B-ARRs extensively overlap. Constructing a primary cytokinin response transcriptional network reveals a recurring theme of widespread cross-regulation between the components of the cytokinin pathway and other plant hormone pathways. The B-ARRs are found to have similar DNA binding motifs, though sequences flanking the core motif were degenerate. Cytokinin treatments amalgamate the three different B-ARRs motifs to identical DNA binding signatures , Y(t/c)) which suggests cytokinin may regulate binding activity of B-ARR family members. Furthermore, we find that WUSCHEL, a key gene required for apical meristem maintenance, is a cytokinin-dependent B-ARR target gene, demonstrating the importance of the cytokinin transcription factor network in shoot development.Cytokinin fulfills its diverse roles in planta through a series of transcriptional responses. We identify the in vivo DNA binding site profiles for three genetically redundant type-B ARABIDOPSIS RESPONSE REGULATORS (B-ARRs): ARR1, ARR10, and ARR12. The expression and genome-wide DNA binding locations of the three  Arabidopsis and provide evidence that cytokinin regulates meristem development by promoting B-ARR binding to WUSCHEL.Cytokinin regulates gene expression by activation of ARR transcription factors. Here, the authors use ChIP-seq to show how three type B-ARRs mediate cytokinin response in  N6-substituted adenine derivative, along with other phytohormones orchestrates almost every aspect of plant growth and development, including meristem function, vascular development, biotic and abiotic stresses, and leaf senescence5. Cytokinin was first discovered for its ability to promote cell division over fifty years ago6. In the past twenty years, its own biosynthesis and signaling pathways and diverse roles in regulating cellular processes have been revealed by both forward and reverse genetic screens12. Cytokinin employs a two-component multi-step phosphorelay for its perception and signaling transduction14. In Arabidopsis, there are three cytokinin receptors  and eleven type-B response regulators 15. In cytokinin signaling cascades, the histidine-containing phosphor-transfer proteins (AHPs) act as phosphor-transfer intermediates for various AHK-AHP-B-ARR modules16. Genetic analysis also revealed that only higher order mutants of each family render pronounced developmental phenotypes, indicating redundancy in the cytokinin signaling pathway8.Cytokinin, an 12. The DNA binding domain and protein nuclear localization signal domain at the C-terminus of B-ARRs are responsible for B-ARRs entering the nucleus and binding to their targets while their activation domain is responsible for the activation of cytokinin transcriptional responses. The presence of the receiver domain in B-ARRs is thought to cause inhibition at low cytokinin levels and may block the upstream phosphorelay to B-ARRs. It is postulated that the receiver domain masks the DNA binding domain of B-ARRs until its conformation is altered by cytokinin, which finally results in the activation of B-ARRs15. In previous genetic analyses, five Arabidopsis B-ARRs were shown to act in cytokinin signaling cascades with ARR1, ARR10, and ARR12 playing critical roles in plant growth and development8. The A-ARRs are cytokinin response genes that are the targets of B-ARR TFs12. However, the B-ARRs are not regulated at the transcriptional level by cytokinin but are post transcriptionally controlled11. Recently, B-ARRs were shown to be regulated at the level of protein stability, at least in part, through the ubiquitin-proteasome pathway17.The cytokinin transcriptional response centrally affects the family of ARRs. Type-B ARRs (B-ARRs) are transcription factors (TFs) with a GARP-like DNA binding domain at their C-termini and a receiver domain at their N-termini. Type-A ARRs (A-ARRs) are similar to the N-termini receiver domain of B-ARRs but do not possess a DNA binding domain. A-ARRs are negative cytokinin regulators but their mechanism of inhibition in cytokinin signaling remains unknown19 and a “golden list” of cytokinin response genes from microarray expression data and RNA-seq data20. However, the identity of which cytokinin responsive genes may be direct targets of the B-ARRs remains unknown. In addition, most experiments have depended upon treatment with high concentrations of cytokinin, since the targets of B-ARRs are almost impossible to identify at the endogenous levels of cytokinin in transcriptomic studies. Therefore, identification of the genome-wide targets of B-ARRs, with and without cytokinin treatment, would facilitate our understanding of the cytokinin responsive DNA regulatory elements, provide insights into cytokinin primary responsive gene expression, and potentially elucidate the mechanism(s) by which cytokinin ultimately regulates diverse physiological responses. Recently, genome-wide binding sites of ARR10 were identified by chromatin immunoprecipitation sequencing (ChIP-seq) of a tagged, over-expressed ARR10 fusion protein21, demonstrating the utility of in vivo DNA binding studies for cytokinin response pathway analysis.Previous in vitro studies have identified candidate binding motifs for the B-ARRs23. First, inhibition of a subset of A-ARRs by WUSCHEL (WUS) has been demonstrated, although the mechanism of this repression remains unknown24. Since A-ARRs are targets of B-ARRs, it can be postulated that the repression of A-ARRs by WUS involves B-ARRs. Second, the arr1/10/12 triple mutant was shown to produce a smaller size shoot apical meristem8. Third, genetic manipulation of cytokinin levels either by loss-of-function mutants of LONELY GUYS, which are involved in the one step conversion of cytokinin precursors to active cytokinin25, or by over-expression of cytokinin oxidase produces meristem defects26. Finally, plant regeneration requires the proper ratio of cytokinin and auxin27. Therefore, a greater understanding of the targets of B-ARRs may provide a link between the cytokinin transcriptional response and important plant developmental processes such as meristem development.Cytokinin plays an important but poorly understood role in the maintenance of the stem cell niche and regulation of meristem size28 to generate plants containing epitope-tagged B-ARRs and employ ChIP-seq to identify their genome-wide binding locations. Extensive targeting of multiple type-B ARRs to a common set of genes reveals a conserved core cytokinin transcriptional response network and extensive cross-regulation of the plant hormone pathways. We also demonstrate that the regulation of WUS by B-ARRs is critical for stem cell maintenance in the shoot apical meristem. These findings provide potential avenues to further explore the mechanism operating downstream cytokinin responses that control diverse growth and development processes.Our study aims to construct a core cytokinin transcriptional response network with a focus on systematic identification of the binding targets of the B-ARRs, the key players in cytokinin signaling. We take advantage of the power of recombineeringarr1/10/12 triple mutant revealed pronounced developmental phenotypes, such as smaller size seedling and adult plants, effects likely due to a smaller shoot apical meristem and insensitivity to cytokinin treatment8. Such studies revealed that ARR1, ARR10, ARR12 are critical components of the cytokinin signaling pathway  regulatory information is maintained, these tagged gene constructs provide a state nearest to the native expression of the endogenous B-ARRs as is currently technically possible in plants28. ARR1, ARR10, and ARR12 tagged lines were generated in the Col-0 background and were used for ChIP-seq experiments. The functionality of these constructs was also confirmed by successful complementation of the arr1/10/12 triple mutant phenotype ). We found that all three B-ARRs had similar pattern expressed in roots, hypocotyl, and cotyledons . Consistent with previous findings, all three B-ARRs were localized in the nucleus31. However, the localization of ARR1 upon 6-BA treatment was more obvious in root tip and cotyledons than in other tissues . Using the same standards, the cytokinin treated samples had higher numbers of targets for all three B-ARRs (5128 (ARR1_BA), 6272 (ARR10_BA), and 6240 (ARR12_BA)). An increase in the number of targets upon cytokinin treatment might result from either protein stabilization or modification by phosphorylation11 or both processes. Interestingly, samples treated with 10 µM 6-BA for three days showed even further increase in the number of targets for ARR1, up to approximately 10,000. B-ARR binding sites detected by ChIP-seq were highly enriched near gene transcription start sites (TSS)  of three key B-ARRs 1, 10, 12) were identified by ChIP-seq using long-day (16-h light/8 h dark cycle) conditions at 22 °C and 3-days-old seedlings growing vertically on plates containing Murashige and Skoog (MS) medium  as nodes  of ARR10 mock targets overlap with 4 h 6-BA targets , well-known cytokinin response genes, were among the targets of B-ARRs  out of 4004 potential targets were also identified in our study , cytokinin receptor (WOL/AtHK4), and cytokinin degradation enzyme (CKX5) and showing a 10-fold enrichment . Upon exogenous cytokinin treatment (6-BA treatment for 4 h to 3 days), B-ARRs show significant increase of binding to A-ARRs . In contrast, there was not much change in binding at the promoter of the EIN3 binding factor (EBF2)  Fig. , a negatets Fig. . Overall36. To find out whether this TF-governed auto-regulation and cross-regulation with other pathways also holds true or not for B-ARRs, we performed gene ontology (GO) analysis of target genes of B-ARRs using the top 3000 genes ranked by IDR score37. These analyses revealed enrichment for similar biological function and processes for all tested B-ARRs . Among the 3373 common targets of the three B-ARRs (ARR1/ARR10/ARR12) tested, we observed enrichment of hormone-related genes  were found in the B-ARR target gene list as being under control of B-ARRs. In addition, B-ARRs were found to bind at cytokinin biosynthesis and degradation pathway genes , and the GH3s genes which mediate auxin conjugation. Auxin transcriptional response regulators, including several ARFs and many Aux/IAAs genes, were also among the list of B-ARR targets , as well as modification enzyme for salicylic acid (SA)  of 158 up-regulated genes in the golden list were identified among the 8770 union binding targets of three B-ARRs, suggesting significant association between B-ARR binding and cytokinin-induced transcriptional responses . When compared to the 3373 common targets of three B-ARRs, the percentage of overlap dropped to 35.4% which is still highly significant . Similarly, 35 (51.4%) of 68 down-regulated genes in the golden list were overlapped among the 8770 union targets of B-ARRs (Fisher’s Exact Test p < 0.001) although only 26.5% of these were among the 3373 common targets of the three key B-ARRs . A small portion (74 of 226 or 32.7%) of genes in the golden list were not found in the list of B-ARRs targets. They might be either indirect cytokinin response genes or the targets of other B-ARRs that were not tested in this experiment. It