Pseudomonas fluorescens 2P24. However, the detailed mechanism underlying GrxD-mediated activation of the production of 2,4-DAPG remains unclear. Here, we found that GrxD directly interacted with IbaG, a BolA protein family member. The mutation of ibaG significantly decreased 2,4-DAPG production. Furthermore, expressing ibaG restored the production of 2,4-DAPG in the grxD ibaG double mutant to wild-type levels in the presence of dithiothreitol, suggesting that IbaG was required for GrxD-mediated regulation of 2,4-DAPG production. Transcriptome sequencing analyses revealed that IbaG plays a global role in gene regulation by affecting the expression of numerous genes throughout the genome. We also demonstrated that IbaG is an important regulator of several cellular processes, including swarming motility, biofilm formation, siderophore production, and acid resistance. Altogether, our data suggest that IbaG has an essential role in 2,4-DAPG production, motility, and biofilm formation. We also propose a regulatory mechanism linking GrxD to 2,4-DAPG production via IbaG.The monothiol glutaredoxin GrxD plays an essential role in the biosynthesis of the antibiotic 2,4-diacetylphloroglucinol  and the biocontrol capacity of the soil bacterium Pseudomonas fluorescens 2P24. However, the regulatory mechanism underlying GrxD-mediated regulation of 2,4-DAPG biosynthesis is mostly uncharacterized. Here, we show the function of the BolA-like protein IbaG in 2,4-DAPG biosynthesis. We also demonstrate that GrxD directly interacts with IbaG and influences the redox state of IbaG. Altogether, this work provides new insights into the role of the highly conserved IbaG protein in regulating 2,4-DAPG synthesis, biofilm formation, and other biocontrol traits of P. fluorescens.The production of 2,4-diacetylphloroglucinol  is positively influenced by the monothiol glutaredoxin GrxD in  Pseudomonas fluorescens belongs to a group of ubiquitous beneficial rhizobacteria that play important roles in inhibiting the growth of phytopathogens, promoting plant growth, and eliciting plant immune responses  has received special attention because of its broad-spectrum antimicrobial activity  from malonyl-CoA. Monoacetylphloroglucinol (MAPG) acetyltransferase, which is encoded by phlACBD, acetylates PG to MAPG and 2,4-DAPG  to search for BolA homologs in P. fluorescens using the psi-BLAST algorithm. We found two proteins homologous to BolA of E. coli. Sequence analyses revealed that C0J56_08545 and C0J56_05010 had 42% and 15% sequence identity, respectively, with BolA of E. coli. Moreover, they exhibited 26% and 32% sequence identity, respectively, with IbaG of E. coli. Thus, we named C0J56_08545 and C0J56_05010 of P. fluorescens BolA and IbaG, respectively, based on their homologs in E. coli. Structural predications of P. fluorescens BolA and IbaG based on E. coli BolA protein (PBD ID 2DHM) indicated 100% coverage and 99% confidence  assays showed that the production of 2,4-DAPG significantly decreased in the ibaG mutant compared with that in wild-type P. fluorescens. Complementation with the wild-type ibaG gene restored 2,4-DAPG production to the wild-type level . Altogether, these data suggested that ibaG, but not bolA, was required for 2,4-DAPG biosynthesis of P. fluorescens.To determine whether BolA and IbaG proteins are involved in 2,4-DAPG biosynthesis in pe level ; Fig. S2ynthesis . Furtherorescens . Next, wain 2P24 . In addiArabidopsis thaliana, and these interactions can influence the redox state of cells  analyses showed that deletion of ibaG significantly increased the expression levels of rsmA and rsmE  . These data suggested that IbaG silenced prophage genes, thus preventing the activation of and lysis by prophages.To identify genes, whose expression depended on IbaG, RNA sequencing analyses (RNA-seq) were performed to characterize the transcriptome of strain 2P24 and the gulated) . All difgulated) . The genibaG mutant. Genes responsible for flagellar assembly (FlgF and FlgC) and the chemotaxis response regulator CheY were also downregulated in the ibaG mutant. Additionally, the ibaG regulon includes genes involved in Fe-S cluster biosynthesis and assembly, secretion system, amino acid metabolism, transporter, signal transduction, and other functions.The BolA protein family is involved in stress response and bacterial motility. Our transcriptomic results also showed that several stress response-associated genes and genes encoding stress proteins, including C0J56_16570, C0J56_24075, and C0J56_19920, were significantly downregulated in the cheY, iscR, gabP, and purU) from the identified DEGs. Transcriptional fusion assays indicated that the expression of cheY and iscR significantly decreased, whereas that of gabP and purU significantly increased in the ibaG mutant compared with that in the wild-type strain, thus validating the RNA-seq data .To verify the RNA-seq data, we randomly selected four genes . Collectively, these results demonstrate that IbaG is involved in siderophore production in P. fluorescens.Siderophore production by strain 2P24 is partially responsible for promoting plant growth . A previD mutant . Therefo strains . This reG mutant . We furtibaG mutant. The swimming motility of the ibaG mutant was significantly reduced compared with that of the wild-type strain .Motility is a key bacterial trait for root colonization and is probably involved in biocontrol activities. We compared the swimming motility of strain 2P24 and e strain . Complemmotility . We thenP. fluorescens 2P24, we conducted wheat root colonization assays under greenhouse conditions. Strain 2P24 and its derivatives were introduced into sterile soil. The ability of the ibaG mutant to colonize the root rhizosphere was significantly impaired 14 days after inoculation compared with that of the wild-type and bolA mutants  the ibaG mutant, like the grxD and ibaG grxD double mutants, produced undetectable amounts of 2,4-DAPG; (ii) GrxD protein directly interacted with IbaG; (iii) the C26 of IbaG and the C29 of GrxD were needed for IbaG-GrxD interaction; (iv) expression of a functional IbaG, but not GrxD, restored 2,4-DAPG production in the ibaG grxD double mutant; (v) DTT, which mimics the function of GrxD, was needed for the function of IbaG. DTT is a known strong reducing agent that can block the formation of disulfide bonds. A previous study has shown that the redox shift pattern of At3g11630 of Arabidopsis thaliana changes in the presence of DTT  are responsible for the stability of BolA protein , suggesting that IbaG and BolA proteins have different functions in P. fluorescens. Such functional differences among the BolA-like proteins have previously been reported in E. coli, V. cholerae, and Sinorhizobium meliloti, indicating that BolA and IbaG may be involved in different regulatory pathways to allow adaptation to the constantly changing environment  in an A mutant . The secA mutant . However pathway .ibaG gene is involved in 2,4-DAPG production mediated by GrxD, the swimming motility, and the acid tolerance of P. fluorescens. This finding highlights the complexity of the regulation of 2,4-DAPG production and provides new insights for fine-tuning the biocontrol traits of P. fluorescens under adverse environmental conditions.Overall, our results demonstrated that the P. fluorescens 2P24 and E. coli DH5α strains were routinely grown in lysogeny broth (LB), KB, or ABM medium at 28°C. Antibiotics were supplemented when required for plasmid maintenance or transformation at the following concentrations: ampicillin (50 µg/mL), kanamycin (50 µg/mL), and tetracycline (20 µg/mL).The strains and plasmids used in this study are listed in P. fluorescens ibaG deletion mutant was constructed as follows. The flanking regions of the ibaG gene were amplified by PCR . Protein structural prediction was performed using the I-TASSER server (https://zhanggroup.org/I-TASSER/).Amino acid alignment of BolA or IbaG homologs was performed using the Clustal Omega multiple sequence alignment tool at the European Bioinformatics Institute (EMBL-EBI) website . Primers are listed in The site-directed point mutant of bolA mutant, and the ibaG mutant were grown overnight on LB agar and then cultured in liquid LB medium to an OD600 of 1.0 (ca. 1 × 108 CFU/mL)  and 1 mL samples were taken. Cells were collected by centrifugation, resuspended in 1× phosphate-buffered saline (PBS) buffer, and lysed by sonication. Samples were separated on a 10% SDS-PAGE gel and blotted on a polyvinylidene difluoride membrane according to the manufacturer’s instructions . The blotting membrane was blocked in blocking buffer (5% milk powder) for 1 h at room temperature and then incubated with an anti-FLAG antibody . After washing with Tris-buffered saline with Tween 20 buffer, the membrane was then incubated with horseradish peroxidase-goat anti-mouse IgG secondary antibody, and the signal was detected using a chemiluminescence detection kit . RNA polymerase beta was used as the reference protein as the internal standard .Strain 2P24, the grxD and ibaG genes were amplified and cloned into the pUC18C and pKT25 vectors in frame with two gene fragments of the adenylate cyclase of Bordetella pertussis. The resulting vectors were then cotransformed into E. coli BTH101 and protein interactions were assessed by β-galactosidase activity  at 30°C for 36 h. The fermentation culture was mixed with 0.1% (vol/vol) formic acid and then extracted with an equal volume of ethyl acetate as previously described . The ethibaG mutant were cultured to the exponential phase (OD600 = 1.0) in LB medium. Three biological replicates were prepared for each strain. The cells (10 mL) were collected and centrifuged for 3 min at 9,600 × g. The total RNA was extracted using the RNeasy minikit . Extracted RNA was treated with Turbo DNase I  to remove genomic DNA contaminants. The rRNA was removed with the Ribo-Zero rRNA removal Kit . The integrity of the total RNA was then assessed with the Agilent TapeStation System . The cDNA library was constructed using the NEBNext UltraTM II RNA Library Prep Kit, which was then sequenced using Illumina HiSeq 4000 in rapid mode at a read length of 100 bp paired ends by Sangon Biotech Co., Ltd. . The RNA-seq raw data were mapped to the genome of P. fluorescens 2P24 (GenBank accession no. CP025542) using HISAT software. The DESeq2 method was used to identify the DEGs of the ibaG mutant compared to the wild type  of <0.05 were used to establish significance. The volcano plots were generated using the R language ggplots 2 package. To identify the potential pathways that the DEGs were involved, KEGG pathway enrichment analysis was performed using DAVID (https://david.cifcrf.gov/). The GO analysis was performed with Blast2GO (BioBam) (http://bowtie-bio.sourceforge.net/index.shtml).Cells of strain 2P24 and its ild type , and a l−ΔΔCt method.The validation of the RNA-seq analysis was performed by RT-qPCR. After RNA isolation and DNase I treatment, reverse transcription was done with cDNA Synthesis SuperMix . RT-qPCR was carried out with a TaqMan PCR master mix . Normalization was performed against the 16S rRNA gene using the 2Siderophore production was determined by CAS assay . BrieflySwimming assays were performed as previously described . BrieflyTriticum aestivum cultivar Yumai 49) were soaked inside the bacterial cultures (108 CFU/mL) before sowing into seedling plates containing sterile soil. After 10 days of growth, ten plants were harvested randomly from each treatment, and bacteria were recovered from the rhizosphere by vortexing the root tips (last centimeter of the main root) for 2 min in a tube containing 5 mL of PBS buffer. The samples were serially diluted and then plated on LB agar plates. The experiment was performed three times with three replicates, and population data, collected as CFU counts, were log10 transformed before statistical analysis.Rhizosphere colonization was performed as described previously . Brieflyt test. Data were presented as mean values ± standard deviations, and P values less than 0.05 were considered statistically significant. All experiments were performed at least three times independently to confirm reproducibility.Statistical analysis was performed by the least significant difference test or the Student’s


