SBP-CNR and NAC-NOR transcription factors, both of which are considered as master regulators in the current tomato ripening model. These plants only displayed delayed or partial non-ripening phenotypes, distinct from the original mutant plants, which categorically failed to ripen, suggesting that they might be gain-of-function mutants. Besides increased DNA methylation genome-wide, the original mutants also have hyper-H3K27me3 in ripening gene loci such as ACS2, RIN, and TDR4. It is most likely that multiple genetic and epigenetic factors have contributed to their strong non-ripening phenotypes. Hence, we propose that the field should move beyond these linear and two-dimensional models and embrace the fact that important biological processes such as ripening are often regulated by highly redundant network with inputs from multiple levels.Tomato is considered as the genetic model for climacteric fruits, in which three major players control the fruit ripening process: ethylene, ripening transcription factors, and DNA methylation. The fruitENCODE project has now shown that there are multiple transcriptional circuits regulating fruit ripening in different species, and H3K27me3, instead of DNA methylation, plays a conserved role in restricting these ripening pathways. In addition, the function of the core tomato ripening transcription factors is now being questioned. We have employed CRISPR/Cas9 genome editing to mutate the  The regulatory circuits that govern the expression of genes required for ripening in tomato plants are highly redundant. Fleshy fruits that use the hormone ethylene to regulate ripening have developed independently multiple times in the history of the angiosperms. Guiqin Qu at China Agricultural University in Beijing and colleagues working on the fruitENCODE project are exploring the genetic and epigenetic basis of this convergent evolution. In tomatoes, three transcription factors have been shown to control ethylene production and regulate ripening. However, when gene editing techniques were used to introduce mutations that interfere with the function of these transcription factors, partial ripening or a delay in ripening was observed. The fact that ripening was not abolished indicates that the ripening process is more robust and complex than previously thought. This enables plants to interact with the coevolving animals that consume the fruits and disperse the defecated seeds to distant locations, and hence increase plant reproductive success2. Charles Darwin has also acknowledged its evolutionary advantage as “beauty serves merely as a guide to birds and beasts in order that the fruit may be devoured and the matured seed disseminated”.For flowering plants, fruits serve as seed dispersal vehicles that have evolved usually from carpels or adjacent floral tissues. Fruit ripening is often described as a developmental process that alters the physiological and biochemical properties of a seed-bearing organ to aid seed dispersalHowever, at the molecular level, transforming an unappealing carpel to a tasty fruit is not a simple task. It requires a complete reprograming of the carpel gene expression network, during which hundreds if not thousands of genes have to be switched on and off in a highly coordinated manner. It is the combined action of these so called “ripening genes” at a precise developmental timing that has transformed nearly every aspect of the carpel tissue, such as color, aroma, flavor, texture, and nutritional content, in order to attract frugivorous as seed dispersers. This process must also be kept under strict regulatory control, as any premature transformation ahead of seed maturation is highly detrimental.Solanum lycopersicum) as a model to elucidate its genetic and epigenetic basis4. The tomato ripening model has three key components: the hormone ethylene, ripening transcription factors , and DNA methylation. However, recent findings from the fruitENCODE project has shown that this model is not universal, as there are at least three different types of transcriptional positive feedback circuits controlling ripening in seven climacteric species5. Only tomato has a genome-wide DNA, CG and CHG demethylation, and CHH hypermethylation, while most species used H3K27me3 to regulate the ripening circuits. The H3K27me3 marks and the ripening genes could be traced back to dry and non-climacteric fruits, in which they regulate the floral organ identity and senescence. In addition, gene silencing and genome editing technology have now enabled us to re-examine the tomato fruit ripening transcription factors, resulting in controversial and sometimes conflicting views of its biological functions8.This ripening regulatory network has fascinated scientists for decades, and also has important implications in agriculture worldwide as it affects yield, nutritional value, and shelf life of our horticultural produces. Significant progress has been made using tomato : MADS-box RIPENING INHIBITOR (RIN), SBP-box COLORLESS NON-RIPENING (CNR), and NAC transcription factor NON-RIPENING (NOR)PG, EXP1, CEL2, aroma biosynthesis genes LoxC, pigment formation genes PSY1, and additional TF genes, such as NOR, CNR, AP2a, FUL1/215. Most importantly, RIN also binds to the promoter of ethylene biosynthesis genes ACS2 and ACO116. Promoter motif analysis and ChIP-Seq also confirmed that ethylene transcription factor EIN3 binds to the promoter of RIN, which completes a positive feedback loop that fits the autocatalytic system 2 ethylene proposed by McMurchie et al., 197214. With this MADS-loop activated in ripening fruit, a small amount of ethylene could rapidly amplify itself and drives the downstream ripening gene expression  that failed to ripen. The rin mutant is caused by a DNA deletion, resulting in a truncated RIN fused to an adjacent MADS gene MACROCALYX12. It was once thought that the chimeric RIN-MC fusion protein could not have biological functional, and rin is a loss-of-function mutant, while recent evidence suggests otherwise. CRISPR/Cas9 knockout and RNAi silencing of RIN in the wild-type tomato only recreated a partial non-ripening phenotype distinct from the complete lack of ripening in the rin mutant6. On the other hand, knockout or RNAi silencing of the chimeric RIN-MC mutant protein in rin background could partially restore ripening. These reults suggest that rin is in fact a gain-of-function mutant8.Much of what we know about the ripening regulation came from the three tomato TF mutants  are sequence-specific DNA binding protein capable of transcription regulation. One of the most important attributes of a TF is the sequence preference, the binding sites to be precise. Therefore, the NAC-NOR and MADS-RIN mutants, where the mutant protein is either truncated or fused to others, that they