is interesting that activators like B-ARRs directly target cytokinin repressed genes which may involve the recruitment of other co-regulators. Only 490 (14.5%) of 3373 common targets of three B-ARRs (ARR1/ARR10/ARR12) were affected transcriptionally either in triple mutant or by cytokinin treatment .Numerous studies have reported that TF binding does not necessarily coincide with changes in gene transcriptionent Fig. . A subseent Fig. . When an52. Second, after cytokinin treatment, all binding motifs strikingly become invariant AGAT((t/a/c)(t/c)) which we have termed the B-ARR-6-BA motif (AGATHY) . This ARR1 version does not possess the receiver domain and represents a constitutive active form of ARR118. ChIP-seq and MEME analysis of ARR1∆DDK uncovered the B-ARR-6-BA motif as the primary binding motif , indicating that cytokinin can directly affect B-ARR binding through the receiver domain. Although previous in vitro-derived B-ARR binding-motifs were identified by Weirauch et al.53, our study reveals that the subfamily-1 B-ARRs share a similar DNA-binding motif and provides direct in vivo evidence of the DNA-binding signatures of B-ARRs. The similarity between 6-BA-treatment-specific DNA binding signature of B-ARRs and that of the constitutive active ARR1∆DDK provides additional insight into the in planta mechanism by which cytokinin modulates TF function. The B-ARR-6-BA motif was enriched in the promoter regions (−1.5 k to + 100 bps surrounding the TSS of 83% (3334/4012) of ARR1 bound genes compared to the whole genome background  is higher in WUS domain than any of the other domains in shoot apical meristem56. Second, cytokinin induces expression of a WUS transcriptional reporter gene22. Interestingly, as mentioned above WUS consistently appears as a low-ranking target gene in ChIP-seq analyses for each of the tested B-ARRs in plants not exposed to exogenous cytokinin. One possibility is that binding of B-ARRs to the promoter of WUS requires an elevated level of cytokinin. When plants were grown with cytokinin, we identified a single binding site in the WUS promoter with the B-ARR-6-BA motif (AGATAT) located at the peak summit  concentrations of cytokinin22. Since a high concentration of cytokinin was required to activate WUS, the effect had been previously thought to be indirect. However, our results demonstrate the presence of a B-ARR-6-BA motif in the WUS promoter, implying that WUS may be an in vivo target of B-ARRs. Interestingly, the B-ARR-6-BA motif is adjacent to HD-ZIP response elements, suggesting possible interactions with other TFs that may be required for stem cell maintenance in the shoot apical meristem  of ARR1 (a B-ARR) has been shown to constitutively activate cytokinin signaling by unmasking the transactivation function of ARR115. The ARR1 deletion construct was fused to the glucocorticoid-inducible artificial transcription factor (GR) and expressed from the cauliflower mosaic virus (CaMV) 35S coat protein gene promoter to generate dexamethasone (Dex) inducible 35S:ARR1∆DDK:GR15. In the absence of dexamethasone (Dex), transgenic plants carrying the 35S:ARR1∆DDK:GR construct were phenotypically normal , a fluorescent reporter for stem-cells and 35S::YFP29-1  and marker for cell boundaries which allows visualization of all SAM cells. We observed that constitutive activation of cytokinin signaling led to expansion of stem-cell domain marked by the pCLV3 reporter upon induction of ARR1∆DDK:GR  was observed. When expression of the transgene was induced by Dex treatment, all ten characterized lines harboring both pCLV3:LhG4 and pMX6xOPs:ARR1∆DDK:GR showed similar phenotypes at the seedling stage  shows a statistically significant overlap with the targets that we identified using recombineered ARR genes . The ARR10 over-expressing ChIP-Seq results shared 2783 gene targets with our ARR10_BA and had 1221 unique targets not identified by ARR10_BA. Interestingly, the 2783 overlapping target genes found by both studies had higher peak scores than unique target genes , short BA treatment binding (targets found in both 4-hour and 3-day BA treatment as 2nd), and longer BA treatment binding (targets found only in 3-day BA treatment as 3rd) . B-ARRs are not regulated at transcriptional level by cytokinin but are post-transcriptionally modifiedWUS gene, which encodes a homeodomain TF that was shown to repress a subset of A-ARRs55. WUS has been suggested to work in conjunction with the cytokinin pathway24 to establish a stem cell niche ensuring early embryogenesis and later for maintenance of the shoot apical meristem55. The study using the new reporter revealed that the WUS expression domain overlaps with SAM regions where cytokinin activity is the highest68. Similarly, the pWUS::GFP-er reporter can be activated in SAM by cytokinin but only at a high concentration of cytokinin. However, subsequent (expected) activation of a pCLV3::GFP-er reporter was not observed22. A previous explanation of why WUS does not robustly respond to low cytokinin levels was that the expression of WUS was only in the a specific (WUS) subdomain of the SAM and that the cytokinin was not accessible to the WUS domain. Our results indicate that most of the potential binding motifs of B-ARRs in the promoter region of WUS were unoccupied. However, we identified one B-ARR-6-BA motif, located precisely at the peak summit of the conservative narrow peak, in multiple B-ARRs data sets. The identification of a strong in vivo B-ARR DNA binding site within the promoter of WUS that requires a high level of cytokinin, provides new direct evidence for this association. In addition, the B-ARR-6-BA motif (−420bp) was found to be within 57 bps as two HD ZIP response elements (−540 to −565) that were previously identified as WUS regulatory sites69. It is possible that the coordination or competition among these TFs is a feature of WUS regulation, which is itself controlled by a WUS-CLV3 negative feedback loop, keeping the meristem size constant in each species. Moreover, the activation of WUS by B-ARRs downstream of cytokinin signaling should also be tightly attenuated in the shoot apical meristem. When a constitutive active form of ARR1 was introduced, removing the possibility of feedback inhibition, the activation of WUS led to an expansion of stem cell domain. Interestingly, ectopic expression of ARR1∆DDK did not lead to an enlarged SAM, indicating the possible presence of an unknown inhibition mechanism from other domains within the SAM. Expression of ARR1∆DDK, under the control of pCLV3 promoter, resulted in an enlarged shoot apical meristem . This phenotype may result from activation of WUS by B-ARRs in the stem cell that cannot be dampened. Since type-A ARRs were highly activated by B-ARRs, increasing the level of type-A ARRs may cause increased expression of CLV3 as ARR7 and ARR15 are required for the expression of CLV323. However, as negative regulators, the inhibition mechanism of type-A ARRs to cytokinin signaling remains unknown11. Similarly, the activation mechanism of type-A ARRs leading to CLV3 expression is also not clear. If CLV3 is the solely interface between B-ARR and A-ARRs leading to activate of CLV3, then transcription of WUS would be expected to shut down.Importantly, we identified a B-ARR binding site in the promoter of WUS and CLV3 expression observed in pCLV3:ARR1∆DDK system suggests that B-ARRs activate the transcription of WUS, then activate CLV3 leading to expand the shoot apical meristem, resulting in the “ball “of SAM. Previous reports of pCLV3:WUS expression resulted in similar phenotypes, further providing functional relevance to our model62. This result is also consistent with the idea that multiple feedback loops exist in the shoot apical meristem to adjust its size22. The introduction of a mutation in the B-ARR binding site in the promoter of WUS would test the idea that WUS is, at least in part, under direct transcriptional control of B-ARRs. Taken together, these findings provide new insights to the role of the cytokinin transcriptional responses in stem cell maintenance.The “ball” of the SAM phenotype and high levels of both 20.Three-days-old seedling tissue was collected for these experiments unless otherwise noted. Seeds were surface-sterilized and sown on agar plates (1.8%) containing Murashige and Skoog salts (pH 5.7) and 1% sucrose. Seedlings were grown vertically for 3 days at 22 °C under long day condition using a 16-h light/8-h dark cycle. The seedling was subsequently treated with liquid Murashige and Skoog medium (MS) pH 5.7 with 0.08% Silwet 77 containing 10 µM 6-BA (in DMSO) or mock treated with an equivalent volume of DMSO. The three-days continuous hormone treatment was orchestrated by germinating the tagged lines on MS with 10 µM 6-BA or MS with the same amount of DMSO as mock. 10 µM 6-BA was chosen based on the range from 1 µM to 20 µM cytokinin treatment28, a Ypet (yellow fluorescent protein) tag was recombineered into a transformable bacteria clone (TAC) clone such that the B-ARR gene was located at the center of the large insert clone. We employed the two-step recombineering method using the Flapase–Fret system. Positive Ypet clones were obtained first by selection against ampicillin as the insert contained an ampicillin resistant gene. This ampicillin marker was later removed. While the technique leaves a small scar between Ypet and the B-ARRs the method is easier than the GalK system28 and the scar serve as a linker between the tag and the B-ARRs. The Ypet gene was added at the C-terminus of each B-ARR gene of the whole B-ARR family but only ARRs 1, 10, 12, 11, 13, and 14 were successful. ARR1 was tagged using isoform ARR1.2 since the previous expression data indicated ARR1.2 as the major splice variant. The tagged TACs were sequenced to confirm sequence fidelity of the B-ARRs, the junction, and the fusion to Ypet within TAC. The tagged TAC clones were transformed into GV3101 strains and transformed wild type Columbia-0 plants using the flower dipping method70. After screening for Basta resistance, putative tagged lines were identified by PCR using forward recombineering test primer and reverse Ypet primers. The PCR products were gel-purified and sequenced to confirm the in planta tagging junction. The expression of the tagged B-ARRs  was then examined under Zeiss confocal microscopy 710 (Zeiss) using identical settings .Based on recombineering techniquesARR1 that was previously described18. The deletion called ARR1∆DDK was cloned into pENTR D/TOPO with GR fusion at the C-terminus and then the entire ARR1∆DDK:GR gene cassette was moved into the pEG104 transformation vector to be in frame of a N-terminus YFP71. The pENTR D/TOPO: ARR1∆DDK:GR was cloned into pMX6xOPs vector and then the transgenic line was combined with the pCLV3:LhG4 line to achieve stem cell-specific expression of ARR1∆DDK.The active form of ARR1 was constructed using a deletion of the receiver domain of 62. The pCLV3:LhG4 line was in Landsberg erecta (Ler-0) background. The pMX6xOPs:ARR1∆DDK:GR was transformed into Ler-0 background. The pMX6xOPs:ARR1∆DDK:GR lines were screened using gentamicin. Ten individual lines were used to cross with the pCLV3:LhG4 line and were tested on MS