We propose that several chromatin-mediated regulatory processes are dominated by source-sink relationships in which factors operate as ‘sources’ to produce or provide a resource and compete with each other to occupy separate ‘sinks’. In this model, large portions of genomic DNA operate as ‘sinks’, which are filled by ‘sources’, such as available histone variants, covalent modifications to histones, the readers of these modifications and non-coding RNAs. Competing occupation for the sinks by different sources leads to distinct states of genomic equilibrium in differentiated cells. During dynamic developmental events, such as sexual reproduction, we propose that dramatic and rapid reconfiguration of source-sink relationships modifies chromatin states. We envision that re-routing of sources could occur by altering the dimensions of the sink, by reconfiguration of existing sink occupation or by varying the size of the source, providing a central mechanism to explain a plethora of epigenetic phenomena, which contribute to phenotypic variegation, zygotic genome activation and nucleolar dominance. Summary: A model in which cellular programming dynamics is governed by source-sink relationships, such that chromatin associated factors (‘sources’) compete to occupy regions of genomic DNA (‘sinks’). Drosophila demonstrated that heterochromatin occurs at some inactive genes , whereas others remain continuously condensed (constitutive heterochromatin).All living organisms package their DNA with chromatin proteins. Chromatin proteins protect and compact the DNA , facilitve genes . ObservaDrosophila, where differing degrees of expression arise through heterochromatin spreading outward from neighboring genomic loci into an eye color governing gene. It was also noted that the extent of variegation depends on the ratio of heterochromatin to euchromatin in the nucleus. The loss of the largely heterochromatic Y chromosome leads to the increase of a variegation in the X0 male compared with the XY male  that occupy discrete parts of genomic DNA. Competition between these factors occurs in a manner dependent on DNA affinity, chromatin turnover and overall factor abundance. Hence, chromatin dynamics revolves around sources of free factors, which flow into a restricted number of DNA sinks . Here, wChromatin results from multiple interactions between its components. The basic unit of eukaryotic chromatin is the nucleosome comprising two copies of each core histone: two heterodimers of H2A-H2B and one tetramer of H3 and H4. Nucleosomes prevent access of transcription factors to DNA, and nucleosome remodelers are ATPases able to weaken the histone-DNA interaction and move nucleosomes to modulate access to transcription factor-binding elements . NucleosAlthough chromatin is not separated by internal membrane compartments, it appears segregated into subdomains that might result from events comparable with phase separation. Various forms of organization have the potential to segment the chromatin into specialized sinks insulated from each other. For example, the nucleolus comprises the repetitive regions encoding ribosomal RNAs (rRNAs) . HeterocNext, we illustrate how such source-sink relationships might provide a unified model to explain several types of epigenetic regulations, including variegation, zygotic genome activation, nucleolar dominance and the effects of mutants deficient in chromatin regulators.Drosophila  in polymorphic Y chromosomes in D. pseudoobscura. This size difference is the result of invasion from TEs and the increased heterochromatin sink  causes expression of Y-enriched TEs, and influences expression of dozens of autosomal and X-linked genes  correspond with delayed zygotic genome activation (Drosophila homolog to H2A.Z (H2Av) accumulates on chromatin during cleavage phase  in stem cells, and the subsequent cell divisions that occur during cleavage phase effectively dilute out these histones to appropriate levels during stem cell formation  Fig. 4)Fig. 4).  earlier . Similar earlier , and onl earlier . Taken ttivation , while tge phase  and promge phase . The abuAlthough histone structure shows little diversity, the sequence of their intrinsically disordered loops and tails are not subject to the same constraints. This has permitted histones to diverge during eukaryotic evolution, resulting in new histone proteins referred to as histone variants , 2021. DOne of the most thoroughly investigated features of H2A variants is their defining sequence motifs located within their C-terminal tails. These motifs are important for the diverse mechanisms ascribed to the variants, typically via interaction with modifiers and accessory proteins, illustrated by the SQE/DΦ, the H2A.X motif . Unique Arabidopsis, H2A.W is found only in constitutive heterochromatin on TEs. By contrast, H2A.Z occupies primarily protein-coding genes repressed by the Polycomb repressive complexes 1 and 2, and H2A and H2A.X occupy the bodies of expressed genes. In absence of H2A.W, this balance is upset with an invasion of heterochromatin by H2A and H2A.X, coupled with limited misexpression of hundreds of protein-coding genes and delocalization of de novo DNA methylation Arabidopsild type .ddm1, and of H2A.X by H2A in h2a.w suggest that the competition between H2A variants for occupation of heterochromatin is regulated by distinct remodelers and chaperones  is paralleled by the deprivation of H3K27me3 from its target protein-coding genes phosphate, or a DNA methylation inhibitor, 5-aza-deoxycytidine, causes H2A.Z to accumulate specifically at transcriptionally active repetitive elements, including DNA transposons and satellite repeats  preprinttrans. Owing to the large proportion of the genome occupied by repetitive DNA elements in many organisms   , it is vtrans effects result from changes in occupations of sinks, consequently affecting occupation of other sinks, which accounts for the often disconcerting broad spectrum of phenotypes in mutants of chromatin regulators.Genetic and genomic studies altogether point to a general source-sink model to explain a large array of epigenetic regulations. Source proteins such as chromatin regulators, histones or transcription factors compete for genomic sinks within the cell. Changing availability of a source protein affects occupation of the sinks by other source proteins. In mutants that affect the wild type equilibrium, These source-sink relationships are used in wild type to regulate sequences of development. Limiting levels of source proteins appears to be an important aspect of developmental transitions because activating new loci results in ancillary silencing of loci that were previously active in progenitor cells. Hence, cells can transition to a new state or developmental lineage more easily and rapidly. If source proteins were instead in excess, then additional silencing machinery would be required to turn off previously active genes. Thus, keeping limits on sources allows the cell to effectively ‘kill two birds with one stone’. The constraints on the levels of source and the limits of the sinks set the boundaries for competition among genomic loci for source proteins, while preserving a finely tunable set of transitions from one equilibrium to another, entraining cell lineages in their respective valleys of the Waddington epigenetic landscape . In thisTEs and repetitive elements play a crucial role as their sink is modulated at the scale of the individual organisms and the evolutionary scale. At the scale of the organism, widespread de-repression of repetitive elements may allow the reservoir to re-fill, sequestering away transcription machinery while the genome undergoes epigenetic reprogramming, thus facilitating epigenetic switching on a dramatic scale. Subsequently, the reservoir can empty as repetitive loci undergo silencing and trans-factors become re-localized to genic regions. Examples of this include reprogramming events after fertilization in mammals, when de-repression of repetitive elements occurs in 2C cells, during primordial germ cell (PGC) specification in mammals when piRNAs are generated from repetitive loci, and during innate immune activation caused by ‘viral mimicry’ during cancer treatment . This paConsidering TEs collectively as a major epigenetic sink adds a layer of complexity to our view of their role in evolution. A sudden expansion of this sink (through chromosome rearrangements or transposon mobility) causes a dilution of the silencing sources, which are then unable to silence not only the new loci but also other TEs. This could explain bursts of transpositions causing mutations, DNA damage and malfunction of ancillary source sink. Increasing the production of source molecules, whether they are silencing or activating factors, is therefore a necessity response for survival. The wide effects of changing several source-sink relationships likely enable wide arrays of combinations of genome-wide changes submitted to selection; this might account for dramatic changes that lead to a rapid series of innovations observed in the evolutionary tree, including, notably, the evolution of sex chromosomes .Another predictable effect is the selective pressure to regulate the size of epigenetic sinks with consequences on genome size. Evolutionary studies have proposed that constraints on genome size limit the degree to which TEs are able to infiltrate genomes . One int


Drosophila zinc finger transcription factor, we show here that the promoter state can also be subject to developmental regulation independently of gene activation. Lola-I is ubiquitously expressed at the end of embryogenesis and causes its target promoters to become accessible and acquire paused Pol II throughout the embryo. This promoter transition is required but not sufficient for tissue-specific target gene activation. Lola-I mediates this function by depleting promoter nucleosomes, similar to the action of pioneer factors at enhancers. These results uncover a level of regulation for promoters that is normally found at enhancers and reveal a mechanism for the de novo establishment of paused Pol II at promoters.While the accessibility of enhancers is dynamically regulated during development, promoters tend to be constitutively accessible and poised for activation by paused Pol II. By studying Lola-I, a  Gene promoters are very often poised for expression by paused RNA polymerase II. Here, Ramalingam et al., identify a mechanism for the de novo establishment of paused Pol II at promoters and study its effects on expression. Since promoters must respond to a large variety of different enhancers, they tend to have constitutively accessible chromatin4. In contrast, distally located developmental enhancers typically only become accessible in the cell lineage in which they become active5. To prevent activation in inappropriate cell types, developmental enhancers tend to be wrapped around stable nucleosomes in their natural state and thus are inaccessible to most transcription factors8. This nucleosome barrier is most often overcome with the help of so-called pioneer factors that recognize their DNA-binding motifs on nucleosomal DNA and initiate chromatin