region that separates these two fragments. This latter conclusion is based on comparison of gel retardations obtained with fragments XI and XII.The  OI) see . Indepenagment X . Togethement XII . InteresLS20 to Fragments I, IV and V that encompass both operators. These fragments gave similar retardation patterns. Interestingly, in these cases, RcoLS20 binding resulted in the appearance of four retarded species. All four of these retarded species could be detected at low RcoLS20 concentrations when most of the fragment had not bound RcoLS20, indicating that RcoLS20 binds cooperatively to multiple sites on these fragments.We next analyzed binding of RcoLS20 operators we used the motif-identification programs MEME II (Fragment III_A), and four times in OI (Fragment XII_A). We named the seven motifs identified in the OII operator a1–a7, and the four motifs in the OI operator b1–b4 .To search for the presence of conserved motifs in the two Rco1–b4 see . WhereasLS20 was obtained by DNase I footprinting (see below) and mutational analysis. Thus, gel retardation assays showed that binding of RcoLS20 is affected in probes containing alterations in one or two motifs in either operator. For instance, RcoLS20 did not bind to a derivative of Fragment III_A containing mutations in both motifs a1 and a7; and binding was affected when only motif a7 was mutated. Similarly, mutation of motif b1 or b4 resulted in the appearance of only one retarded species instead of two observed for corresponding fragments without mutations (LS20rco-gene 28 region contains two RcoLS20 operators that are separated by 75 bp. Operator OII overlaps with promoters rP/cP and the other region is located 75 bp towards the direction of gene 28. Each region contains repeats of a motif whose consensus sequence is 5′-CAGTGAAA-3′ and which forms (part of) the binding site of RcoLS20. Motifs in OI are located on the lower strand, and except for one, motifs in OII are located on the upper strand.Evidence that the identified motif constitutes (part of) the binding site for Rcoutations . In summLS20 to operators OI and OII was confirmed by DNase I footprinting. The results presented in LS20 binds to a region that overlaps with the rP/cP promoters and to another region located about 75 bp downstream of the cP promoter. The combined in vitro results are in line with the in vivo results presented above.Binding of RcoI, -located at a distance of more than 75 bp from rP/cP-, is needed for proper regulation of these promoters. This and other data presented above, suggest that proper regulation of the rP/cP promoters involves DNA looping mediated by RcoLS20 bound to operators OI and OII. Due to the intrinsic stiffness of DNA, loops are generally longer than 90 bp because the curvature energy required to make smaller loops is too large, unless the DNA region separating the two operator sites is bent I and OII are separated by only 75 bp. Several periodically spaced A/T tracts can result in formation of a static bent Operator OLS20-mediated DNA looping occurs then it is expected (i) that RcoLS20 will form oligomers thereby creating a DNA binding unit able to bind simultaneously to OI and OII, and (ii) that the two operators are in phase such that the RcoLS20 binding sites have a spatial orientation that is optimal for RcoLS20 binding. We tested both predictions. The oligomerization state of RcoLS20 was studied by two complementary analytical ultracentrifugation approaches  was compatible with an elongated protein tetramer . Promoter cP was not efficiently repressed by RcoLS20 however, when lacZ was fused to Fragment Ic+5 (strain GR191). Thus, colonies of pLS20cat-harboring cells were blue when grown on Xgal-containing plates , because the curvature energy required for forming such small loops becomes too great. For such short loops to occur specific features like intrinsic static bending or binding of an additional protein inducing bending are required. In the case of pLS20, in which the operators OI and OII are separated by only 75 bp, we show that the spacer region contains a static bent.The DNA loop in the E. coli ara operon in 1984 E. coli R6K plasmid Enterococcus faecalis plasmid pCF10 it has been proposed that regulation of its conjugation system involves DNA looping mediated by the pheromone-responsive transcriptional regulator PrgX E. coli genome for the presence of putative “simple DNA looping systems” in which looping would involve a single regulator . Under these restrictive settings, this survey identified 48 genes/operons in which DNA looping mediated regulation is likely to play a role. Interestingly, fourteen of them involve divergently oriented promoters. In the context of our studies, it is worth mentioning the regulation of the conjugation genes located on the integrative and