medium with 10 µM Dexamethasone (Dex). MS medium with the same amount of ethanol was used as control. All ten lines showed the enlarged apical meristem phenotype. Initial observation of phenotypes was done by germinating seeds directly on Dex plates. The pCLV3:LhG4/pMX6xOPs:WUS:GR line was provided by Dr. Reddy’s lab and the same induction method was used.The two-components inducible system was used36. Chromatin immunoprecipitation (ChIP) was performed as previously described36 with modifications, including the use of Bioruptor sonicator . Bioruptor settings used were: Low, 10 cycles of 25 s on, 120 s off. Sonication was performed in auto-cooling system with water bath at 4 °C. A small amount of chromatin (10 µl) was evaluated for shearing; the size range of chromatin was 150–450 bps, most fragments at 250 bps.Seedlings were dried by paper towel and transferred in 1% formaldehyde solution. Cross-linking occurred under 5-10-5-min vacuum cycles with a quick vacuum release in between each cycle. A final concentration of 125 mM glycine was applied for 5 min to deactivate the remaining formaldehyde. Cross-linking resulted in translucent seedling tissue. Tissue was then liquid nitrogen cooled and either stored in −80 °C degree or directly ground and an extraction of chromatin was performed as previously described36 at 4 °C overnight (16 h). The Dynabeads were washed using a low stringent wash buffer followed by a high stringent buffer36, a quick rinsing, and 5 min rotating at 4 °C. A final wash buffer was applied to clear the detergent from previous buffers followed by another 5 min rotating. During each wash, the tubes were quickly centrifuged before returning to the magnetic stand and trace amounts of buffer were removed to avoid non-specific binding carryover. The resulting ChIP DNA was collected in two elution buffers (100 µl each) at 65 °C and combined. The proteinase K digestion occurred at 55 °C. The reverse of cross-linking was done at 65 °C overnight (16 h). The ChIP DNA was then purified by extraction using phenol:chloroform:isoamyl alcohol (25:24:1)  twice in a phase lock gel system before the ethanol precipitation step using glycogen for the pellet observation. The pellets were then washed with 70% cooled ethanol and dried in a speed-vac. The ChIP DNA was then dissolved in 50 µl water for subsequent library preparation.The commercial anti-GFP antibody  was used for the immunoprecipitation reactions. Five microgram antibody and 50 µl of Dynabeads M-280 Sheep anti-Rabbit IgG  were consumed for each reaction. The incubation was performed using previously described bufferThe single-end read libraries were prepared using the TruSeq ChIP kit from Illumina , with DNA size selection made with lab-made Serapure magnetic beads. Library concentration were assayed using the Qubit 2.0 Fluorometer (Thermo Fisher Scientific) and normalized to 1.7 Nano gram per microliter. Six multiplexed libraries were sequenced per lane on a HiSeq 2000.72. MACS2 was used to call peaks compared to input using q_value_thresholds = 0.05, 0.01, 0.001. The aligned reads with at least two biological replicates were processed using the irreproducibility discovery rate (IDR)73. The p-value cutoff was 1e-16. Peaks were then annotated using ChIPpeakAnno package. In order for us to classify a peak as associating with a gene, the peak summit must have been within 1.5 kb upstream and downstream of the gene’s annotation. The gene length was normalized to 1 kb to describe average enrichment of peaks around gene body TSS. The B-ARR-6-BA motif matrix was used by Homer74 to identify the locations of the motif sequence on the whole genome. Promoter regions were defined as −1500 to +100 bps relative to the TSS. Gene ontology analysis was conducted using DAVID GO the top 3000 genes ranked by IDR peak score of each experiment were evaluated for GO term overrepresentation . Total 200 bps flanking the binding summits of 1000 top-ranking peaks for each experiment were also used for identification of each DNA binding motif using MEME-ChIP suite75. The list of genes associated with cytokinin pathway was downloaded from Gene Ontology Consortium (http://www.geneontology.org)76. The heatmap of the B-ARRs’ target genes was hierarchical clustered based on the Euclidean distances, calculated from the number of shared targets between each factor for different conditions. A directed network was constructed based on the relationship between TFs and their binding targets by igraph (http://igraph.org)77 with nodes representing either type-A ARR or TFs with significant transcriptional changes defined by the meta-analysis 78. DAP-Seq data were downloaded from the website  and only samples without “amp” label were used for this analysis33.ChIP-seq data analyses were carried out using a suite of pipelines developed in our lab to run the alignment and analysis. Briefly, alignment was performed using Bowtie1 with parameters “ -m 1–best–strata -S–chunkmbs 200”arr1/10/12 triple mutant with the wild type, three-days-old seedlings were collected without any treatment. For cytokinin treatment, 10 µM 6-BA or an equal volume of ethanol was applied to each sample. RNA was isolated using RNeasy plant kit  and libraries were prepared by NeoPre Library Prep System (Illumina). Alignments were done by Tophat 279 and differential expression was called by CuffDiff 80. The significantly differentially expressed genes used 1.6-fold change and q-value <= 0.05 as cutoff.To compare the GSE94486. The authors declare that all other data supporting the findings of this study are available within the manuscript and its supplementary files or are available from the corresponding author upon request.Raw and processed data can be found with GEO deposition accession number Supplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Supplementary Data 6Supplementary Data 7Supplementary Data 8Supplementary Data 9Supplementary Data 10


Gene control systems sometimes interpret multiple signals to set the expression levels of the genes they regulate. In rare instances, ligand-binding riboswitch aptamers form tandem arrangements to approximate the function of specific two-input Boolean logic gates. Here, we report the discovery of riboswitch aptamers for phosphoribosyl pyrophosphate (PRPP) that naturally exist either in singlet arrangements, or occur in tandem with guanine aptamers. Tandem guanine-PRPP aptamers can bind the target ligands, either independently or in combination, to approximate the function expected for an IMPLY Boolean logic gate to regulate transcription of messenger RNAs for de novo purine biosynthesis in bacteria. The existence of sophisticated all-RNA regulatory systems that sense two ancient ribonucleotide derivatives to control synthesis of RNA molecules supports the hypothesis that RNA World organisms could have managed a complex metabolic state without the assistance of protein regulatory factors. Life on Earth is magnificently complex. The instructions to make this complexity are stored within the genes of living cells. For many genes, this process involves two steps. First, the cells copy, or transcribe, the information in the gene – which is part of a DNA molecule – into a more portable but shorter-lived molecule called RNA. The RNA can then be transported to other parts of a cell where its information is decoded to make a protein. Proteins are like molecular machines and making proteins is how many genes contribute to keeping life going.RNA molecules can fold into intricate shapes including structures called riboswitches. These are parts of an RNA molecule that can control how and when the RNA is used to make proteins. Riboswitches detect the presence of other chemicals inside a cell and respond by changing their shape. Binding to the right molecule will move a riboswitch from an “off” to an “on” position, or vice versa. Riboswitches can help to ensure that some proteins are only made when they are needed. For example, a protein that helps to make a certain chemical is only produced when that chemical is in short supply.Facklamia ignava. The investigation revealed a new riboswitch structure that detects phosphoribosyl pyrophosphate, or PRPP, which is a building block for RNA and, eventually, DNA molecules. Further examination revealed that these riboswitches are often linked to others that detect guanine molecules – another component of RNA and DNA. This double riboswitch is similar to a logic gate in computing, activating or shutting down protein production in response to changes in the concentrations of both PRPP and guanine.Sherlock et al. have now studied riboswitches in a species of bacteria called The findings of Sherlock et al. reveal a system where RNA controls its own production in response to the availability of two of its critical building blocks. This entirely RNA-dependent system adds weight to the so-called ‘RNA world’ hypothesis which suggests that the earliest forms of life on Earth depended solely on RNA. Molecules of RNA are able to store data like DNA while also behaving as molecular machines like proteins. This potentially helps to explain how the first life could have arisen from simple, non-living chemicals. Riboswitch systems like this are likely to be relics of this ancient time. Also, because they are involved in fundamental processes in living bacteria, riboswitch systems also provide potentially interesting targets for new antibiotics. A variety of riboswitch classes regulate gene expression in response to the binding of specific metabolite or inorganic ion ligands . PreviouykkC orphan riboswitch candidate because no ligand had been identified . Upon further investigation of the RNA sequences, the phylogeny of organisms containing these RNAs, and the genes associated with each instance of the RNA, we concluded that ykkC subtype 2 RNAs were populated by multiple riboswitch classes that sensed distinct ligands.With these considerations in mind, we evaluated the possible functions of variants of recently validated riboswitches for guanidine . Collectentified . After rykkC RNA motif to create revised consensus models for the RNAs within each gene association group. In some cases, additional conserved nucleotides were observed immediately 5´ and 3´ of the three hairpin structures  that are characteristic of ykkC motif RNAs. This region located downstream of the guanidine binding site of subtype 1 RNAs were found to be unnecessary for guanidine-I riboswitches. In total, these distinct representatives were sorted into four candidate riboswitch classes called subtypes 2a through 2d.Subtype 2 representatives were sorted according to the gene located immediately downstream from the ykkC RNAs are involved in forming tertiary contacts within the larger folded structure that positions other nucleotides to form the ligand-binding pocket. However, nucleotides that change identity and are characteristic of each subtype are almost exclusively found in close proximity to the known binding pocket of guanidine-I riboswitches , a gene involved in pyrimidine salvage that is known to be repressed by the purine guanine   as the lynthesis . In addi guanine , is alsoykkC subtype 2b RNAs distributed throughout bacteria of the phylum Firmicutes. Of