remodeling9. Once enhancers are accessible, other transcription factors can bind and drive enhancers toward activation.Gene regulation during development depends on the coordinated action of enhancer and promoter sequences. Enhancers respond to specific developmental signals and transmit the information to the core promoter where transcription of the gene begins12, while others are actively kept accessible through constitutively expressed pioneer factors, which antagonize the main nucleosome over the promoter. Examples of such constitutively expressed promoter pioneer factors are Reb1 or Abf1 in budding yeast, GAGA factor (GAF) in Drosophila, and SP1 in mammals17.Promoters on the other hand use a variety of mechanisms to maintain their accessibility across cell types and conditions. Some inherently have a low nucleosome barrier due to nucleosome-disfavoring sequences 24. At such promoters, Pol II initiates transcription and transcribes for 30–50 bp before going into a paused state, and the presence of paused Pol II poises genes for robust induction during development26. Upon induction, paused Pol II is released into productive elongation and new Pol II initiates at high frequencies, which results in a burst of transcription29.A hallmark of accessible promoters is the presence of paused RNA polymerase II (Pol II), which is present even in cell types where the genes are inactive22, promoters could nevertheless be regulated, by acquiring paused Pol II only in a specific developmental context or at alternative start sites. Indeed, how alternative start sites are regulated is generally not known30. The mechanisms by which promoters might undergo such a transition and how this might affect the target gene expression has not been studied.While the majority of promoters are constitutively accessible and have paused Pol IIDrosophila, we previously observed that a subset of promoters had no Pol II occupancy during the beginning of embryogenesis , but acquired paused Pol II gradually over time until the end of embryogenesis 19. Since Drosophila is a well accessible experimental system, this provided us with an opportunity to discover the mechanisms by which promoters acquire Pol II de novo during development. Here, we identify one of the responsible transcription factors as Lola-I and found that Lola-I functions at promoters analogous to pioneer factors at enhancers. By binding to its DNA-binding motif near the nucleosome edge, it makes its target promoters broadly accessible throughout the embryo, without directly causing gene activation. In this manner, Lola-I orchestrates the de novo acquisition of paused Pol II over the course of embryogenesis, producing poised promoters ready for tissue-specific transcriptional bursts during differentiation. These results illustrate a new mechanism for the regulation of promoters, thus providing a basis for studying promoter regulation in mammals.By analyzing promoters with high levels of paused Pol II in 19. As a control, we used promoters with high levels of paused Pol II throughout embryogenesis (constant set)19. The most highly enriched motif was AAAGCT , we considered that the process is regulated by a transcription factor and searched for DNA-binding motifs that are enriched at these promoters (opening set)4–17 h, wlola locus32. All Lola proteins code for transcription factors that share the same N-terminal BTB/POZ domain, but have isoform-specific C-terminal zinc-finger domains with distinct DNA-binding specificities and developmental roles35. Notably, the RNA of Lola-I is upregulated during the later stages of embryogenesis36, consistent with our proposed role for Lola-I.Lola-I is encoded by one of the more than 25 different splice isoforms from the Drosophila embryos (14–17 h). Among the top 750 regions, 725 had a Lola-I motif within 200 bp of the peak summits , confirming that the antibodies were specific for Lola-I.To test whether Lola-I indeed specifically binds to the promoters in the opening set, we raised polyclonal antibodies specific for the Lola-I isoform .To test whether Lola-I is the factor responsible for causing these promoters to change over developmental time, we analyzed a previously identified ain Fig. . A low anal Fig. , 4c. Thelola-I−/− embryos, we found that Pol II occupancy was specifically reduced at Lola-I targets , but not at control promoters  from an equivalent stage. As expected, Lola-I target genes showed Pol II occupancy very broadly among cell types, as is known for paused genes 18 Fig. . This sulola-I−/− and wild-type embryos, we found that the expression of Lola-I target genes, but not that of control genes, was significantly reduced in lola-I−/− embryos , followed by a period of inactivity (OFF state)44. Given this two-state model, Lola-I could regulate the rate of gene activation (through the activation rate constant Kon) and increase the burst frequency. This is typically how enhancers regulate transcription47. Alternatively, Lola-I could regulate the burst size (through the rate constants Kprod and Koff), which can be a promoter-specific property50. If Lola-I affects the burst size, we would expect that most cells in lola-I−/− embryos show a similar proportional reduction of Lola-I target gene expression. On the other hand, if Lola-I affects the burst frequency, we would expect a more heterogeneous reduction of transcripts in lola-I−/− embryos, where some cells in the mutant embryos show high levels of RNA similar to wild-type, while others show very few or no RNA.If Lola-I is not a traditional activator and primarily affects the promoter state, we wondered how Lola-I regulates the ability of promoters to produce tissue-specific transcripts. Lola-I’s recruitment of paused Pol II could be a requisite for the activation process, or it could be affecting how many transcripts are produced upon gene activation. The transcription of most genes occurs in bursts, characterized by a period of transcriptional activity (ON state), during which Pol II produces a burst of multiple nascent RNAs , Gip was expressed at high levels and localized to the same cells as PPO1/PPO2   Fig. . Consistype Fig. . Hence, Gip expression was due to developmental delays, we performed the same single-molecule FISH experiments in wild-type and lola-I−/− embryos over several time points . This confirmed the reduced number of cells with Gip expression in lola-I−/− embryos across all time points  to the ratio of cells with and without nascent transcripts. The two-state model produced a reasonably good fit . We note that the model also suggests a reduction in the burst size, which would be consistent with previous observations that the burst size moderately decreases with lower transcriptional levels56. However, we note that very few cells are producing detectable levels of Gip in the mutant, thus it is difficult to accurately assess Kprod or burst size , were reconstituted with nucleosomes in vitro and incubated with different concentrations of full-length Lola-I protein  sequence, a hallmark of promoters with Pol II pausing, increases the burst frequency66. However, Pol II pausing is highly correlated with promoter accessibility, thus we cannot rule out that some of these properties are at least in part due to increased chromatin accessibility. For example, budding yeast does not have Pol II pausing, and yet promoters that have nucleosome-repelling sequences or motifs for pioneer factors have increased promoter strength68. These results suggest that paused Pol II and promoter accessibility could both increase the rate of gene activation.An obvious explanation for the increased responsiveness of Lola-I target promoters to activation signals is that Lola-I promotes the acquisition of paused Pol II. Paused Pol II is associated with more synchronous and robust gene activation69.The importance of promoter accessibility became apparent when we analyzed the mechanism by which Lola-I establishes paused Pol II. Rather than recruiting Pol II, we found that Lola-I functions at promoters primarily as a pioneer factor that depletes nucleosomes. This is supported by our observation that Lola-I also depletes nucleosomes at distal regions where paused Pol II is not detected. Furthermore, incubation of Lola-I protein with nucleosome-bound sequences in vitro suggested that Lola-I preferentially binds to its motifs near the nucleosome edge when the DNA is partially unwrapped. While this in vitro assay is based on a strong nucleosome-positioning sequence and may not necessarily reflect Lola-I’s binding in vivo, Lola-I’s binding preference in this assay is consistent with those of other pioneer factors71, and no effect on paused Pol II prior to transcription has been reported. Notably, the establishment of poised Pol II is largely unaffected in Zelda mutants70. Thus, Lola-I has the function of a promoter pioneer factor but its expression is developmentally regulated and thus it changes the state of Pol II pausing at promoters over developmental time.We conclude that Lola-I functions similarly to constitutive promoter pioneer factors. Constitutively expressed GAF also preferentially binds to promoters (like Lola-I ~ 60%) and promotes Pol II pausing. In contrast, pioneer factors that regulate enhancers, such as Zelda or Opa, do not bind promoters to the same extent as Lola-I (Zelda ~40% and Opa 35%)72. Moreover, certain BTB domain containing proteins such as GAF have been shown to affect 3D chromatin organization73. If so, this could pinpoint to a role of Lola-I and paused Pol II in 3D chromatin organization.Since this type of promoter regulation during development has not been observed before, it is interesting to speculate why the Lola-I target genes, which affect metabolism and ion transport, are regulated in this way. One possibility is that Lola-I serves as a timer that makes sure that the target genes are not expressed precociously, while still allowing tissue-specific enhancers to control their expression patterns. Another possibility is that having these promoters open in the early embryo would hijack early patterning enhancers and hinder the activation of patterning genes. This would be consistent with the recent observation that enhancer-promoter contacts change in the late embryolola locus has many isoforms that are expressed in various patterns during development32. They all have the same BTB domain for protein-protein interactions but have different sequence-specific DNA-binding domains. Motifs for some of the isoforms have recently been suggested to regulate chromatin accessibility in the Drosophila brain74. Thus, it is conceivable that other BTB-domain-containing transcription factors also target promoters and help establish paused Pol II in specific tissues or stages of development.While Lola-I’s role as promoter pioneer factor appears unusual, it is possible that other pioneer factors affect promoters in a developmentally regulated way. For example, only a subset of the opening set promoters are regulated by Lola-I. The Drosophila because the gene structure is simpler, promoters have high levels of Pol II pausing, and time course experiments can be easily performed. However, the strength of promoters in mammals is also influenced by transcription factor motifs75, which could modulate the level of accessibility. Furthermore, an opening of promoters has been observed during early mouse development76. An even more intriguing possibility is that pioneer factors regulate the usage of alternative start sites. At least 15% of protein-coding genes in the human genome use alternative tissue-specific promoters that are enriched for specific transcription factor binding motifs77, but the mechanism underlying this promoter selection is not known30. It is