conjugative element ICEBs1 that is present in several B. subtilis strains. The gene encoding the transcriptional regulator ImmR, and the excision and conjugation genes are expressed from two divergently oriented promoters that are separated by ∼130 bp. At low concentrations, the ImmR protein can bind to six regions, three being proximal to each promoter. It has been suggested that repression of the immR promoter might involve cooperative interactions between ImmR molecules bound to binding sites proximal to both promoters, i.e. DNA looping The first experimental demonstration that a DNA loop can play a crucial role in transcriptional regulation was reported for the LS20 is not an exception-, transcriptional regulators are produced in limited amounts per cell. Low numbers of regulators enhance the possibility of transcriptional fluctuations between individual cells within a population. In addition, the intrinsic stochasticity of transcription, -also referred to as noise-, affects the temporal effectiveness of transcriptional regulation; again this is especially prominent when the number of regulatory proteins involved is low. Recent evidences indicate that DNA looping contributes importantly to controlling temporal transcriptional noise, as well as dampening transcriptional fluctuations between cells within a population What are the benefits of DNA looping in general and for the regulation of the conjugation genes of pLS20 in particular? A major consequence of DNA looping is that it results in a high local concentration of the transcriptional regulator at the right place, which would increase its specificity and affinity LS20 and overlapping divergent promoters of different strength.For some differentiation processes, cell-to-cell or stochastic variability in levels of transcriptional regulators form the basis for activation of these processes, resulting in different behavior of genetically identical cells within a population Escherichia coli phage λ RP and LP. A third operon, which encodes amongst others the CI transcriptional regulator, is located in between the two early lytic operons such that the promoter of gene cI, RMP, flanks the divergently oriented RP promoter driving expression of one of the two early operons. In several aspects, functional analogies exist between CI and RcoLS20 although they share only 16% of identity at their primary protein sequence level. Both RcoLS20 and CI stimulate and repress their own promoter at low and high concentrations, respectively, resulting in a self-sustaining positive feedback loop while keeping the transcriptional regulator in a low concentration range. Above, arguments have been given that for pLS20 this situation, together with the effects of the DNA loop, is important for the tight repression of the cP promoter during conditions in which conjugation is not favourable, while maintaining the sensitivity to be able to respond rapidly to switch on the conjugation genes when appropriate conditions occur. The transcriptional regulation of λ appears to serve a similar purpose. Thus, on the one hand the lytic genes are tightly repressed since spontaneous switching to the lytic cycle occurs less than once every 108 generations cI expression impair prophage induction LS20 and CI, can form higher order oligomers, permitting them to bind cooperatively to multiple sites distributed in two operators, effectively resulting in DNA looping which plays an important role in the genetic regulation of the conjugation and the lytic operon, respectively. Taking the analogy further, it is interesting to note that these regulatory systems both control a process of horizontal gene transfer.A well-studied genetic switch involving DNA looping is the one that governs the switch from the lysogenic to the lytic state of the RMP promoter. At present, we do not have such detailed insights in transcriptional regulations at the molecular level for RcoLS20. However, instead of dimers, RcoLS20 forms tetramers in solution, which probably means that the molecular mechanism by which the pLS20 promoters rP and cP are regulated is distinct from the way CI regulates λ promoters RP and RMP. Another argument supporting this assumption is the different configuration of the divergent promoters and the binding sites for the regulator protein. In pLS20, the position of promoters cP/rP overlaps and the RcoLS20 binding sites in OII overlap and flank these core promoters. In λ the binding sites for CI regulator in one operator overlap the RP promoter and are located upstream of the RMP core promoter sequences. Finally, a major difference between the DNA looping involved systems of pLS20 and λ is how the switches are induced. In λ, the switch is induced by an SOS response which results in RecA-mediated CI autocleavage. In the case of