these, 127 examples occur in a tandem arrangement  and subtype 2b RNAs are remarkably alike, including many similarities at the ligand binding site, suggesting that their ligands might also share some chemical features. Particularly notable was the observation that a long-range C-G base-pair present in guanidine-I riboswitches  is absenpurC gene of the bacterium Facklamia ignava  or for the guanine riboswitch (guanine) are added. Importantly, the structural changes selectively brought about by PRPP largely occur in regions equivalent to those previously implicated in ligand binding by guanidine-I  is less than 100 µM  and ΔpurF Bacillus subtilis cells. Deletion of the purF gene, which encodes the enzyme that catalyzes the first committed step of de novo purine biosynthesis (lacZ gene is expressed (blue) in glucose minimal medium in the ΔpurF strain, but not (clear) in the parental B. subtilis strain with a functional purF gene  in the terminator stem expresses ongation .codA gene of Bacillus megaterium  confirm the OFF and ON function of the guanine and PRPP aptamers, respectively. Specifically, the mutations in M3 alter two highly-conserved nucleotides in the core of the guanine aptamer that are known to be essential for forming its ligand-binding core . As expeis cells .The versatility of this tandem riboswitch system is best demonstrated by varying the concentrations of both guanine and PRPP in a series of in vitro transcription termination assays. The data from these reactions yields a 3D landscape demonstrating again that the relative concentrations of guanine and PRPP dictate the fraction of RNA transcripts that reach full length . Thus, tAt the single molecule level, each riboswitch aptamer in this tandem construct favors either transcription termination or elongation based on the binding of its cognate ligand. In this regard, each RNA molecule carrying this tandem aptamer arrangement functions as a Boolean IMPLY logic gate. The truth table for an IMPLY logic gate  indicateWhen transcription termination or gene expression is observed as the average output from a population of RNA constructs, the resulting data do not represent a perfect Boolean function because the output yields intermediate values between zero and maximal values. Whereas each of the individual RNAs operates as a binary logic gate that is either on or off, a population of these RNAs exceeds the function of an individual Boolean logic gate. Together, the output of the tandem riboswitch population functions more like a molecular rheostat to tune gene expression based on relative ligand concentrations, rather than as an all-or-nothing binary switch. Regardless, when ligand concentrations are at their extreme values, the output of the tandem system  approachA situation in cells in which both guanine and PRPP are scarce would indicate an extremely starved state. Ordinarily, one might expect guanine and PRPP levels to be negatively correlated, such that an excess of guanine would deplete PRPP as a result of the synthesis of 5′-guanosine monophosphate, and vice versa, either through de novo purine biosynthesis or the purine salvage pathway . If trueDuring the stringent response in Firmicutes, nutrient starvation is signaled by the presence of uncharged tRNAs that activate the ppGpp synthase RelA . ppGpp sUnder these conditions the guanine-PRPP tandem riboswitch still turns on expression of its downstream genes, despite the mechanisms in place to keep GTP levels low. At first this appears counterintuitive, but the genes associated with the tandem riboswitch system only include those for catalyzing the steps of de novo purine synthesis up to the production of inosine monophosphate (IMP), the branch point between AMP and GMP production . Thus, tS-adenosylmethionine (SAM) and adenosylcobalamin (AdoCbl or coenzyme B12) riboswitches  to approximate the function of a Boolean AND gate . In thisMost experimentally validated riboswitch classes sense and respond to ligands originating from RNA metabolism pathways, and therefore it seemed reasonable to consider the likelihood that riboswitches for PRPP would exist. PRPP is an essential precursor for the modern biosynthesis of RNA monomers in organisms from all three domains of life, and is likely to have been important for the production of ribonucleotides very early in evolution. If PRPP was used by RNA World organisms to generate the building blocks of this major biopolymer, then RNA aptamers that selectively bound this biosynthetic precursor in ancient times might have persisted to serve as riboswitch aptamers in modern cells.https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2011097027&recNum=27&docAn=US2011000204&queryString=(riboswitch)%2520&maxRec=179; Intriguingly, we had representatives of PRPP riboswitches on our list of riboswitch candidates over a decade ago , but dueykkC motif RNAs whose ligands have been identified, the chemical differences in the ligands can be substantial. The ppGpp (subtype 2a) and PRPP (subtype 2b) riboswitch ligands differ by the presence or absence of a guanine nucleobase and by the location and number of phosphate moieties. Guanidinium, the ligand for guanidine-I (subtype 1) riboswitches is entirely distinct from PRPP, and this moiety forms only a portion of the purine ring of ppGpp. The ligands sensed by subtypes 2 c and 2d, which are associated with genes encoding nucleoside diphosphate linked to X (NUDIX) hydrolases of unknown specificity and a transporter of unknown function associated with phosphonate utilization (phn_DUF6), respectively, still remain to be discovered.For the subtypes of ykkC subtype 2c and 2d RNAs each have more prominent changes compared to subtype 2a versus 2b at nucleotide positions that are known to be involved in ligand recognition , ligand concentrations for riboswitches can be highly variable. Therefore, given the nature of ligand binding to receptors (each described by an equilibrium constant equation), differences in ligand concentrations will result in differences in the probabilities that the two riboswitch aptamers will be occupied. This occupancy distribution will be reflected in the transcriptional output of the RNA population, but each individual transcript still yields a binary output (terminated or full-length product) that is biased by the concentrations of ligands present.The IMPLY gate described herein involves a riboswitch RNA with aptamers for the RNA nucleobase guanine and the RNA precursor PRPP. While tandem riboswitch systems have previously been described , the gua32P]-ATP and [α-32P]-UTP were purchased from Perkin Elmer and used within two weeks of receipt. Bulk chemicals were purchased from J.T. Baker and enzymes from New England Biolabs, unless otherwise noted. All solutions were prepared using deionized water (dH2O) and were either autoclaved or filter sterilized  prior to use. DNA oligonucleotides were purchased from Sigma-Aldrich and Integrated DNA Technologies. A list of oligonucleotides used in this study can be found in Chemicals were purchased from Sigma-Aldrich with the exceptions of phosphoribosyl pyrophosphate (Santa Cruz Biotech) and guanosine 5′,3′-bisdiphosphate (TriLink Biotech). -UTP was added per 8 µL reaction and transcription was allowed to proceed at 37°C for 10 min, leading to formation of a stalled polymerase complex at the first cytidine nucleotide of each transcript. For each 8 µL transcription reaction, 1 µL of 10x elongation buffer  as well as 1 µL of a 10x solution of the ligand of interest were added sequentially. Transcription reactions were then incubated at 37°C for an additional 60 min.Approximately 2 pmol of the resulting, purified DNA template was added to a transcription initiation mixture  10% PAGE and visualized using a phosphorimager . Fraction full length values were calculated by varying the ligand concentration in separate reactions and quantifying the changes in band intensity of both full length (FL) and terminated (T) transcription products using the formula (FL)/(FL +T). The TB. subtilis lysC gene promoter and either the H. modesticaldum or B. megaterium riboswitch fused upstream of the E. coli lacZ gene were inserted into the vector pDG1661 as described previously (amyE locus of wild-type WT (1A1 strain 168 Δtrp) or ΔpurF strains (BKE06490 [erythromycin resistant] and BKK06490 [kanamycin resistant]) obtained from the Bacillus Genetic Stock Center, The Ohio State University) B. subtilis  or kanamycin-resistance gene (ΔpurF::kan strains) as expected. Mutations were incorporated into the B. megaterium reporter construct by using Quikchange site-directed mutagenesis (Agilent Technologies).Riboswitch-reporter constructs consisting of the eviously  and intesubtilis . The resB. subtilis strains into Lysogeny Broth (LB) with appropriate antibiotics and growing overnight at 37°C. For experiments with the singlet H. modesticaldum reporter, the bacteria were diluted into lysogeny broth (LB), grown for an additional 4–6 hr until culture growth was visible, centrifuged, supernatant removed, the resuspended in glucose minimal media (GMM). For experiments with the tandem B. megaterium reporter, bacteria were diluted directly into either LB or GMM, as indicated, and grown for 6 hr with or without supplemented guanine. Synthetic DNAs used in cloning are listed in Reporter experiments were performed by inoculating various  In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by Timothy Nilsen as Reviewing Editor and Naama Barkai as the Senior Editor. The following individual involved in review of your submission has agreed to reveal his identity: Ailong Ke (Reviewer #1).Thank you for submitting your article "Tandem riboswitches form a natural Boolean logic gate to control purine metabolism in bacteria" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.eLife. Since all of the points raised by the referees can be addressed by fairly minor textual changes, the entire reviews are included. Please address all comments.There was a consensus that this work reports a novel type of RNA-based logic gate that uses a regulatory mechanism distinct from previously described tandem riboswitches. The work was performed rigorously and all the reviewers felt that it was, in principle, suitable for publication in Reviewer #1:The manuscript by Sherlock et al. describes the function and mechanism of a riboswitch class that resembles the guanidine-I riboswitch in structure, but senses a different ligand, PRPP, to regulate the purine synthesis pathway. In the first half of the study, the specificity of these riboswitches for PRPP is proven through in-line probing analysis. This combined with transcription read-through assays and in vivo expression studies provides convincing evidence that these riboswitches only respond to PRPP and not guanidine. In the second part, the authors describe the IMPLY logic gate that PRPP riboswitches form in tandem with guanine riboswitches to regulate purine biosynthesis in starvation conditions. The regulatory properties of these tandem riboswitches in response to different ratios of PRPP and guanine is thoroughly characterized with in vitro and in vivo experiments. This is a novel type of RNA-based logic gate that utilizes