tempting to speculate that alternative promoter selection during mammalian development is regulated by pioneer factors with analogous roles to Lola-I.This likely also holds true for mammalian development. We likely discovered promoter regulation in 30. However, the constitutive accessibility of promoters and the more dynamic accessibility of enhancers have still been considered to be distinctive features of this class of elements5. The fact that promoters can also be dynamically regulated during development further supports the idea that enhancers and promoters share fundamentally similar characteristics with each other.Whether or not the developmental regulation of promoters is widespread, our finding that promoters can be regulated at the level of accessibility further blurs the distinction between promoters and enhancers. The traditional separation between promoters and enhancers has already been challenged in recent years since their function is often not cleanly separable in reporter assaysOregon-R was used as the wild-type strain. Lola-I mutant lines were obtained from Bloomington stock center (ORC4 - 28267) and from Edward Giniger (ORE50). Homozygous lola-I mutant files were non-viable and were maintained over a CyO-GFP balancer to allow sorting of the homozygous mutant embryos that are GFP-. Lola-I rescue lines were generated as follows: a construct with an Actin promoter driving full-length lola-I cDNA and marked by mini-white was integrated into the attP40 locus on 2 L and then crossed with the lola-IORC4 line (lola is on 2 R) to obtain females that recombine the second chromosome in the germ line. After crossing in a CyO balancer, several males harboring the mini-white marker were selected. After mating single males with a CyO balancer stock, each male was screened for the lola-IORC4 mutation by amplifying the relevant portion of the lola locus by PCR and sequencing. Meiotic recombinants that had both the rescue construct and the lola-IORC4 mutation were viable as homozygotes. For the INTACT experiments, embryos from fly stocks expressing tissue-specific RAN-GAP-mcherry-FLAG-BirA with the help of tissue-specific Gal4 driver lines were collected as described18. To isolate tracheal or gut cells from lola−/− embryos, fly lines containing RAN-GAP-mcherry-FLAG-BirA on the second chromosome (expressing in either trachea or gut) was recombined with the lola-IORC4 chromosome and maintained over a GFP-marked CyO balancer 5, lolaORC4/CyO-GFP) 5, lolaORC4/CyO-GFP). Homozygous embryos for the recombinant chromosome were obtained by sorting for GFP-negative embryos.18. Briefly, embryos were collected and matured at 25 °C, then dechorionated for 1 min with 67% bleach and cross-linked for 15 min with 1.8% formaldehyde . For the single-molecule FISH experiments, the embryos were cross-linked in 1x PBS in DEPC-treated water. Homozygous lola-I mutant embryos were obtained by sorting for GFP-negative embryos in PBT (PBS with 0.05-0.1% Triton). Embryos used for ChIP-seq experiments were flash frozen in liquid nitrogen and stored at −80 °C. For ATAC-seq and scRNA-seq experiments, the embryos were processed immediately after dechorionation without crosslinking. For immunostainings, antibodies were used in the following dilutions: Lola-I (custom-made by Genescript) 1:750, α-tubulin antibodies  at 1:500, Lamin , α-MHC (1:500), Elav (7E8A10 from DSHB) at 1:30, and Repo (8D12 from DSHB) at 1:10. For Western blot experiments, antibodies against Lola-I (custom-made by Genescript) were used 1:2000, those against Pol II  at 1:2000.Adult fly maintenance and embryo collections were performed as described38, as described in ref. 18.Nuclei isolation was performed using modified versions of previously published protocolsDrosophila Pol II subunit Rpb3  is also used.Antibodies were raised against the Lola-I-specific portion (455-877 AA), thus excluding the BTB domain. It also excludes the DNA-binding domains of Lola-I. The antigen for antibody production was expressed in E.coli with a his-tag and purified using a Ni column. The antibody production was done by inoculating the antigens in rabbits and the antibody serum was antigen-affinity purified. Rabbit polyclonal antibodies against the full-length ChIP-seq experiments were performed as follows. ~100 mg embryos were used per ChIP, and 5 µg chromatin was used for tissue-specific ChIP-seq experiments. Fixed embryos were homogenized by douncing in an ice cold A1 buffer (15 mM HEPES (pH 7.5), 15 mM NaCl, 60 mM KCl, 4 mM MgCl, 0.5% Triton X-100, 0.5 mM DTT, protease inhibitors) and A2 buffer (15 mM HEPES (pH 7.5), 140 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 0.5% N-lauroylsarcosine, protease inhibitors) in a tissue grinder for 10–15 times in A1 and A2 buffer each. Then the sonication of the chromatin was performed with a Bioruptor Pico for four-five rounds of 30 s on and 30 s off cycles. The sonicated chromatin was cleared by centrifugation and the supernatant was used for ChIP. Chromatin was incubated with antibodies pre-bound to Dynal magnetic beads (IgA or IgG) overnight with end-to-end rotation at 4 °C and washed with an ice-cold RIPA buffer (50 mM HEPES (pH 7.5), 1 mM EDTA, 0.7% sodium deoxycholate, 1% NP-40 (IGEPAL CA-630), 0.5 M LiCl). Eluted, reverse cross-linked DNA was then purified using phenol-chloroform-isoamylalcohol phase separation and ethanol precipitation. ChIP-seq libraries were prepared from 5 to 15 ng ChIP DNA or 100 ng input DNA according to the manufacturer’s instructions (NEBNext ChIP-Seq Library Prep kit).OregonR embryos. The transposition of the nuclei was performed as described in78. Computational filtering for fragments of size 0–100 bp was done to capture signals from the accessible regions.ATAC-seq was performed using ~500–2000 embryos of stage 14–17 h AED. Nuclei were isolated by douncing the embryos in the HBS buffer as described above in the Isolation of tissue-specific nuclei section. Whole embryo ATAC-seq was performed without the selection of nuclei from a specific tissue using the 79. Briefly, chromatin was extracted from 0.1 mg of Oregon-R or lola-I mutant embryos per replicate by douncing embryos in the NPS buffer , 5 mM MgCl, 1 mM Cacl, 1 mM beta-mercaptoethanol) using a tissue homogenizer, then digested with a concentration gradient of MNase  in NPS buffer for 30 min at 37 °C. All digestion concentrations were run on a gel and the concentration to be sequenced was chosen such that the digestion is complete, characterized by the presence of only mononucleosomes, but the samples are not over digested . Libraries were prepared from purified MNAse digested DNA using the NEBNext DNA Library Prep kit following the manufacturer’s instructions and then paired-end sequenced on an Illumina HiSeq 2500 sequencing system. Computational filtering for fragments of size 100–200 bp to analyze the nucleosome occupancy.MNase digestion was performed similarly to previously published protocolslola-I sequence was synthesized and sub-cloned into the Flag-TAG expression vector F1 and expressed in Sf9 insect cells using a recombinant Bacmid. Sf9 cells were grown in Sf‐900 II SFM Expression Medium in Erlenmeyer Flasks at 27 °C in an orbital shaker. Cells were seeded in 6 wells, transfected the next day by adding DNA mixed with Cellfectin II at an optimal ratio, and incubated for 5–7 days before harvesting P1 and P2 viral stock. The Sf9 cells (1 L) containing 5% FBS were infected with the P2 virus at MOI = 3 and harvested at 48 h post-infection. Cells were sonicated in 50 mM Tris, pH 8.0, 150 mM NaCl, 5% Glycerol containing protease inhibitors. Cell pellets were harvested and lysed, and the supernatant was incubated with Flag Columns to capture the target protein. Fractions were pooled and dialyzed with 50 mM Tris-HCl, 150 mM NaCl, 5% Glycerol, pH 8.0 followed by 0.22 um filter sterilization. Proteins were analyzed by SDS‐PAGE and Western blot by using standard protocols for molecular weight and purity measurements.Full-length Lola-I protein was expressed using baculovirus and purified by Genescript. Briefly, the 62. Templates were designed by altering the right side of the Widom 601 nucleosome-positioning sequence and placing Lola-I binding motifs with increasing distance to the dyad axis. Four translational settings were tested—dyad (at superhelix location (SHL) R0, R0.5), intermediate , edge , and linker, which is outside the nucleosome (SHL 8). The rotational setting of each motif was designed such that it is either outside on the solvent accessible side  or concealed  based on the nucleosome crystal structure formed on the Widom 601 sequence80. To explore cooperativity, a template with two neighboring motifs was designed (SHL R6 + R7), as well as one with two motifs further apart as a control (SHL L6 on left +R7). All templates were compared to non-specific binding to the Widom 601 sequence.The nucleosome binding assay was performed as previously described81, purified from free DNA with 7–20% sucrose gradient centrifuge, and concentrated by 50 K centrifugal filter units .All 11 synthesized DNA templates were amplified via PCR with the primer pair 5′-GATGGACCCTATACGCGGC-3′ and 5′-GGAACACTATCCGACTGGCA-3′, and the products were column-purified (QIAGEN), quantified, and pooled equally. In vitro nucleosomes were generated from H2A/H2B dimer and H3.1/H4 tetramer (NEB). The pool of 11 nucleosome sequences were added to histones at octamer/DNA molar ratios of 1.25:1 in 2 M NaCl solution. Nucleosomes were reconstituted by salt gradient dialysis as described inFor the protein-nucleosome binding assays, each of 0.25 pmol of purified nucleosomes were incubated with increasing concentrations of Lola-I protein  in 7 µl DNA-binding buffer  for 10 min on ice and then for 30 min at room temperature. Protein binding was detected by mobility shift assay on 4% (w/v) native polyacrylamide gels  in 0.5 × Tris Borate-EDTA buffers at 100 V at 4 °C. After electrophoresis, DNA was imaged by staining with SYBR Green (LONZA).All visual bands were excised from the gel, as well as the bands at the same locations in the other lanes. Each gel slice was processed separately for a total of 20 samples from 2 replicate experiments. In order to extract DNA from polyacrylamide gel, the chopped gel slices were soaked in diffusion buffer , and incubated at 50 °C overnight. The supernatant was collected, residual polyacrylamide removed with glass wool, and DNA purified with QIAquick Spin column (QIAGEN). The DNA concentration for each sample was determined by qPCR by comparing it to a standard curve generated from the control 601 sequence. Based on this concentration, the samples were amplified by PCR using Illumina primers (the cycle number ranged from 8 to 12) and then indexed in a second round of PCR using Nextera dual index primers, followed by clean up with AMPure XP beads (Beckman Coulter). The samples were multiplexed and sequenced on an Illumina MISeq using 2 × 150 bp paired-end sequencing. Sequencing and quality control were performed at the University at Buffalo Genomics and Bioinformatics Core.82. After obtaining the amount of the reads from each band, the data from each of the nucleosome sequences N were normalized to the Widom 601 control sequences: The results were then analyzed relative to the 601 control sequence and the non-specific binding in the input lane without any Lola-I. Relative shift is determined from the non-shifted nucleosome bands and controls for the technical variability introduced by gel-excision, PCR, NGS-library construction, or NGS sequencing. In this method each specific nucleosome sequence or the super-shifted sequence is measured relative to non-specific binding (601 fragment without a Lola-I binding site):High-quality sequence reads were mapped to each specific starting sequence using VSearchDrosophila melanogaster genes Gip, PPO1, PPO2, and lola-I using the Stellaris probe designer, and purchased with a label ready C-term TEG-Amino tag from Biosearch Technologies. 