pLS20, the switch is dictated ultimately by intercellular quorum sensing signaling involving the signaling peptide Phr*LS20 that regulates the activity of RapLS20, the anti-repressor of RcoLS20LS20 will build up when the majority of the cells that surround a donor cell already contain pLS20, and this will inactivate RapLS20 and hence block activation of the conjugation genes.However, there are also several differences between the two systems. For instance, whereas regulation of pLS20 conjugation genes involves a short loop of 75 bp, regulation of the λ lytic genes involves a long loop of 2.3 kb. A second difference is that CI protein forms dimers in solution. A pair of CI dimers tetramerizes when binding to the binding sites in one operator and another dimer pair does the same when binding to the other operator. Upon DNA looping, interaction between the two tetramers constitutes a functional octamer. In addition, when a loop is formed another pair of dimers may bind to additional binding sites present in both operators, and this additional bridge is responsible for repressing 28 is predicted to form complex secondary structures, which might modulate expression of the downstream genes in a variety of scenarios. Currently, a study to elucidate a possible role of this long 5′ untranslated region is carried out in our laboratory.Besides those described here, it is possible that the pLS20 conjugation genes are regulated by additional mechanism(s). For example, the conspicuously long 5′ untranslated region upstream of gene rP promoter by the transcriptional regulator at low and elevated concentrations, respectively, combined with simultaneous repression of the divergent strong conjugation promoter, and (iii) DNA looping mediated by binding of RcoLS20 regulator to two operators separated by a short loop. Most likely, the combination of these different layers causes tight repression of the main conjugation promoter cP when conditions for conjugation are not optimal, while allowing the system to switch rapidly to high expression of the conjugation genes when appropriate conditions occur.In summary, in this work we have provided evidence that regulation of the conjugation genes present on pLS20 is based on a unique genetic switch that combines at least three levels of control. These include (i) overlapping divergent promoters of different strengths, (ii) auto-stimulation and repression of the weak B. subtilis and E. coli, respectively), chloramphenicol (5 µg/ml), spectinomycin (100 µg/ml), and kanamycin (10 µg/ml). B. subtilis strains used. All of them are isogenic with B. subtilis strain 168. Plasmids and oligonucleotides used are listed in Bacterial strains were grown in LB liquid medium or on 1.5% LB agar plates E. coli cells were transformed using standardized methods as described in Singh et alB. subtilis transformations, competent cells were prepared as described by Bron B. subtilis strains containing a copy of lacZ fused to the entire or part of the LS20rco-gene 28 intergenic DNA region. First, the region of DNA to be cloned was amplified using appropriate primers . The LS20rco gene was amplified from pLS20cat by PCR using primers oPKS14N and oPKS8. The purified PCR product was digested with NcoI and SalI and cloned into the vector pET28b+ digested with the same restriction enzymes to produce plasmid pRcoLS20-His. B. subtilis strain GR90 contains the LS20-hisrco(6) under the control of the spankP promoter at the amyE locus. To construct this strain LS20-hisrco(6) was amplified from pRcoLS20-His by PCR using primers oGR3 and oGR4. The PCR product was digested with NheI and SphI and cloned into the vector pDR110 digested with the same enzymes to generate pspankPrcoLS20-His. This plasmid was used to transform competent B. subtilis cells selecting for spectinomycin resistance. Double cross-over events were selected by loss of amylase gene.The following strategy was used to construct a translational fusion of β-galactosidase activities were determined as described previously spankP promoter was studied as follows. Overnight cultures were diluted in prewarmd LB supplemented with IPTG at the indicated concentrations to an OD600 of ∼0.05. Next, samples were taken at regular intervals to determine OD600 and were subjected to matings with proper recipient cells.Conjugation was carried out in liquid medium as described previously Preparation of total RNA samples, RNA sequencing and Bioinformatic analysis of RNAseq data was done as described previously E. coli BL21 (DE3) cells carrying plasmid prcoLS20-His were used to inoculate 1 litre of fresh LB medium supplemented with 30 mg/ml kanamycin and grown at 37°C with shaking. At an OD600 of 0.4, expression of LS20-his(6)rco was induced by adding IPTG to a final concentration of 1 mM and growth was continued