a regulatory mechanism distinct from other tandem riboswitches described previously. Therefore, this work has significance to the riboswitch and RNA biology fields.eLife. There is a major point I would ask the authors to consider.Overall, the manuscript is polished with only a few areas needing some proof-reading, the conclusions are sound, and the significance qualifies it for the broad readership of While the authors convincingly show the regulatory effects of the guanine-PRPP tandem switch, the mechanism by which the two aptamers communicate to alter expression is not given. The authors suggest steric hindrance as a possible mechanism; would extending the linker between the guanine and PRPP domains change their regulatory outcome? Is it possible to make a mutant that retains binding activity in both aptamers, but loses the ability to repress transcription in response to guanine? Furthermore, the authors show in their in-line probing experiments that an AAAAA sequence between the guanine and PRPP domains has altered scission depending on if guanine or PRPP is present. Can the authors suggest a possible role for this sequence?Reviewer #2:This riboswitch-focused manuscript includes a full range of the methods that are typically used to validate new riboswitch classes; therefore, this represents a thorough validation of a new and important riboswitch class. It is particularly interesting as it discovers PRPP as the ligand for a subclass (ykkC-2b) of a previously identified riboswitch class.eLife as opposed to other more specialty journals. A subset of the reasons that it does are as follows:It is a well-written manuscript, with a thorough Discussion and effective figures; I have no criticisms or substantive reservations with the manuscript as written. Therefore, the only real question is whether the manuscript warrants publication in - This manuscript is effectively a meaningful "sequel" to a prior publication, which suggested that some bacteria might use tandem riboswitches that respond to combinatorial inputs. The current manuscript reveals how these original tandem riboswitches function, and how they are likely to influence bacterial stress responses.- The model for the logic behind the tandem riboswitch  makes perfect sense. Moreover, it meaningfully contributes to the general understanding of the types of cellular responses that occur during ppGpp signaling.- While some riboswitch classes have been found to have members that respond to a ligand that is modestly different than is sensed by the overall riboswitch family, the "ykkC" riboswitch class is truly remarkable in this regard. This manuscript demonstrates that the evolutionary malleability of the "ykkC" structural scaffold is incredible, with subclasses that have been shown to respond to guanidinium, ppGpp, and now PRPP, and still with several more uncharacterized classes. This important data reveals that riboswitch classes must be carefully analyzed by bioinformatics techniques in order to try and identify meaningful subclasses. In other words, the emerging "ykkC" story serves as a precautionary reminder that one cannot assume that all members of a riboswitch class behave the exact same, without further bioinformatic or biochemical analysis.- This manuscript adds substantially to the discussion on how combinations of riboswitches can act as different types of Boolean logic gates. This is important for understanding the complex regulatory signals that underlie genetic regulation of biological functions in bacteria, but also because it helps set the stage for development of synthetic riboswitch RNAs.The only mark against the manuscript is that it is unable to provide a molecular description of the exact mechanism of allosteric connectivity between the guanine and PRPP aptamers; however, I agree with the authors that this is well beyond the scope of this current manuscript.Reviewer #3:ELife. The first major finding is identification of a cognate ligand for a subtype of the orphan ykkC riboswitch. Surprisingly, this ligand, PRPP, is very different from guanidine, a ligand for another subtype of the same riboswitch type. Therefore, the PRPP and guanidine riboswitches are the first example of riboswitches that appear to share the same or very similar structures but recognize ligands with distinct physicochemical properties.The study by Sherlock and coauthors presents several findings, which will be of interest to the broad readership of Another major finding is characterization of a tandem riboswitch arrangement involving guanine and PRPP sensors. Integration of the two sensors and a single expression platform results in a complex biological readout that approximates the Boolean IMPLY logic gate. Such tandem riboswitch arrangement adds to the growing collection of complex regulatory outputs from the RNA-only regulatory elements. The study is performed according to the highest standards of the Breaker laboratory and I do not have any major concerns.I would only recommend the authors to make additional analysis of the linker between guanine and PRPP sensors in the tandem riboswitches. The tandem riboswitch requires overlapping sequences of the sensors to participate in the formation of mutually exclusive ligand-binding structures in order to function as the IMPLY gate. While the majority of the linkers are short (≤5 nt), as reflected in the consensus sequence, a number of them are longer than 5 nt, with ~20% being 7 nt long. The in-line probing experiments were performed on the riboswitch with a 6-nt linker, and while there was no sequence overlap between the sensors, formation of the PRPP sensor affected the 3' nucleotide of the guanine sensor, thereby explaining the IMPLY mechanism. With a 7-mer linker, the IMPLY mechanism may not be efficient and I wonder whether there is a simple explanation for having such outliers. Is the P1 helix of these riboswitches longer than 7 base pairs? If not, these riboswitches could be an interesting system to test in future studies, and the authors may comment on this observation. Reviewer #1:[…] Overall, the manuscript is polished with only a few areas needing some proof-reading, the conclusions are sound, and the significance qualifies it for the broad readership of eLife. There is a major point I would ask the authors to consider.While the authors convincingly show the regulatory effects of the guanine-PRPP tandem switch, the mechanism by which the two aptamers communicate to alter expression is not given. The authors suggest steric hindrance as a possible mechanism; would extending the linker between the guanine and PRPP domains change their regulatory outcome? Is it possible to make a mutant that retains binding activity in both aptamers, but loses the ability to repress transcription in response to guanine? Furthermore, the authors show in their in-line probing experiments that an AAAAA sequence between the guanine and PRPP domains has altered scission depending on if guanine or PRPP is present. Can the authors suggest a possible role for this sequence?This is the major topic for comments from all three reviewers. We had initially judged that a careful assessment of the mechanism of inter-aptamer communication would take considerable biochemical and biophysical research campaign. As a result, in the current manuscript, we only discussed two clues regarding how these aptamers interact . We felt that a deeper understanding of the mechanism was beyond the scope of our report of the discovery and initial validation of this new riboswitch class. Specifically, we noted that the two aptamers are always located immediately adjacent to each other. In addition, the relative flexibility of the conserved 5’ portion of the PRPP aptamer is dependent on the ligand occupancy of the two aptamers. These two observations allow us to tentatively conclude that this region is likely to be critical for the mutually-exclusive formation of the two aptamers. It now turns out that our speculation is accurate, but we must consider two additional concerns when preparing our revised manuscript as described below:First, both reviewer 2 and reviewer 3 note that the distance between the two aptamers is not perfectly conserved, and that some natural representatives have one nucleotide more or less compared to the construct we tested. This is true, and therefore we needed to soften our statement in the paragraph summarizing the clues regarding the mechanism of inter-aptamer communication. The relevant sentences now read as follows:“First, guanine aptamers are always located almost immediately adjacent to the conserved nucleotides at the 5ˊ terminus of the PPRP aptamer in tandem arrangements . Specifically, there are typically only 5 to 7 nucleotides separating the P1 stems of the guanine and PRPP aptamers, suggesting this juxtaposition is important for logic gate function.”Second, our Yale colleagues in the laboratory of Scott Strobel have now generated an atomicresolution model of a PRPP aptamer, which demonstrates that the linker sequence indeed is an integral part of the PRPP aptamer structure formed upon binding ligand. Thus, our finding that the guanine riboswitch aptamer (when bound to guanine) destabilizes the structure of this adjacent linker sequence is entirely consistent with a model wherein the two aptamers form in a mutually-exclusive fashion. Because this atomic-resolution structure of the PRPP aptamer was completed independently by the Strobel laboratory, and given that this work has not yet been submitted for publication, we really must restrict our comments on the mechanism. This part of the story should be described by the Strobel laboratory, and therefore we have not added more discussion regarding the mechanism of logic gate function to our revised manuscript.Reviewer #2:[…] The only mark against the manuscript is that it is unable to provide a molecular description of the exact mechanism of allosteric connectivity between the guanine and PRPP aptamers; however, I agree with the authors that this is well beyond the scope of this current manuscript.See the response point above.Reviewer #3:[…] I would only recommend the authors to make additional analysis of the linker between guanine and PRPP sensors in the tandem riboswitches. The tandem riboswitch requires overlapping sequences of the sensors to participate in the formation of mutually exclusive ligand-binding structures in order to function as the IMPLY gate. While the majority of the linkers are short (≤5 nt), as reflected in the consensus sequence, a number of them are longer than 5 nt, with ~20% being 7 nt long. The in-line probing experiments were performed on the riboswitch with a 6-nt linker, and while there was no sequence overlap between the sensors, formation of the PRPP sensor affected the 3' nucleotide of the guanine sensor, thereby explaining the IMPLY mechanism. With a 7-mer linker, the IMPLY mechanism may not be efficient and I wonder whether there is a simple explanation for having such outliers. Is the P1 helix of these riboswitches longer than 7 base pairs? If not, these riboswitches could be an interesting system to test in future studies, and the authors may comment on this observation.See the response point above.