4 nMol of PPO1 and PPO2 probe sets were combined and labeled with two units of AF647 amine reactive succinimidyl ester Decapacks (ThermoFisher) and the Gip probe set was labeled similarly, with AF555 and lola-I with AF647. Labeling was overnight at 4 °C in 0.1 M sodium tetraborate at pH 9. HPLC was used to purify labeled from unlabelled probes as described83.Stellaris single-molecule FISH probes were designed for the The single-molecule FISH technique was optimized and adapted from the Stellaris website and based on Dr. Shawn Little’s protocol . Embryos were collected, dechorionated with bleach and crosslinked in 4% formaldehyde in a 1:3 mixture of PBS and heptane. Embryos were then sorted in PBT (0.1% Triton), and then devitellinated with a 1:1 mixture of methanol and heptane, equilibrated in methanol and stored at −20 °C. After gradual rehydration, embryos were post-fixed, treated with proteinase K (0.5 ug/ml) for 1 h on ice and 1 h at 37 °C, and crosslinked once more. The permeabilized embryos were washed with a series of PBT and Stellaris WashA buffers. The embryos then underwent prehybridization in the Stellaris Hyb buffer for one day at 37 °C. Hybridization with 1 μM concentration of probes was performed at 25 °C overnight. After several washes with WashA buffer and one with PBS, DAPI staining was performed in PBS buffer (5 µg/ml) for 5 min, followed by washes with PBT buffer to remove unbound DAPI. Embryos were mounted in Prolong gold and cured at room temperature (22 °C) for 3 days to a week.lola-I transcripts, images of whole-mount Drosophila embryos were acquired on a PerkinElmer Ultraview spinning disk confocal microscope equipped with an EM-CCD camera , using Volocity software (PerkinElmer). An Apochromat 63×, 1.46 NA oil immersion objective was used with a 405/488/561/640 nm multiband dichroic. Dual color images of DAPI and lola-I-Af647 were acquired with 405 nm and 640 nm laser lines, respectively, with 415–475 nm emission filter for DAPI and a 660–750 nm emission filter for Af647.For analyzing Drosophila embryos were acquired from a Nikon 3PO spinning disc, with a W1 disc, sitting on a Nikon Ti Eclipse base, controlled by Nikon Elements. Data were collected either with a 40×1.1 NA Plan ApoChromatic long working distance water objective (for overviews), or a 100X, 1.4 NA Plan-Apochromatic oil objective . AF647, AF555, and DAPI were excited with 640 nm, 561 nm, and 405 nm lasers, respectively, through a 405/488/561/640 nm main dichroic. Emission filters included a 700/75- and 455/50 nm dual band pass filter for the far red and DAPI channel, and a 605/70 nm emission filter for the red channel. An ORCA-Flash 4.0 V2 digital sCMOS camera was used, Z-step spacing was 1 micron for the 40× overview data, and 0.3 microns for 100× data. For 40× data, prior to display, gut autofluorescence was subtracted from the red channel using a reference in the green channel.For the expression analysis, images of whole-mount lolaORC4 embryos of 12–14 h. To calibrate transcripts per cell from integrated intensity, single transcripts were fit to a two-dimensional Gaussian. The total integrated intensity per cell was then divided by the average integrated intensity of single transcripts to get the number of transcripts per cell. Likewise, to find the intensity of nascent sites, nascents were identified and fit to a two-dimensional Gaussian. The integrated intensity of the Gaussian was then divided by a single RNA spot intensity to yield the number of RNAs per nascent.For quantification of transcripts per cell for transcriptional modeling, the 100× data were used. A Gaussian blur of radius 1 pixel was applied, followed by a rolling ball background subtraction with a radius of 50 pixels. To integrate the total signal over each cell, a z bin of 7 was applied for a final spacing of 2.1 microns. Cell outlines were manually drawn in FIJI, and integrated intensity was taken over 3 of the binned z slices, for a total cell size in z of 6.3 microns, after an intensity threshold was applied to remove the background. This background intensity was found from areas with no visible transcripts. Total cells counted were 184 from 7 wild-type embryos, and 340 cells from 14 52:HG is the confluent hypergeometric function of the first kind, Kon is the activation rate constant, Koff is the inactivation rate constant, and Kprod is the production rate constant, all expressed as ratios to the degradation rate constant. Given the difficulty in fitting these complex distributions, we took advantage of the fact that we can observe nascent transcripts for transcriptionally active cells. That lets us measure the ratio of active to inactive cells and therefore the ratio of activation to inactivation rates:Koff from Kon. Fitting was accomplished with the scipy optimize curve_fit function in python utilizing the trf (trust region reflective) algorithm85. Because the variance is higher for more frequent histogram bins and those are the bins that are most confidently determined for our data set, fitting was accomplished without weighting. Errors are determined by Monte Carlo analysis. This analysis assumes that the data distribution is reasonably well described by the fit and simulates random distributions repeatedly from the fit distribution and fits those distributions to obtain the error distribution for the fit parameters. For wild-type data, the fit produced the following parameters relative to the degradation rate constant: an activation rate constant, Kon, of 2.7 ± 0.42 and a production rate constant, Kprod, of 1524 ± 58. From the images, we know the ratio of active/inactive is 0.69. Taking these data together yields an inactivation rate constant, Koff, of 3.7. For the mutant, the fit produced a Kon of 0.19 ± 0.12 and a Kprod of 571 ± 207.8. From the images we know that the ratio of active/inactive is 0.036. These values yield a Koff of 5.1. Supplementary Data The fit of the distributions of transcripts per cell to the simple two-state model shown in Supplementary Fig. 86 with isolated RNA from entire embryo or single cells from 14 to 14.5 h AED Oregon-R embryos or lola-I mutant embryos sorted for homozygous mutant embryos that are GFP-. Total mRNA was extracted from non-cross-linked embryos using the Maxwell Total mRNA purification kit . mRNA from single cells for the scRNA-seq experiments was isolated using the 10x Genomics instrument.mRNA-seq and scRNA-seq experiments were performed as describedLola-I (custom-made by Genescript), α-tubulin antibodies , Lamin , α-MHC , Elav (7E8A10 from DSHB), Repo (8D12 from DSHB), Pol II .Drosophila melanogaster genome (dm6) using Bowtie (v 1.1.2)87, allowing a maximum of two mismatches and including only uniquely aligned reads. Coverage files were created by extending the aligned reads to the estimated insert size or the actual size for the paired-end libraries. For the bulk mRNAseq samples, pseudo-alignment was performed using the Kallisto package (0.46.0)88, to calculate the gene expression values. For the scRNA-seq samples, alignment and separations of reads from different cells and quantification of gene expression were done using the Cell Ranger pipeline (v 2.1.1) from 10× Genomics.Reads from the ChIP-seq data and ATAC-seq experiments were aligned to the lola/−− scRNAseq data by performing the canonical correlation analysis (CCA) using the Seurat package90. Gene expression in a particular tissue/ single-cell cluster is measured either using the median absolute normalized expression counts or using the percentage of cells with any detectable transcripts for each gene in each tissue. Expression of each Lola-I target gene was calculated in the presumed target tissue (sc-cluster with the maximum expression for each gene was considered as the expressing tissue for that gene) and the presumed non-expressing tissues (the five least expressing sc-clusters for each gene were considered as other tissues for that gene).The wild-type scRNAseq data were aligned to the n = 330) and distal sites (n = 233) based on whether the binding sites are present within +50 to −450 bp of an annotated TSS (flybase version r6.21) (Supplementary Data 71) we used the same promoter and distal site definitions for our analysis or directly used the frequencies from the publication when the definitions are comparable (Zelda6). Lola-I targets are defined as genes with a Lola-I binding peak  in both the Lola-I ChIP-seq replicates in the promoter region (Supplementary Data n = 329). Lola-I targets genes are further filtered for at least a twofold change in pol II occupancy between wild-type and mutant embryos for the scRNAseq analysis.Control genes are a randomly chosen 250 gene-subset of the constant set genes  (Supplementary Data De-novo identification of the Lola-I motif (position weight matrix) found at the opening-set genes in Fig. P values in Figs. P values in Fig. P values in Supplementary Data Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Reporting SummarySource Data


FAAH-OUT long non-coding RNA (lncRNA) gene, which was found from studying a pain-insensitive patient with reduced anxiety and fast wound healing, regulates the adjacent key endocannabinoid system gene FAAH, which encodes the anandamide-degrading fatty acid amide hydrolase enzyme.Chronic pain affects millions of people worldwide and new treatments are needed urgently. One way to identify novel analgesic strategies is to understand the biological dysfunctions that lead to human inherited pain insensitivity disorders. Here we report how the recently discovered brain and dorsal root ganglia-expressed FAAH-OUT lncRNA transcription leads to DNMT1-dependent DNA methylation within the FAAH promoter. In addition, FAAH-OUT contains a conserved regulatory element, FAAH-AMP, that acts as an enhancer for FAAH expression.We demonstrate that the disruption in FAAH-FAAH-OUT axis, thus providing a coherent mechanistic basis to understand the human phenotype observed.Furthermore, using transcriptomic analyses in patient-derived cells we have uncovered a network of genes that are dysregulated from disruption of the FAAH is a potential target for the treatment of pain, anxiety, depression and other neurological disorders, this new understanding of the regulatory role of the FAAH-OUT gene provides a platform for the development of future gene and small molecule therapies.Given that  et al. identify the molecular basis of FAAH-OUT-associated pain insensitivity, in which painlessness is accompanied by reduced anxiety and accelerated wound healing. The FAAH-OUT long non-coding RNA gene regulates FAAH expression via two transcription-dependent mechanisms, providing a platform for future gene therapy.Mikaeili  N-acyl phosphatidylethanolamine phospholipase D (NAPE-PLD)] and metabolizing [fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL)] enzymes.6 The expanded eCBS includes oleoylethanolamide (OEA) and palmitoylethanolamide (PEA) lipid mediators, their receptors (e.g. TRPV1 and PPARα) and metabolic enzymes. Components of the eCBS are potential therapeutic targets for a wide range of neurological conditions including chronic pain, anxiety and depression, as well as neurodegenerative conditions such as Alzheimer’s and Parkinson’s diseases.5The endogenous cannabinoid  affects a diverse array of key physiological functions including anxiety and stress responses, pain modulation, learning and memory, wound healing and development.N-acyltaurines (NATs).7-9 FAAH is particularly enriched in the liver, brain and also expressed within