for 2 h. Cells were further processed as described previously 2, 7 mM β-mercaptoethanol, 50% v/v glycerol) and stored in aliquots at −80°C. Bradford assay was used to determine the protein concentrations.gene 28 and LS20rco were amplified by PCR using pLS20cat as template. The resulting PCR fragments were purified and equal concentrations (300 nM) were incubated on ice in binding buffer  without and with increasing amounts of purified RcoLS20His(6) in a total volume of 16 µl. After careful mixing, samples were incubated for 20 min at 30°C, placed back on ice for 10 min, then loaded onto 2% agarose gel in 0.5XTBE. Electrophoresis was carried out in 0.5X TBE at 50 V at 4°C.Finally, the gel was stained with ethidium bromide, destained in 0.5XTBE and photographed with UV illumination.In essence, the gel retardation assays were carried out as described before cP was 5′-ttctagttctttttacac, while that used for promoter rP was 5′-tctctattgcccacttat. Oligonucleotides were end-labeled with [γ-32P]-ATP and T4 polynucleotide kinase as recommended by the supplier (New England Biolabs). The 186 bp DNA fragment that served as sequence ladder was PCR amplified with primers 5′-acggtctagcgcttacaat and 5′-ttctagttctttttacac, the last one labeled at its 5′ end.Determination of the transcription start sites by primer extension was performed essentially as described cP/rp promoter encompassing region was amplified by PCR using primers p28_Δ16 and Prom28UpBam, and pLS20cat as template. One of the ends was radio-labeled by digesting the fragment with BamHI and subsequently filling in the end with exo− Klenow fragment in the presence of [α-32P]-ATP.DNaseI footprinting assay was carried out as described Presence of conserved motifs was searched by using motif-identification programs MEME 2, 1 mM EDTA and 100 mM glycerol, pH 7.4, were loaded (320 µL) into analytical ultracentrifugation cells. The experiments were carried out at 43–48 krpm in an XL-I analytical ultracentrifuge (Beckman-Coulter Inc.) equipped with UV-VIS absorbance and Raleigh interference detection systems. Sedimentation profiles were recorded at 280 nm. Sedimentation coefficient distributions were calculated by least-squares boundary modelling of sedimentation velocity data using the continuous distribution c(s) Lamm equation model as implemented by SEDFIT 14.1 s values were corrected to standard conditions  using the program SEDNTERP s values .Sedimentation velocity assay. Samples in 20 mM Tris-HCl, 250 mM NaCl, 10 mM MgClSedimentation equilibrium assay. Using the same experimental conditions as in the SV experiments, short columns (90 µL) SE experiments were carried out at speeds ranging from 7,000 to 10,000 rpm and at 280 nm. After the last equilibrium scan, a high-speed centrifugation run  was done to estimate the corresponding baseline offsets. Weight-average buoyant molecular weights of protein were determined by fitting a single species model to the experimental data using the HeteroAnalysis program Figure S1LS20rco – gene 28 intergenic region contains a strong promoter that is inhibited by the pLS20cat encoded protein RcoLS20. Strains were streaked on Xgal-containing LB plates and incubated for 16 hours at 37°C. When indicated, plates were also supplemented with 10 µM IPTG in the case of PKS5. Strain PKS3 contains a cassette at the thrC locus in which the lacZ gene is preceded by the 570 bp rcoLS20-gene 28 intergenic region . PKS8 is a derivative of PKS3 harboring pLS20cat. PKS5 is a derivative of PKS3 containing the spankP-LS20rco cassette at amyE. The negative control strain PKS7 contains a promoterless version of lacZ at the thrC locus.The (TIF)Click here for additional data file.Figure S2I and OII is predicted to contain a static bent. The global 3D structure of a 256 bp DNA region encompassing operators OI and OII was predicted according the dinucleotide wedge mode using the online webpage http://www.lfd.uci.edu/~gohlke/dnacurve/. For clarity, sequences corresponding to promoters cP/rP and motifs in operators OI and OII are presented as space filling. Positions of the promoters and RcoLS20 binding motifs are given in blue and purple respectively.The 75 bp region separating operators O(TIF)Click here for additional data file.Figure S3I and OII with half a helical turn affects RcoLS20-mediated inhibition of promoter cP. Strains containing F_Ic and F_Ic+5 fused to lacZ  and their derivatives harboring pLS20cat  were spread on Xgal-containing LB agar plates and photographed after 24 hours incubation at 37°C.Enlarging the distance between operators O(TIF)Click here for additional data file.Table S1Strains used.(DOCX)Click here for additional data file.Table S2Plasmids used.(DOCX)Click here for additional data file.Table S3Oligonucleotides used.(DOCX)Click here for additional data file.