Hylocereus polyrhizus) fruit is a high-value, functional food, containing a high level of betalains. Several genes potentially related to betalain biosynthesis, such as cytochrome P450-like (CytP450-like), have been identified in pitaya fruit, while their transcriptional regulation remains unclear. In this work, the potential involvement of a WRKY transcription factor, HpWRKY44, in regulating CytP450-like1 expression in pitaya fruit was examined. HpWRKY44, a member of the Group 1 WRKY family, contains two conserved WRKY motifs and is localized in the nucleus. HpWRKY44 also exhibits trans-activation ability. Gene expression analysis showed that the expression of HpCytP450-like1 and HpWRKY44 increased steadily during pitaya fruit coloration, which corresponded with the production of elevated betalain levels in the fruit. HpWRKY44 was also demonstrated to directly bind to and activate the HpCytP450-like1 promoter via the recognition of the W-box element present in the promoter. Collectively, our findings indicate that HpWRKY44 transcriptionally activates HpCytP450-like1, which perhaps, at least in part, contributes to betalain biosynthesis in pitaya fruit. The information provided in the current study provides novel insights into the regulatory network associated with betalain biosynthesis during pitaya fruit coloration.Red pitaya ( L-tyrosine. More than 50 natural betalains have been reported and they are present only in species within the order Caryophyllales, such as beets, Amaranthus, prickly pear cactus and red pitaya fruit.1–3 The red-violet betacyanins and the yellow betaxanthins, are the two types of betalains.4 These pigments are not produced in the same plant or at the same time when anthocyanins, another major pigment group, are present.5 Betalains are widely used as economically important natural food colorants and functional foods.7 Therefore, understanding the regulatory mechanisms responsible for betalain biosynthesis could provide information to assist the development of biotechnological approaches aimed to improve betalain production in plants.Betalains are naturally occurring, water-soluble pigments produced from amino acid 8–10 Enzymes involved in betalain biosynthesis, such as tyrosinase (TYR), 4,5-dihydroxy-phenylalanine (DOPA)-dioxygenase (DOD) and glucosyltransferases (GTs) have been identified in higher plants.8–10 It has been proposed that TYR converts tyrosine to L-3,4-dihydroxyphenylalanine (L-DOPA), which in turn is converted to betalamic acid by DOD. GTs, the last family of enzymes in the pathway, make betalains stable and diversify their composition by transferring sugar residues.8–10 Three cytochrome P450-type enzymes, CYP76AD1, CYP76AD5 and CYP76AD6, have been well documented to be associated with the hydroxylation of tyrosine to form L-DOPA. In addition, CYP76AD1 has also been shown to function exclusively in the formation of 5,6-dihydroxyindoline-2-carboxylic acid (cyclo-DOPA) from L-DOPA in red beet.11–13 Genes encoding these enzymes have been identified in plant species within the Caryophyllales and their expression levels have been demonstrated to increase with betalain accumulation, suggesting that betalain biosynthesis is regulated by the transcription level of the genes encoding these enzymes.1 Recently, a betalain biosynthetic gene, CYP76AD1 has been demonstrated to be transcriptional regulated by a MYB-family transcription factor (TF).14 This is the first TF reported to be involved in the regulation of the betalain biosynthesis. Although thousands of TFs have been identified in plants, however, the identification and role of other TFs regulating betalain biosynthesis remains largely unknown.The biochemical pathway of betalain biosynthesis from tyrosine has been extensively studied.15 Numerous WRKY genes have now been isolated and characterized in many plants. For instance, >197, 100, 74, 287 and 62 WRKY members have been identified in Glycine max, Oryza sativa, Arabidopsis, Brassica napus and Fragaria vesca, respectively.16–20 Two highly conserved domains are found in WRKY proteins, one is WRKY with WRKYGQK sequences, and the other is a C-terminal zinc-finger motif of Cys and His residues.21 Plant WRKY proteins are well-known to be involved in various stress responses, hormone signaling, morphogenesis of trichomes and embryos, and senescence.22–24 WRKYs are also reported to regulate plant metabolite biosynthesis, including phenylpropanoids, alkaloids and terpenes, by regulating metabolite biosynthetic genes.25–28 Betalains are also secondary metabolites, however, the involvement of WRKY TFs in betalain biosynthesis, especially in economical fruits, has been largely unexplored.As a large family of plant-specific TFs, the first WRKY protein is identified in sweet potato.Hylocereus in the order Caryophyllales, and is commercially cultivated due to its nutritional value.29 There are several species of pitayas, while the most widely cultivated are Hylocereus undatus and Hylocereus polyrhizus, which have white and red colored pulp, respectively.30 As red pulp pitaya contains high levels of betalains, most of the previous studies of this fruit have focused on purification and characterization of betalins.31 Recently, several putative genes, including TYR, DOD-like, CytP450-like and GT-like, were identified in red pulp pitaya using RNA-seq, and associated with betalain biosynthesis.10 In the present work, the possible association of a Group I WRKY TF, HpWRKY44, with the direct activation of HpCytP450-like1 is reported, providing new information on the transcriptional control of betalain production in pitaya fruit.Pitayas, also called as dragon fruit, belongs to the genus Hylocereus polyrhizus cv. Dayeshuijing) were grown under field conditions at a local commercial plantation in Guangzhou, China. Fruits were sampled on the 16th, 21st, 26th, 30th, 35th, 40th and 49th day after artificial pollination (DAAP). The sample dates were selected to provide a set of samples covering pulp color changes in the fruit. At each sampling, internal fruit tissue (pulp tissue) from three fruits from three different plants were sampled, sliced, and frozen in liquid nitrogen immediately. Samples were stored at −80 °C for future analysis.Red pitaya plants  solution. The amount of betacyanin and betaxanthin in the solutions were measured by spectrophotometry  at 538 and 483 nm, respectively, and reported as mg/100 g fresh pulp.The level of betalains in the pulp tissue of each sample was determined as previously described.Total RNA was extracted from pitaya pulp samples using the Quick RNA Isolation Kit  following manufacturer’s instructions. RNA quality was assessed by gel electrophoresis and spectrophotometry. The extracted RNA was used as template to synthesis cDNAs using a PrimeScript RT reagent Kit with gDNA Eraser .10 a WRKY TF was found to be significantly up-regulated during pulp coloration. As this WRKY displays high degree of sequence homology to Arabidopsis thaliana WRKY44, thus it was termed as HpWRKY44. HpWRKY44 was cloned  and GeneDoc software. A phylogenetic tree was constructed using the Neighbor–Joining method in MEGA5.0.On the basis of the generation of a previous RNA-seq database,10 The PCR was conducted on a Bio-Rad CFX96 Real-Time PCR System. The cycling began with an initial denaturation step at 94 °C for 5 min, followed by 40 cycles of 94 °C for 10 s, 60 °C for 30 s, and 72 °C for 30 s. A no-template control and melting curve analysis was included in every PCR run. Relative gene expression was normalized according to the cycle threshold (Ct) value using actin as the reference gene. Primers used in the RT-qPCR analysis are listed in Gene expression was analyzed by reverse transcription-quantitative PCR (RT-qPCR) using GoTaq qPCR Master Mix Kit  as described previously.HpWRKY44, its full-length was cloned into pEAQ-GFP for fusion with a GFP reporter gene (primers are listed in Agrobacterium tumefaciens strain GV3101 cells carrying pEAQ-HpWRKY44-GFP or the GFP positive control were infiltrated into tobacco (Nicotiana benthamiana) leaves as previously described.33 Transient expression of GFP was recorded using a fluorescence microscope (Zeiss Axioskop 2 Plus) at 500 nm after two days of infiltration.To determine the cellular localization of 34 The HpCytP450-like1 promoter was isolated with a Genome Walker Kit (Clontech) using a nested PCR approach. The sequence of HpCytP450-like1 promoter was subjected to the Plant-CARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) for the prediction of conserved cis-element motifs. Specific primers are listed in A CTAB-based method was used to extract genomic DNA from pitaya pulps.HpWRKY44, including the WRKY domain (from 574 to 1194 bp), was inserted into pGEX-4T-1 , Beijing, China) to construct a GST-HpWRKY44 expression vector, and introduced into Escherichia coli strain BM Rosetta (DE3). GST-HpWRKY44 fusion protein was purified using Glutathione-Superflow Resin  after induction by 1 mM isopropyl thio-β-D-galactoside (IPTG) at 30 °C for 6 h.The N-terminus of 35 DNA fragments, including an oligonucleotide harboring two W-box (TTGAC) motifs and the HpCytP450-like1 promoter with 74 bp containing two consensus W-box motifs were biotin-labeled at the 5′ end  was used to perform EMSA as previously described.36 The minimal TATA box of the CaMV 35S promoter plus five copies of the GAL4 DNA-binding element (5×GAL4) were placed in front of the firefly luciferase (LUC). CaMV 35S-driving Renilla luciferase (REN) in the same vector was considered as an internal control. For the effector vector, the full-length of HpWRKY44 fusing with the yeast GAL4 DNA-binding domain (GAL4BD) was droven by CaMV 35S.Dual-luciferase transient expression system in tobacco leaves was adopted to investigate the transcriptional activity of HpWRKY44. The reporter vector was constructed based on the pGreenII 0800-LUC vector.HpCytP450-like1 promoter, the HpCytP450-like1 promoter (1532 bp) was inserted into the pGreenII0800-LUC vector,36 while HpWRKY44 was inserted into the pEAQ vector as an effector. Primers for all constructs are listed in In order to assess the specific binding and activity of HpWRKY44 to 33 The transcriptional activity of HpWRKY44, as well as the trans-activation of HpCytP450-like1 by the HpWRKY44, were calculated as the ratio of LUC/REN. For each pair, at least six independent replicates were assessed.A total of 2.5 μg plasmid DNA of the reporter and the effector were co-transformed into tobacco leaves in each assay, as described above. Two days after inoculation, LUC and REN luciferase activity was quantified as described previously.P<0.05 and P<0.01.At least three individual biological replicates were utilized in all of the conducted analyses. Data represent the mean±s.e.m. A one-way ANOVA was performed to determine the significance of experimental means at 1 As illustrated in In pitaya fruit there are two types of betalains which are reddish violet (betacyanins) and yellow (betaxanthins).CytP450 genes (HpCytP450-like1 and HpCytP450-like4), and one DOD gene (DODA-like2), increased gradually during the development of the red pulp color, and that the gradual increase in the expression of these genes corresponded with the accumulation of betalain during the transition of pulp color from white to red.10 A database query indicated that the amino acid sequence of HpCytP450-like1 and DODA-like2 shared high identity with BvCYP76AD1 (74%) and