trigeminal and dorsal root ganglia (DRG).10-13 Within the brain, FAAH is found in regions that are significant for nociceptive transmission and modulation including the thalamus, periaqueductal grey (PAG) and amygdala. Faah is expressed in approximately a third of rat DRG neurons, with ∼70% of these being TRPV1-positive.12 Following sciatic nerve axotomy, expression of Faah is also induced in large diameter DRG neurons.12 Over the past 20 years many FAAH-inhibiting drugs have been developed, although none has yet successfully reached the clinic after human trials.14-17 Unfortunately, a lethal toxic cerebral syndrome was precipitated by a recently trialled FAAH inhibitor (BIA 10-2474) that was later shown to be related to off-target effects.19A key target in the eCBS is fatty acid amide hydrolase, an important catabolic enzyme that degrades AEA, OEA, PEA and other lipids such as 20 Recently we reported a new pain insensitivity disorder after studying a female patient (Patient PFS) who, in addition to being pain insensitive, also presented with additional clinical symptoms including a happy, non-anxious disposition, fast wound-healing, reduced stress and fear symptoms, mild memory deficits and significant postoperative nausea and vomiting induced by morphine.21 This phenotype was consistent with enhanced eCB signalling and genetic analyses showed two distinct mutations: (i) a microdeletion in a DRG and brain-expressed long non-coding RNA (lncRNA)-expressing pseudogene, FAAH-OUT, which is adjacent to the FAAH gene on human chromosome 1; and (ii) a common functional single-nucleotide polymorphism in FAAH, conferring reduced FAAH expression and activity.23 These mutations result in enhanced levels of anandamide and other bioactive lipids, that are normally degraded by FAAH.21A powerful way to identify novel human-validated analgesic drug targets is to study rare individuals with intact damage-sensing neurons that present with a congenital pain insensitive phenotype.FAAH being a heavily researched gene, the FAAH-OUT gene locus and how it regulates FAAH expression have been overlooked. Here we set up to elucidate how the ∼8 kb microdeletion that is distinct from and begins ∼5 kb downstream of the 3’ end of the currently annotated footprint of the FAAH gene disrupts its function. Potential key mechanisms we considered included (i) the microdeleted genomic sequence contains important regulatory elements needed for normal FAAH expression (e.g. an enhancer); and (ii) the FAAH-OUT lncRNA transcript has an epigenetic/transcriptional role in regulating FAAH expression.Despite FAAH. We also demonstrate that the FAAH-OUT lncRNA is enriched in nuclei and its transcription positively correlates with expression of FAAH, bearing all the trademarks of a positive regulator. The reduction in FAAH-OUT transcription leads to enhanced DNA Methyltransferase 1 (DNMT1)-dependent DNA methylation of the CpG island within the FAAH gene promoter, resulting in transcriptional shutdown of FAAH. FAAH-OUT therefore appears to regulate FAAH expression via preventing DNMT1-dependent DNA methylation of the FAAH promoter, thus maintaining its transcriptional potential.Here we show by gene editing in human cells that the ∼8 kb region that is deleted in Patient PFS results in reduced expression of FAAH-OUT microdeletion region contains a conserved regulatory element within the first intron of FAAH-OUT, FAAH-AMP, that behaves as an active enhancer regulating FAAH expression. Editing or silencing the FAAH-OUT promoter region or the short evolutionarily conserved FAAH-AMP element leads to reduced FAAH mRNA in human cells.Furthermore, we show that the FAAH − FAAH-OUT axis, we used microarray analysis of Patient PFS-derived fibroblasts to uncover a network of key molecular pathways and genes that become dysregulated as a result of activity disabling mutations in the FAAH and FAAH-OUT genes.Finally, to narrow in on the key functional targets downstream of the 2 incubator with 92–95% humidity for another 24 h.Human embryonic kidney 293 cells  were cultured in Dulbecco’s modified Eagle medium  with 10% foetal bovine serum . Mouse catecholaminergic neuronal tumour cells  were cultured in DMEM:HAMS F12 (1:1) with 2% glutamine and 8% FBS. Lipofectamine 3000 (Invitrogen) was used for DNA transfections  was added directly to the cells and was incubated for 5 min at room temperature. Cell lysate was passed through a pipette up and down several times. RNA was extracted using PureLink™ RNA Micro Scale Kit (Invitrogen) according to the manufacturer’s procedures. Genomic DNA was isolated using the DNeasy Blood and Tissue kit (Qiagen) and used as template to confirm gene editing . TRIzol editing .HEK293 cells were transfected using Lipofectamine 3000 with SaCas9-IRES-AcGFP1 plasmids containing guide pairs HMa or HMb  or a no FAAH (Hs01038660_m1), FAAH-OUT (Hs04275438_g1), BDNF (Hs03805848_m1), ACKR3 (Hs00604567_m1), WNT5B (Hs01086864_m1), GABBR2 (Hs01554996_m1), DKK1 (Hs00183740_m1), SFRP2 (Hs00293258_m1), SERPINF1 (Hs01106937_m1) and ACTB (Hs01060665_g1). Mouse TaqMan probes used were: Faah (Mm01191801_m1) and Actb (Mm01205647_g1). The expression level of target genes was normalized to the housekeeping Actin gene mRNA. Relative gene expression [relative quantities (RQ) value] was determined using the 2−ΔΔCt equation in which control unaffected individuals or empty vector cDNA samples were designated as the calibrator. All RT–PCR data are expressed as mean ± standard error of the mean (SEM) with significance indicated by *P ≤ 0.05, **P ≤ 0.01 and ***P ≤ 0.001 (two-tailed Student’s t-test).Reverse transcription was performed using oligo d(T) and Superscript III first-strand synthesis system (Invitrogen) according to the manufacturer’s conditions. TaqMan real-time PCR was carried out using the following probes for human genes: Chromatin immunoprecipitation (ChIP) assays were performed according to the manufacturer’s protocol using the Chromatrap Enzymatic ChIP-seq kit. Immunoprecipitations were performed overnight at 4°C using antibodies against H3K27ac (Abcam 4729), H3K4me1 (Abcam 8895), H3K4me3 (Abcam 8580), H3K9me3 (Abcam 8898), H3K27me3 (Active Motif 39157), DNMT1 (Active Motif 39204) and DNMT3A (Active Motif 39206). Rabbit IgG were used as control for ChIP and primers within a gene desert on chromosome 16 were used as a negative control for qPCR.All ChIP experiments were performed in triplicates using two independent chromatin preparations. The immunoprecipitated DNA and the input DNA were analysed by real-time PCR using the ΔΔCt method and the primers are listed in FAAH (CpG Island 100530) . Methylation-sensitive (EPHS115450-1A) and methylation-dependent (EPHS115451-1A) digest control assays were performed to test the cutting efficiency of the restriction enzymes. Samples were analysed as recommended by the manufacturer (Qiagen).EpiTect methyl II PCR primer assay (Qiagen) was performed according to the manufacturer’s protocol. Briefly, 250 ng of genomic DNA was used to set up the four independent restriction enzyme digests: (i) methylation-sensitive; (ii) methylation-dependent; (iii) methylation-sensitive and methylation-dependent double digest; or (iv) mock digest. Q-PCR was performed as per the manufacturer’s protocol, using commercially available primers for human Ethical approval was granted by University College London REC and written informed consent was provided by Patient PFS and four gender-matched healthy control subjects. A punch skin biopsy (3–6 mm) was taken from the outer upper arm of each individual and primary cultures of dermal fibroblasts were passaged in DMEM (Thermo Fisher) supplemented with 10% FBS (Hyclone) and 1% penicillin/streptomycin (Thermo Fisher).Total RNA was isolated from the primary fibroblast cultures derived from Patient PFS and four healthy unrelated gender-matched control subjects  using the PureLink RNA Micro Kit (Invitrogen) and run by Eurofins on the human Clariom D transcriptomic array (Thermo Fisher) using the GeneChip WT Plus labelling kit reagent. Expression data were RMA normalized and analysed using the Transcriptome Analysis Console (TAC) software (Thermo Fisher) and Ingenuity Pathway Analysis (Qiagen). Microarray data have been deposited at Gene Expression Omnibus Array Express with reference number E-MTAB-11809.www.zyagen.com) via AMS Biotechnology (https://www.amsbio.com) , human brain cerebral cortex frozen sections , human cerebellum frozen sections  and human prostate frozen sections  were obtained commercially from Zyagen (bio.com) .For the mouse DRG sections , adult CIn situ hybridization (ISH) was performed using the RNAscope assay (Advanced Cell Diagnostics) following the protocol for fresh-frozen samples for human cerebral cortex, human cerebellum and human prostate tissue samples, and mouse DRG samples using Multiplex Fluorescence Kit v2. Human DRG paraffin sections were treated according to the ACD’s FFPE-fixed samples protocol.hsNEFH (#448141-c4), hsCNR1 (#591521-c4), hsFAAH (#534291-c2) and hsFAAH-OUT (#534301-c3). RNA localization was detected with either AF488 or Opal 520 (green), Opal 570 (red) and Opal 650 (far-red) fluorochrome dyes (Perkin Elmer) compared to DAPI staining (nuclei) or TS-coumarin  used for NEFH or CNR1. ISH slides were mounted using Prolong Gold (ThermoFisher Scientific #P36930). Mouse RNAscope probes included mmFaah (#453391) and mmNefh (#443671-c4).Probes included Fluorescence was detected using Zeiss LSM 880 Airyscan microscope. Images were taken at 10 × and 20 × magnifications with 4 × averaging. Tiles were stitched when more than one was used to image the area, Airyscan processed and exported as 16-bit uncompressed tiff files for further basic editing in Adobe Lightroom v6 (Adobe) on a colour calibrated iMac (X-Rite) retina monitor. Final images were exported as jpeg files with 7200 pix on longest side at 300 ppi.n referring to the number of samples tested per group, as indicated in the figure legends.Data were analysed using GraphPad Prism 9 , and results presented as mean ± SEM with Microarray data have been deposited at Gene Expression Omnibus Array Express with reference number E-MTAB-11809. All data are available in the main text or the FAAH gene , a novel 13-exon lncRNA that is classed as a FAAH pseudogene and which has a similar tissue expression profile to FAAH.21Patient PFS carries a 8131 bp heterozygous microdeletion on chromosome 1  that begins ∼4.9 kb downstream of the end of the AAH gene .21 The mFAAH-OUT microdeletion on FAAH gene expression, we used the CRISPR/Cas9 system to edit human embryonic kidney cell lines (HEK293) to mimic the patient’s microdeletion. HEK293 cells were transiently transfected for 48 h with an SaCas9 plasmid bearing a guide pair  that flank the microdeletion, highlighting that FAAH expression is affected by the induced downstream deletion  technology—RNAscope assay and confocal microscopy. To ensure the specific detection of FAAH and FAAH-OUT transcripts, we used probes that target different regions of each transcript.We have shown previously that FAAH and FAAH-OUT transcripts in fresh-frozen (FF) and formalin-fixed paraffin-embedded (FFPE) human tissue samples  provided direct evidence that FAAH mRNA and FAAH-OUT lncRNA were expressed within the same cells and predominantly localized in the cytoplasm and nucleus, respectively  delete the putative FAAH-OUT promoter region; or (ii) epigenetically silence the promoter using CRISPR interference via targeting of a nuclease-deficient form of SaCas9 (dSaCas9) fused to a Krüppel-associated box (KRAB) repressor to the FAAH-OUT promoter.26 When localized to DNA, dSaCas9-KRAB recruits a heterochromatin-forming complex that causes histone deacetylation and methylation (H3K9 