BvDODA1 (63%), respectively; two genes that are known to be involved in betalains biosynthesis in red sugar beet.14 These homologies indicated that HpCytP450-like1 and DODA-like2 might be related to betalains biosynthesis in pitaya fruit. Further biochemical and molecular evidence are required, however, to support their direct involvement in betalain biosynthesis, and to identify where they belong in the betalain biosynthetic pathway. In the current study, an attempt was made to isolate the promoters of all three of these genes, however, only the cloning of the HpCytP450-like1 promoter was successful. A motif scan of the 1532 bp-long HpCytP450-like1 promoter was conducted using Plant-CARE database in order to identify cis-acting elements. In addition to the core cis-acting elements, such as a TATA box and CAAT box, three typical W-box motifs with a core sequence (C/T)TGAC(C/T)   were ideentified .25–28 Betalains are secondary metabolites and the HpCytP450-like1 promoter contains a motif suitable for the binding of WRKY TFs (WRKY gene that was up-regulated during color development in pitaya fruit pulp. The full-length sequence of this WRKY gene exhibited high similarity to AtWRKY44 (40%), and so was named HpWRKY44. The Open Reading Frame of HpWRKY44 is 1371 bp in length, and encodes a polypeptide of 457 amino acids, with a calculated molecular weight of 50.89 kDa, and a pI of 9.02. HpWRKY44 has two highly conserved amino acid sequences of WRKYGQK, which is a WRKY domain and a defining characteristic of WRKY TFs.21 HpWRKY44 also contains two putative zinc-finger motifs (C-X4-CX23–24-H-X1-H)  .21 A phylogenetic tree was constructed using the amino acid sequence of HpWRKY44, sugar beet, Arabidopsis thaliana, tomato, and rice WRKYs. HpWRKY44 was clustered with Group I WRKYs, along with the BvWRKY44 from sugar beet, and the AtWRKY44, AtWRKY25 and AtWRKY33 from Arabidopsis thaliana , and group II can be further subdivided into five subgroups (IIa–e).thaliana . NotablyHpCytP450-like1 and HpWRKY44 during the coloration of the pulp was determined by RT-qPCR in order to examine their relationship to betalain biosynthesis during fruit development. Results indicated that the transcript levels of HpCytP450-like1 and HpWRKY44 were relatively low from 16 to 21 DAAP -HpWRKY44 fusion protein was prokaryotic induced and purified successfully (HpCytP450-like1 promoter containing a single W-box motif (HpCytP450-like1.Previous studies have reported the direct binding of WRKY TFs to the W-box motif within target gene promoters.essfully . Resultsox motif . Formatiox motif . As expeox motif . CollectHpCytP450-like1 to activate the transcription of HpWRKY44 was determined using transient dual-luciferase assays in tobacco leaves (HpCytP450-like1 relative to the corresponding empty control. These results indicate that HpWRKY44 activated the transcription of HpCytP450-like1 and supports the premise that HPWRKY44 has a functional role in the regulation of HpCytP450-like1. In red beets, a MYB1 TF, BvMYB1, has been reported to be involved in betalain biosynthesis, as silencing of BvMYB1 results in the down-regulation of betalain biosynthetic genes and pigmentation.14 BvMYB1 can also target the CYP76AD1 promoter.14 Interestingly, MYB and AREB/ABF TFs binding sites are also present in the promoter of HpCytP450-like1. Therefore, whether or not HpCytP450-like1 is also targeted by MYB and AREB/ABF TFs needs to be examined. The identification of additional TFs involved in the regulation of betalain biosynthesis genes in pitaya fruit also need to be identified. Moreover, it has been well documented that regulatory proteins, such as TFs, seldom act alone. Numerous studies have demonstrated that WRKY TFs physically interact with a wide range of proteins that have functional roles in signaling, transcription and chromatin remodeling. These protein interactions can affect the DNA-binding and transcription-regulatory activity that WRKY TFs have with their targets genes.43 For example, MaWRKY1 and MaWRKY2 in banana fruit cooperate with a NAC TF, MaNAC5, to activate the expressions of a specific set of PR genes associated with disease response.44 In addition, MaVQ5 in banana fruit antagonizes MaWRKY26 in the activation of JA biosynthesis in response to cold stress.40 Therefore, in future studies, it would be interesting to determine whether or not HpWRKY44 can coordinate with MYB AREB/ABF TFs, or other proteins to regulate betalain biosynthesis.The ability of o leaves . HpWRKY4o leaves . As showHpCytP450-like1 expression by binding to its promoter. To the best of our knowledge, this is the first report of the involvement of WRKY TFs in regulating HpCytP450-like1 in fruits. Overall, our findings provides new insights into the transcriptional regulation of genes associated with betalain biosynthesis in pitayas; which are economically important and nutritious fruit.In summary, a Group I WRKY TF, HpWRKY44, was cloned and characterized from pitaya fruit. HpWRKY44 activated


Streptomyces cinnamoneus DSM 40646 produces the Class II lantibiotic cinnamycin which possesses an unusual mechanism of action, binding to the membrane lipid phosphatidylethanolamine (PE) to elicit its antimicrobial activity. A comprehensive analysis of the cinnamycin biosynthetic gene cluster has unveiled a novel mechanism of immunity in which the producing organism methylates its entire complement of PE prior to the onset of cinnamycin production. Deletion of the PE methyl transferase gene cinorf10, or the two-component regulatory system (cinKR) that controls its expression, leads not only to sensitivity to the closely related lantibiotic duramycin, but also abolishes cinnamycin production, presumably reflecting a fail-safe mechanism that serves to ensure that biosynthesis does not occur until immunity has been established. Streptomyces cinnamoneus DSM 40646  and expressed it heterologously in Streptomyces lividans [cinA was proposed to encode the cinnamycin precursor peptide, cinM the lanthionine synthetase responsible for the introduction of the lanthionine and methyl-lanthionine bridges, cinX the hydroxylase required for modification of residue D15, cinTH an ABC transporter involved in the export of the cinnamycin, cinKR a two-component regulatory system of unknown function, and cinR1 the activator of the putative operon encoding the cinnamycin biosynthetic machinery. Some of these functions have since been confirmed experimentally [S. cinnamoneus that has unveiled a novel mechanism of immunity that exerts a controlling influence on whether cinnamycin production occurs or not.Cinnamycin Fig. a is a Cl646 Fig. b and bel646 Fig. c, a mechlividans . Likely,mentally . Here, wS. cinnamoneus by PCR-targeting [cinorf7AMXTHRKorf10R1 abolished cinnamycin production (determined by bioassays of culture supernatants using Bacillus subtilis EC1524 as indicator organism and confirmed by Matrix Assisted Laser Desorption Ionization Time-of-Flight (MALDI-ToF) mass spectrometry; data not shown), deletion of cinX resulted in the production of the inactive, non-hydroxylated form of cinnamycin (deoxycinnamycin), consistent with its role as an α-ketoglutarate/iron(II)-dependent hydroxylase [cinYZorf8orf9orf11  had no effect on cinnamycin biosynthesis and the reason for their presence in the cin cluster is not known.Previous work  had idenargeting . While iroxylase . Productcin gene cluster and to ultimately gain insights into the mechanisms underlying the regulation of cinnamycin production, nuclease S1 protection studies were carried out revealing at least nine transcriptional start sites . cinR1, at the right end of the cin cluster, encodes a member of the SARP family of regulatory proteins [cinorf7 transcriptional start site , and introduced into Escherichia coli BL21 (DE3). Thin layer chromatography (TLC) analysis of lipid extracts from the induced cells revealed an additional spot with a similar mobility to monomethylphosphatidylethanolamine (MMPE) observed in lipid extracts of E. coli cells expressing the functionally characterised pmtA genes of either Bradyrhizobium japonicum [Sinorhizobium meliloti [pmtA produces predominantly MMPE and a smaller amount of dimethyl phosphatidylethanolamine (DMPE) when expressed in E. coli, while cells expressing the sinorhizobial pmtA accumulate predominantly trimethyl phosphatidylethanolamine (TMPE), more commonly known as phosphatidylcholine (PC), although some mono- and dimethylated phosphatidylethanolamine could also be detected. Further evidence that the novel lipid spot observed in E. coli cultures expressing cinorf10 did, indeed, contain MMPE was obtained by mass spectrometry  activity. To address this possibility, the aponicum  or Sinori BL21 DE. Thin lai BL21 DE. Thin lai BL21 DE. Thin lacinorf10 coding sequence was next cloned into the integrative Streptomyces vector pIJ10257 [ermE* promoter and introduced into S. lividans 1326 by conjugation. TLC analysis of lipid extracts of the cinorf10-containing strain again revealed an additional lipid spot, the mobility of which was again consistent with MMPE , but shares an identical mode of action [cinorf10 in the heterologous host appeared to confer complete resistance to the lantibiotic ; the ability of constitutively expressed cinorf10 to suppress the non-producing phenotype of the mutants indicates that the only essential role for CinKR in cinnamycin biosynthesis is to regulate cinorf10 expression.The mutational analysis described earlier had shown that deletion of cin Fig. b, consiscinR, cinK, and cinorf10 in MMPE accumulation and duramycin resistance, segments of the cin cluster containing all three genes or just cinorf10 (with its own promoter) were introduced into S. lividans 1326 using the integrative vector pMS81 [cinorf10 remained sensitive to duramycin, the derivative containing all three genes appeared completely resistant  that lacks antimicrobial activity and that is thought not to bind to PE. In cinnamycin, P9 contributes to the PE-binding pocket and may interact directly with the ethanolamine head group of the phospholipid , 19. RepStreptomyces roseosporus and whose lipophilic tail is believed to insert into the bacterial cell membrane causing rapid membrane depolarization and potassium ion efflux, and thus membrane stress [When cultures were treated with daptomycin, a cyclic lipopeptide antibiotic produced by e stress , at conccinorf10 and establishment of immunity to cinnamycin might be a pre-requisite for cinnamycin production. To investigate this relationship further, S. lividans containing the entire cin cluster present in pIJ10109 [cinorf10, and that establishment of immunity was necessary for biosynthesis to occur. Consistent with this hypothesis, deletion of cinA from the heterologous gene cluster in S. lividans resulted in loss of PEMT activity as well as the expected abolition of cinnamycin production; PEMT activity was restored by the exogenous addition of sub-inhibitory concentrations of duramycin  analysis . Activation of transcription of cinorf7 and cinM was essentially abolished in the cinR1 mutant, consistent with the latter’s proposed role as the direct transcriptional activator of the cinorf7AMX operon. Deletion of cinA caused a marked reduction in the levels of induction, but did not completely abolish it, commensurate with a role for cinnamycin in promoting maximal levels of cinorf7AMX transcription. Surprisingly, since the mutant fails to produce the lantibiotic, deletion of cinorf10 had no effect on cinorf7 or cinM transcription.Since deletion of sis Fig. . Cinnamysis Fig. a, corresned Fig. b. The lecinorf7 is essential for cinnamycin biosynthesis in S. cinnamoneus. More recently, co-expression studies in E. coli have shown that the same gene was required for lysino-alanine bridge formation and that the latter was required for antibacterial activity [S. cinnamoneus in which S6 or K19 were replaced with A. Production of both variants  was readily confirmed by MALDI-ToF mass spectrometry, but both failed to inhibit B. subtilis in bioassays, confirming the previous E. coli expression studies.Our earlier deletion analysis had shown that activity . In a cocin cluster is initiated by nutrient limitation that results in a low level of transcription of cinR1 (1). This results in a low level of expression of the proposed cinorf7AMX operon and the production of a small amount of cinnamycin that is exported from the cell by CinTH (2). Cinnamycin then interacts with the membrane-associated sensor kinase CinK which results in phosphorylation of CinR and activation of transcription of cinorf10 encoding the PEMT (3). CinR-P may also further activate transcription of cinR1  (4), resulting in higher levels of transcription of the cinorf7AMX operon (4). In the meantime, Cinorf10 proceeds to methylate PE in the membrane (5) and only when all of the PE has been modified does high-level production of cinnamycin occur (6). Intriguingly, while deletion of cinorf10 abolishes cinnamycin production, it appears to have no effect on the level of transcription of the cinorf7AMX operon, implying the existence of post-transcriptional regulatory feedback mechanism that functions to ensure that cinnamycin production only occurs once immunity to the lantibiotic has been established. The existence of an apparent fail-safe mechanism such as this that presumably serves to prevent the strain from deleterious antibiotic production before the establishment of immunity was also observed in microbisporicin biosynthesis in Microbispora corallina, where deletion of an apparent immunity transporter also abolished lantibiotic production [S. cinnamoneus from cinnamycin produced either by itself or other members of the microbial community in which it exists in nature.The results described here allow us to present a model for the regulation of cinnamycin biosynthesis Fig. . We propt shown) , resultimembrane  and only


DEFB1 gene, which encodes the antimicrobial peptide human β-defensin-1, in vaginal keratinocyte VK2/E6E7 cells. We investigated whether exposure to Lactobacillus gasseri and Lactobacillus reuteri would trigger the epigenetic modulation of DEFB1 expression in VK2/E6E7 cells in a bacterial species-dependent manner. While enhanced expression of DEFB1 was observed when VK2/E6E7 cells were exposed to L. gasseri, treatment with L. reuteri resulted in reduced DEFB1 expression. Moreover, L. gasseri stimulated the recruitment of active histone marks and, in contrast, L. reuteri led to the decrease of active histone marks at the DEFB1 promoter. It was remarkable that distinct histone modifications within the same promoter region of DEFB1 were mediated by L. gasseri and L. reuteri. Therefore, our study suggested that one of the underlying mechanisms of DEFB1 expression in the vaginal ecosystem might be associated with the epigenetic crosstalk between individual Lactobacillus spp. and vaginal keratinocytes.Vaginal commensal lactobacilli are considered to contribute significantly to the control of vaginal microbiota by competing with other microflora for adherence to the vaginal epithelium and by producing antimicrobial compounds. However, the molecular mechanisms of symbiotic prokaryotic-eukaryotic communication in the vaginal ecosystem remain poorly understood. Here, we showed that both DNA methylation and histone modifications were associated with expression of the  Lactobacillus  containing six CpG sites or a primer set specific for the “distal” promoter region of DEFB1 (nucleotides −1000 to −600 upstream from the TSS) containing six CpG sites. The sequences of the PCR primers used were as follows: “proximal” region, forward (5′-TTGGTAGGGTTGAAGTGGGAG-3′) and reverse (5′-TAAAACCCTAATACCAACTCCTC-3′); “distal” region, forward (5′-TTAAGGAAAATTTGAGGGATATTTGG-3′) and reverse (5′-CAAATATCCCTCAAATTTTCCTTAATTC-3′). Cycling conditions were as follows: initial denaturation at 94 °C for 10 min; 45–50 cycles of denaturation at 94 °C for 30 s, annealing at 52 °C for 30 s, and extension at 72 °C for 30 s; and a final extension at 72 °C for 10 min. The PCR products were purified and subcloned into the pGEM-T Easy Vector (Promega) for subsequent sequencing. The nucleotide sequences of 20–25 independent clones were analyzed.Genomic DNA was extracted from the cell lines using the QIAamp DNA Mini Kit (Qiagen). Bisulfite modification of the genomic DNA was performed using the EZ DNA Methylation-Lightning™ Kit (Zymo Research), according to the manufacturer’s instructions. The bisulfite-converted genomic DNA was amplified using either a primer set specific for the “proximal” promoter region of 5 VK2/E6E7 cells were plated in a 100-mm culture dish the day before bacterial exposure. After 48 h of incubation, formaldehyde-treated cells were resuspended in SDS lysis buffer, and the cell lysates were sheared by sonication. The chromatin fragments were immunoprecipitated overnight with antibodies against AcH3, H3K4me3, and H2A.Z . Precipitated chromatin was then washed, reverse-crosslinked, and digested with RNase A and proteinase K. The purified DNA was analyzed by quantitative PCR using the Rotor-Gene Q 5plex HRM system (Qiagen). The sequences of the PCR primers used were as follows: “ChIP 1” region (nucleotides −320 to −186 upstream from the TSS), forward (5′-GTTTGTCTTGCAGGAAGACAATC-3′) and reverse (5′-AACAGGCAGTTCACACTGGAG-3′); “ChIP 2” region (nucleotides −500 to −392 upstream from the TSS), forward (5′-ACTTTCTGAGGAGTGCCCTTTG-3′) and reverse (5′-CCTTCTCATCTCTCCCCTTATG-3′). Thermal cycling was performed using the default conditions of the Rotor-Gene Q Series Software (Qiagen), which consisted of 5 min at 95 °C followed by 40 rounds of 5 s at 95 °C and 10 s at either 58 or 60 °C for “ChIP 1” or “ChIP 2,” respectively.ChIP analysis was carried out using an EZ ChIP kit  following the manufacturer’s protocol. Briefly, 4 × 10t test. A P value of <0.05 was considered statistically significant.Data are presented as the mean ± standard error of the mean (SEM) and analyzed using Student’s DEFB1 gene occurs in human vaginal keratinocytes, we first examined the transcriptional restoration of DEFB1 in VK2/E6E7 cells after treatment with either the DNA methyltransferase inhibitor DAC or the HDAC inhibitor TSA  located at the distal region of DEFB1. Since a single nucleotide polymorphism (rs2978863) is present in the CpG 8 locus within the DEFB1 promoter region in VK2/E6E7 cells, the five CpG dinucleotides (CpGs 3 to 7) other than the CpG 8 site were subjected to bisulfite sequencing. As shown in Fig. DEFB1 was preferentially affected by the DNA-demethylating agent DAC. However, no significant difference was observed in the methylation profile of the “distal” promoter region of DEFB1 in the VK2/E6E7 cells treated with DAC. These results suggested that the differentially methylated CpG dinucleotides, i.e., CpG 3–CpG 7, within the DEFB1 promoter region might be particularly important for the transcriptional regulation of DEFB1 in VK2/E6E7 cells.To identify the site-specific CpG dinucleotides that are important for FB1 Fig. , the proDEFB1 promoter region indicated a subtle difference in the VK2/E6E7 cells exposed to L. gasseri compared to that in the untreated control cells  and three CpG dinucleotides , were analyzed using the ChIP assay  [DEFB1 promoter upon exposure to L. gasseri, while reduction in the level of this protein was detected in response to L. reuteri. Finally, the deposition of the histone variant H2A.Z, which is enriched in regulatory elements at the 5′ ends of many genes and may destabilize nucleosomes to facilitate transcriptional initiation [DEFB1 promoter were observed following exposure to L. gasseri, but a consistent decrease in the level of this histone mark was found upon exposure to L. reuteri. Collectively, these data indicate that these proximal promoter regions of DEFB1 might undergo distinct Lactobacillus spp.-dependent histone modifications to achieve epigenetic regulation of DEFB1 expression in VK2/E6E7 cells in a mutually exclusive manner.To investigate the modulation of histone occupancy at the H3K4me3) , 18. We itiation , was alsDEFB1 expression in vaginal cells were associated with both promoter CpG methylation and histone modifications. Our findings also showed that distinct species of the same genus of commensal bacteria could affect host gene  expression differently by mutually antagonistic processes involving epigenetic alterations. We noted that significant changes in histone marks occurred in the proximal promoter region of DEFB1, in which critical CpG sites were affected by treatment with a DNA demethylating agent, in a bacterial species-dependent manner. Moreover, our data implied that L. gasseri and L. reuteri could control DEFB1 expression by modulating the recruitment of active histone marks within the DEFB1 promoter region.An epigenetic trait is defined as a stably heritable phenotype resulting from changes in a chromosome without alterations in the DNA sequence . EpigeneLactobacillus represents a large number of species and strains of lactic acid bacteria (LAB) that produce lactic acid as the main end-product of carbohydrate metabolism [L. gasseri EV1461, was characterized as a vaginal probiotic candidate [Genus tabolism . There htabolism . Potentitabolism . It has tabolism . Recentlandidate . In the TLR4 gene might be affected by commensal bacteria in the large intestine of mice [Lactobacillus genus may have developed molecular mechanisms by which the expression of the host gene DEFB1 can be modulated by species-specific histone marks in mutually exclusive ways. Additionally, the overexpression of AMPs in vaginal fluid has been detected during infections of the female genital tract [Candida albicans [Chlamydia trachomatis or Neisseria gonorrhoeae infections [L. gasseri and L. reuteri, could help better understand the inducible changes in AMPs in the vaginal ecosystem. Furthermore, genomic and empirical evidence supports the probiotic application of L. gasseri for maintenance of vaginal homeostasis [Lactobacillus spp. may help establish and maintain a healthy vaginal ecosystem that is beneficial for host innate immunity.The tissues of the female reproductive tract are vulnerable to a large number of infectious agents, and the stratified squamous epithelium of the vagina represents a physical barrier to pathogens. Mucosal surfaces of the vaginal tract are equipped with a homeostatic balance between immunity to harmful pathogens and tolerance for maintaining useful bacteria that represents a unique regulatory challenge for the mucosal immune system . In addi of mice . In addi of mice , suggestal tract . For insalbicans , and botfections . Our ongeostasis . We antiDEFB1 might be affected by commensal lactobacilli through species-specific, opposing histone modifications in vaginal keratinocytes.In conclusion, our findings provided the novel evidence that the specific promoter regions of