trimethylation).27To explore what effect FAAH-OUT promoter and cloned into an SaCas9-expressing vector. HEK293 cells were transiently transfected and the activity of each sgRNA-pair was assessed 72 h after transfection by RT-qPCR for FAAH and FAAH-OUT mRNA expression. Both FAAH and FAAH-OUT had markedly reduced expression when the FOP2- and FOP3-guide pairs were used to induce a deletion in the FAAH-OUT promoter compared to cells transfected with SaCas9 only  system to recruit a strong transcriptional activator to the FAAH-OUT putative promoter region and activate the gene in cis. We successfully increased FAAH-OUT expression more than 2-fold in transiently transfected HEK293 cells which lead to a ∼60% increase in FAAH expression, as measured by RT-qPCR  was identified in the first intron of FAAH-OUT , H3K4 tri-methylation (H3K4me3) and H3K27 acetylation (H3K27Ac).Several studies have shown that active enhancer regions are enriched in specific histone modifications including histone 3 lysine 4 (H3K4) methylation and histone 3 lysine 27 (H3K27) acetylationFAAH-OUT putative promoter with a gene desert control region by ChIP-qPCR, we observed that both the FAAH-AMP region and the FAAH-OUT upstream region showed strong enrichment in H3K27ac and H3K4me1 and a low level of H3K4me3  in ide-pair .FAAH gene expression compared to control  to recruit dSaCas9-KRAB to FAAH-AMP to enforce inhibition of the region’s regulatory elements without cutting out the FAAH-AMP sequence. Upon transient expression of the FOC1 sgRNA with CRISPRi in HEK293 cells, we observed significant repression of  control . Taken t binding , furtherFAAH-OUT transcription, either by induced promoter deletion or epigenetic inhibition, leads to reduced FAAH expression  could be sufficient to shift the balance of DNA methylation and chromatin modification towards FAAH promoter inactivation.CpG-rich promoters are typically unmethylated, marked with histone modifications such as H3K4me3, and are highly active. If the FAAH-OUT expression affects the local epigenomic profile at the FAAH promoter region, we used DNA methylation and ChIP-qPCR assays to screen for levels of methylated DNA at the FAAH promoter in a heterozygous HEK293 FAAH-OUT+/− cell line. As shown in FAAH promoter are balanced between 40% methylated and 60% unmethylated DNA, with methylation rising sharply by ∼60% in heterozygous (HTZ) cells with reduced levels of FAAH-OUT expression, reversing the methylated versus unmethylated ratio. This suggests that lower levels of the FAAH-OUT lncRNA due to one allele loss leads to local epigenetic changes that drive FAAH expression down. Furthermore, the epigenetic inactivation of the FAAH promoter is enhanced by a rise in H3K9me3 modification  between the Patient PFS line and four control subjects. Ingenuity Pathway analyses highlighted groups of gene products which take part in WNT-induced signalling, wound-healing, brain-derived neurotrophic factor (BDNF)-signalling and G-protein signalling  expression of the FAAH-OUT lncRNA that may play a role as a positive regulator of FAAH; and (ii) opening up of chromatin in the FAAH-AMP enhancer region, which improves accessibility to the region for proteins that in turn modulate efficiency of FAAH transcription and potentially allow local looping between FAAH and FAAH-OUT genes for co-ordinated transcription  form when a nascent transcript or a lncRNA invades and makes a complex with a DNA duplex and are widespread at the GC-rich regions of promoters, protecting CpG islands from DNA methylation and preventing silencing.55 Both FAAH and FAAH-OUT promoters are GC-rich with >100 CpG pairs within the FAAH promoter sequence and about half of those are clustered in large CpG islands. R-loop formation could be achieved by locking RNA onto the DNA strand via 4xG repeats using a velcro-type interaction between them and quadruple Cs on the other strand.56 The FAAH promoter sequence has ∼10 4xGs whereas the FAAH-OUT lncRNA has 20 4xGs.Further work will help to understand exactly how FAAH-OUT promoter region were similar to those at the FAAH-AMP region and resembled those of an active enhancer (enriched in H3K27Ac and H3K4me1) rather than a promoter (enriched in H3K4me3 and H3K27Ac)  . This suA levels .21 In the original case report, we also noted that her dental surgeon observed, most unusually, that her saliva dissolves the fixative for a temporary denture after just 90 min.21 Interestingly, there is a recent report of lipidomic profile differences in the submandibular gland of FAAH knockout versus wild-type mice.59 It will be interesting to assay the salivary lipidomic profiles of Patient PFS in potential future studies.We further aimed to understand how decreased levels of FAAH and higher levels of anandamide (and other substrates) translate into the patient’s pain insensitivity syndrome, which is characterized by the absence of postoperative pain, painless burns and bone fractures, a happy, non-anxious disposition, fast wound healing, fear and memory deficits, and significant postoperative nausea and vomiting induced by morphine.60 with a major clinical trial with the irreversible FAAH inhibitor PF-04457845 failing to induce effective analgesia in patients with pain due to osteoarthritis of the knee.17 Whether this is a compound specific problem and/or whether efficacy would be achieved for another pain disorder remains to be fully determined. FAAH inhibitors in rodent models are analgesic and genetic knockout of Faah in mice causes hypoalgesia, but not complete painlessness.61-63 Our results highlight the importance of the FAAH-OUT lncRNA to the human pain insensitive phenotype observed in Patient PFS. Long non-coding RNAs are known for their exquisite spatial and temporal sensitivity in regulating gene expression.65 In future research, we aim to determine at the single-cell level the specific cellular populations that are contributing to the symptoms observed in Patient PFS and how FAAH-OUT dynamically regulates FAAH expression in these cells. In particular, we plan to determine whether FAAH-OUT acts in trans to regulate gene expression at other genomic loci, similar to other lncRNAs such as lincRNA-Cox2 and Firre.66FAAH inhibitors have yet to reach the clinic as analgesics,FAAH—FAAH-OUT axis, we used microarray analysis of patient-derived fibroblasts to uncover a network of key molecular pathways and genes that become dysregulated as a result of disrupting FAAH-OUT. There were 797 genes upregulated and 348 genes downregulated  between the Patient PFS line and four gender-matched controls. Pathway analyses showed major changes in expression level of genes which take part in WNT-induced signalling, wound healing, BDNF-signalling and G-protein signalling  shift from a canonical to non-canonical type. Importantly, WNT pathways have been previously linked to wound healing and both upregulated Wnt5b and Wnt16 have also been linked to bone regeneration.67-69 In addition to gene expression changes highlighted in 53In this study, to narrow in on the key functional targets downstream of the gnalling . Thus, s71 Furthermore, pharmacological inhibition of FAAH activity has been reported to lead to an increase in BDNF levels in rats.46-48 Our gene expression analyses in patient-derived fibroblasts show a significant upregulation in BDNF expression, although whether this is replicated in other patient tissues remains to be tested. Nevertheless, we have shown that in mice, pharmacological inhibition of the FAAH enzyme also upregulates hippocampal BDNF, providing further evidence for the FAAH-BDNF link.Interestingly, WNT-dependent signalling has been previously reported to be connected to levels of BDNF, which modulates mood and is directly linked to anxiety and depression through TrkB receptor signalling.GABBR2 which encodes receptor subunit GABAB2 which forms an active heterodimeric complex with GABAB1 in the GABAB receptor.72 GABAB receptors are abundant in the brain, where they are localized in many neuronal cell types including interneurons and some glial cells.72 GABBR2 inhibits neuronal activity via G-protein coupled secondary messenger systems and its low levels were implicated in reduced analgesic effects of oxycodone.73 Furthermore, GABAB receptor knockout mice data indicate a role for GABAB receptors in nociception and anxiety, with GABAB knockout mice showing increased anxiety.75 Interestingly, low levels of GABBR2 expression were shown to be a valid biomarker for patients with chronic migraine.76 Thus high levels of GABBR2 expression could be consistent with the pain- and anxiety-free phenotype of Patient PFS.21Another gene that is significantly upregulated is ACKR3  to higher availability of endogenous opioid peptides for the classical opioid receptors  by which the awad098_Supplementary_DataClick here for additional data file.


Saccharomyces cerevisiae promoter and terminator were employed to design a synthetic bifunctional element. The promoter strength of the synthetic element is apparently regulated through a spacer sequence and an upstream activating sequence (UAS) with a ~5-fold increase, and the terminator strength could be finely regulated by the efficiency element, with a ~5-fold increase. Furthermore, the use of a TATA box-like sequence resulted in the adequate execution of both functions of the TATA box and the efficiency element. By regulating the TATA box-like sequence, UAS, and spacer sequence, the strengths of the promoter-like and terminator-like bifunctional elements were optimally fine-tuned with ~8-fold and ~7-fold increases, respectively. The application of bifunctional elements in the lycopene biosynthetic pathway showed an improved pathway assembly efficiency and higher lycopene yield. The designed bifunctional elements effectively simplified pathway construction and can serve as a useful toolbox for yeast synthetic biology.The construction of a genetic circuit requires the substitution and redesign of different promoters and terminators. The assembly efficiency of exogenous pathways will also decrease significantly when the number of regulatory elements and genes is increased. We speculated that a novel bifunctional element with promoter and terminator functions could be created via the fusion of a termination signal with a promoter sequence. In this study, the elements from a  This promoter directs high levels of transcription by RNA polymerase II in eukaryotic cells. The copy number of the UAS was found to be another important factor. The intensity of the promoter could be increased 400 times by changing the copy number of the UAS [E. coli, S. cerevisiae, and B. subtilis, which demonstrated normal function in a range of different hosts.A key concept in synthetic biology is the development of standardized multifunctional components . Specifiapparent ,4,5,6. Wapparent  describe the UAS . Sen et  the UAS  construcTranscription terminators are another important element for regulating gene expression in synthetic gene networks. The terminator sequence in eukaryotic genes are located in the 3′ untranslated region, and its activity is directly related to mRNA stability, and thus the regulation of gene expression . Guo et The assembly of regulatory elements and genes is a basic procedure. However, in yeast, standard parts, such as promoters and terminators, are repeatedly used for genetic circuit construction, which may lead to the rearrangement of repetitive sequences in multigene assemblies ,16,17,18In this study, we thus aimed to establish a regulatory element capable of simultaneously regulating the expression of two genes in yeast. That is, an element in a gene circuit not only performs the function of transcriptional termination of upstream genes but also possesses the ability to recruit RNA polymerase II to induce transcription by acting on the promoters of downstream genes. The terminator–promoter bifunctional element simplifies the construction process in gene circuits and pathway engineering, serving as a useful toolbox for the development of yeast metabolic engineering.S. cerevisiae, TATATAA is recognized as being the optimal binding site sequence for recognition by the TATA-box binding protein (TBP) and is conserved [CP, was assembled by connecting the BRE, TATA box, Inr, MTE, and DPE via linker sequences. Additionally, an upstream activating sequence (UAS) that contains yeast transcription factor binding sites was designed and added upstream of the core promoter DCP   site . The seqT) D. After T) .The synthetic promoter consisting of a core promoter region and UAS was designed to guide the expression of green fluorescent protein in plasmid pRS-Exp1 with CYC1t as a terminator to indirectly characterize its activity in terms of fluorescence intensity . ConservCP1, consisting of the BRE element (located at −37 to −32 bp of the transcription start site), TATA box (−31 to −26 bp), Inr (+1), MTE (+18 to +27 bp), and DPE (+28 to +32 bp), was first tested. In order to simplify the sequence composition, a short DCP1 was also designed by shortening the spacer sequence between the DCP1 elements to 5 bp. The super core promoter (SCP) and the core promoter regions of the natural promoters CYC1 and TYS1 were used as positive controls to verify the basic transcriptional ability of the core promoter element [S. cerevisiae. The results show that the core promoter designed in this study successfully promotes expression of the fluorescent protein. Compared with the native core promoter TYS1, the expression of DCP1 reaches 88%. However, the fluorescence intensity of short DCP1 decreased by 67%, indicating that the length of the spacer sequence appears to affect the intensity of the core promoter element , it failed to increase the expression. This may because the mutations in the TATA box resulted in it no longer being recognized by RNA polymerase II , which confirms that the UAS-only sequence is unable to initiate transcription. TYS1 was used to control the transcriptional initiation of fluorescent proteins [T1, DT2, and DT3 were constructed with the efficiency elements TATATA, TACCTA, and TATGTC, respectively. Furthermore, because the linker 1 and linker 2 sequences also have a notable effect on its activity, the linker sequences TCCCTTTTCT and GAAAGGGC for the strongest terminator were used [T1 with efficiency element TATATA demonstrated the same relative fluorescence levels as the natural terminator CYC1 (TUB1 (as shown in T2) or 35% (DT3) relative to that of DT1. Moreover, if the efficiency element TATATA is mutated to GAGAGA (DTMU), the relative fluorescence decreases significantly to only 21% that of DT1. Similarly, the vector pRS-Exp4 with blue fluorescent protein mTagBFP2 was applied to verify the fluorescent reporter protein levels with similar results. In short, the transcription termination function of the synthetic terminators can be finely regulated by the efficiency element.The vectors pRS-Exp2 with eGFP and pRS-Exp4 with mTagBFP2 were constructed  to verifproteins ,41,42. Tproteins ,44,45. Tproteins . In partproteins ,29. Thusere used . The DT1tor CYC1 A. FurtheCP2 and the strong terminator DT1. The direct assembly of DT1 and P-DCP2 generated new elements Bif-a1 (DT1-P-DCP2) or Bif-a2 (P-DCP2-DT1). For functional validation, pRS-Exp3 was also constructed  can act as a synthetic transcriptional amplifier to increase promoter activity , a seconIn addition, to suppress undesirable homologous recombination events that are present in genetic circuits in yeast, another library was constructed by inserting different spacer sequences between the individual motifs of the bifunctional element A. The reS. cerevisiae. Because yeast contains the MVA pathway to produce the intermediate product geranylgeranyl diphosphate (GGPP) for lycopene synthesis, exogenous phytoene synthase Crt B and phytoene desaturase Crt I need to be introduced to S. cerevisiae [Crt B—Bif-UAS4—Crt I—Bif-U7, pRS—Bif-TATTTA—Crt B—Bif-UAS9—Crt I—Bif-U6, and pRS—Bif-3*TA—Crt B—Bif-UAS5—Crt I—Bif-U2 into S. cerevisiae dubbed as Sl-1, Sl-2 and Sl-3, respectively  showed that the transcription of Crt I and Crt B were significantly higher in strains Sl-2 and Sl-3 than in strain Tl-1  was used as the host strain. Yeast transformants in this study were selected on YPD plates , peptone (2%), glucose (2%), agar (2%), and 500 mg/L hygromycin B). Yeast strains were cultured in YPD liquid medium (containing yeast extract (1%), peptone (2%), glucose (2%), and with corresponding antibiotics) at 30 °C and 220 rpm.E. coli DH5a  was used for clone and routine transformations after routine propagation in lysogeny broth (LB) medium (yeast extract (0.5%), tryptone (1%), NaCl (1%), and 100 μg/mL ampicillin). Oligonucleotides were synthesized by GENEWIZ . DNA polymerase Phanta Max was obtained from Vazyme . Restriction enzymes and T7 DNA ligase were purchased from New England Biolabs . The kits for plasmid extraction were from TIANGEN . The kits for DNA purification were from Vazyme . The chemical reagents were all of analytical grade. A spectrophotometer  was used to determine the cell concentration by measuring the optical density at 600 nm (OD600). Crt B, accession no.KC954270.1), phytoene desaturase  were cloned from the W1 strain constructed by Lixia et al. [Two fluorescence genes, eGFP and mTagBFP2 were synthesized by GENEWIZ after codon optimization . The gena et al.  Yeast prNotI and SacI to form the scaffold plasmids pRS-Exp1 . Specificallmand); the ligated products were then inserted into the pRS41H plasmid linearized by pRS-Exp1 . Five scCrt B—Bif-UAS4—Crt I—Bif-U7 was constructed via Gibson assembly [S. cerevisiae. Specifically, the primer pairs for adjacent fragments were designed with flanking overlap homology sequences, as shown in Crt B and Crt I were amplified using primer pairs, Crt B-F/Crt B-R and Crt I-F/Crt I-R, respectively. Bif-UAS4 was amplified using the primer pair, Bif-UAS4−F/Bif-UAS4-R. Strains Sl-2, Sl-3, and Tl-1 were constructed similarly to Sl-1. The gene expression plasmid pRS—Bif-TATTTA—Crt B—Bif-UAS9—Crt I—Bif-U6, pRS—Bif-3*TA—Crt B—Bif-UAS5—Crt I—Bif-U2, and pRS—CYC1p—Crt B—ADH1—TYS1—Crt I—ADAL were ligated by a similar strategy and were transformed into S. cerevisiae, respectively. Sequences were verified by Sanger sequencing .The construction of lycopene producing strain Sl-1 was taken as an example. First, the gene expression plasmid pRS—Bif-2*TA—assembly . Plasmidg for 1 min. The collected cells were washed with sterile water (2 × 1 mL) and then gently resuspended in 0.1 M LiAc (1 mL). After centrifugation at 844× g for 5 min, the supernatant (900 mL) was discarded, and the remaining cells were used as competent cells. Then, the components, including the DNA fragments (600 ng), linearized plasmid (400 ng), 10% denatured DNA from salmon sperm , poly(ethylene glycol) , and 1 M LiAc (72 µL), were added, according to the indicated sequence, to the competent cells, and fully mixed using a high-speed spiral mixer for about 1 min. After incubation for 30 min at 30 °C, DMSO (72 µL) was added to the above system. The cells were immediately heat-shocked at 42 °C for 15 min, and the supernatant was discarded after centrifugation at 1180× g for 1 min. Finally, 5 mM CaCl2 (200 µL) was used to resuspend the yeast cells. After centrifugation, the yeast cells were re-suspended in double-distilled H2O (100 µL) and coated on the selective medium for incubation.Yeast was cultured overnight in YPD medium and then transferred into YPD medium (50 mL) with a concentration ratio of 10% and continually cultivated for 4–6 h. The yeast broth was collected in 1.5 mL centrifuge tubes, and the medium was discarded after centrifugation at 1844× 2 were cultured in YPD medium in shaking flasks at 30 °C for 12 h and then collected by centrifugation, washed with 1× PBS (pH = 7.4), and diluted to 1 × 106 cell/mL for fluorescence analysis . The green fluorescent protein eGFP  and the blue fluorescent protein mTagBFP2  were detected using a FITC-A detector and PB450-A detector, respectively. Finally, the data were processed using CytExpert software . The FI values for at least 10,000 cells in each sample were measured. The FI of eGFP and mTagBFP2 was obtained by normalization relative to that of the FI of S. cerevisiae CEN.PK2-1C with the plasmids TYS1p-eGFP-CYC1t and TYS1p-mTagBFP2-CYC1t, respectively. All experiments were repeated 3 times. The standard error was estimated from the square root of the estimated error variance of the quantity.The element strength is indirectly indicated by the fluorescence intensity (FI) of eGFP and mTagBFP2. Yeast cells containing the genes of eGFP and mTagBFP® 96 (Roche). TUB1 was used as the reference gene. The primers were as follows: 5′-CCAAC TGGTT TCAAG ATCGG TA-3′ and 5′-TCCAC AGTGG CCAAT TGTGA-3′ for qPCR analysis, 5′-ACGCT GGTTC TCAAA TGCAC-3′ and 5′-AAGTG TCGTC CAA TT GAGAG-3′ for the Crt B gene, and 5′-GGTAA GGACG GTTTC GACAG-3′ and 5′-CGATG AATTG CAATC TCAAG-3′ for the Crt I gene. The PCR conditions consisted of 40 cycles at 95 °C for 5 s, 60 °C for 20 s, and 72 °C for 1 min, with a final extension at 72 °C for 10 min. The transcription levels of the function genes in recombinant S. cerevisiae were calculated based on the level of TUB1 mRNA.Fresh samples of the cell culture were used to determine the relative expression ratio of genes via qPCR. The total RNA in the mid-log-phase yeast cells extracted was quantified by measurement of the absorbance at 260 nm with a NanoDrop 2000c spectrophotometer . Removing DNA Contamination from RNA Samples by treatment with DNase I . cDNA was transcribed from 500 ng total RNA using a high-capacity cDNA reverse transcription kit . qPCR was performed with a SYBR Premix Ex Taq II (TaKaRa) system and LightCyclerThe fluorescence was recorded by flow cytometry. Data represent the mean fluorescence intensity with error bars  from three independent experiments. Statistical analysis was performed using Origin 64 software . Independent univariate analysis of variance (ANOVA) was used for the comparison of multiple books. The difference of 0.05 was statistically significant.18 column  and a photodiode array (PDA) detector was used to measure lycopene. The detection wavelength was 470 nm. The mobile phase was acetonitrile–methanol–dichloromethane solution (21:21:8) with a flow rate of 1.0 mL/min. All experiments were repeated three times. The standard error was estimated from the square root of the estimated error variance of the quantity.All engineered yeasts were grown at 30 °C and 220 rpm for 36 h as a seed solution. Then, they were inoculated into unbaffled 150 mL culture flasks containing 100 mL medium and cultured in the same conditions for four days. Lycopene was extracted using the modified hot HCl/acetone method. The acetone phase containing extracted lycopene was filtered for HPLC analysis. An HPLC system (LC-2030 plus) equipped with an Inertsil ODS-3 CA class of terminator–promoter bifunctional elements was designed and constructed. Three libraries of bifunctional elements were established by adjusting the TATA sequence, transcription factor binding site, and spacer region. The results show that the promoter and terminator activities of the bifunctional elements could be finely regulated by adjusting the TATA sequence, transcription factor binding site, and spacer region. The application of bifunctional elements was shown to not only improve the efficiency of pathway construction, but also increase the yield of lycopene.