retained their putative DNA binding domains. Although they might lack transcriptional activation function, they could block other TFs from binding to the same sites, leading to a gain-of-function non-ripening phenotype.This is further complicated by the fact that TFs also need to signal other proteins like the Mediator and RNA polymerase II to initiate transcription or recruit chromatin modifier to indirectly activate or repressive transcription. Hence, the co-factors also influence the TF’s biochemical properties, not to mention that a TF could lack effector function on its own, and instead act by steric mechanisms. It is likely the case for the original 21. However, the unparalleled resolution of ChIP-Seq presents a new challenge for the study of TF function, and misinterpretation of the ChIP-Seq data often created more problem than it solved. The most common mistake is to define potential TF targets based on the present of ChIP-Seq binding, and then only cherry-pick the genes with altered expression when the TF is knocked out or over-expressed. We should learn from the mammalian research, where hundreds of TF ChIP-Seq experiments have been performed to map binding sites of all highly expressed TFs in different cells and tissues23. These large-scale experiments showed that TF could bind to tens of thousands of sites and each gene is targeted by multiple TFs. Although TF binding is strongly associated with gene transcription, knockdown of specific TF could only impact a small number of its targets, meaning that the regulatory network is highly redundant.The technologies for studying TF have also evolved. First developed for mapping in vivo binding sites of human TFs genome-wide, ChIP-Seq represents a dramatic improvement compared to other analysis methods such as EMSA, ChIP-PCR, and ChIPchip24. Among those, 25 are NAC-class, 16 are MADS-class, and 7 are SBP-class TFs , there are 2026 tomato protein coding genes with putative DNA binding domain(s) that might be considered as transcription factors, and 516 of them are expressed (RPKM > 10) in the ripening tomato fruitFs Table . Over 405, we have counted the potential TF binding sites in each DHS regions using known plant TF motifs from JASPAR and TRANSFAC database  for nearly all tomato genes have been now defined by DNaseI-Seqase Fig. . On averase Fig. . We shouEvolution of ripening is a transcription reprogram in the carpel tissue5. Hence, the plants did not invent new genes to transform the carpel into a fleshy fruit. Instead, they just rewired their gene regulatory network to express existing genes in the carpel tissue. With the exception, however, that genome or gene duplication could create additional genetic materials for plants without the need for repurposing the old ones.Ripening fleshy fruit is a product of convergent evolution. It is difficult to fully understand ripening regulation if we do not look beyond the tomato model. We have compared the climacteric fruit ripening genes with their orthologues in dry and non-climacteric fruits. It turns out that all these transcription factors, hormone signaling genes, ripening genes, and epigenetic factors are already present in the non-climacteric and dry fruit speciesArabidopsis can produce a respiration burst during carpel senescence25. Most intriguingly, it is also ethylene dependent and requires a NAC transcription factor AtNAP1, which is a homolog of the tomato NOR, as well as other species’ NAC TFs involved in the ripening positive feedback loops.We propose that perhaps the first step of evolving a climacteric fruit is to re-direct the ethylene signaling pathway to regulate “ripening genes” in the carpel tissue, which should be a logical choice as ethylene is well known to be synthesized during carpel senescence. It also has the advantage of being a gas signal molecule that could diffuse from cell to cell without the need of a transporter. In addition, there has been evidence suggesting that even the dry fruited cis-regulatory elements in the promoter open chromatin regions of hundreds or thousands of genes. Therefore, these ancestral plants faced a difficult choice regarding the TF and its regulatory network topology. The shortest route might be to directly couple the “ripening genes” to the ethylene signaling pathway. There are conserved TFs controlled by ethylene, such as EIN3 and ERF class TFs. However, if the plants want to restrict ripening to matured fruit tissues, genes directly regulated by these ethylene TFs must be repressed in other tissues, where ethylene signal could be accidentally switch on by biotic or abiotic stresses. Evolving new epigenetic regulations such as methylation or H3K27me3 to repress thousands of ripening genes could be costly. The fruitENCODE project showed that all 7 climacteric fruits took a longer route, which is to create a “new” TF controlled by ethylene, and then couple the ripening genes to it. By taking this longer route, plants only need to control a single or a few TF genes to achieve a ripening fruit-specific gene expression program.After committing the hormone ethylene to regulate ripening, plants still have to assign TFs to control the ripening genes. Although it sounds easy, it actually requires gaining The three ethylene positive feedback circuits discovered so far are perhaps the most optimal designs that have survived the process of natural selection Fig. . However27.Next generation sequencing technologies have enabled genome-wide analysis of biological processes at multiple dimensions. While addressing the same underlying questions, these big data has different properties that requires a complete overhaul of the data analysis and interpretation methods. For example, DNA methylation is often considered as a repressive epigenetic mark. This concept originated from observation that high DNA methylation is associated with inactive transcription. The transposable elements in the genome are heavily methylated, while global demethylation in endosperm tissues are associated with their transcriptional reactivation29. This suggests that DNA methylation is involved in ripening control. However, we still do not know which gene(s) is directly controlled by DNA methylation. There are over ten thousand DNA demethylation sites (hypo-DMRs) identified in the ripen vs. immature tomato fruit pairwise comparison4. The DMRs are enriched at the 5′ end of genes, and many of them are associated with transcription factor RIN binding sites. This result is robust to the choice of the distance used to define the putative promoter region and TF binding regions, as well as different statistical models and thresholds used to define DMR. Hence, it is tempting to suggest that DNA methylation in the promoter region could affect TF binding leading to repression of ripening genes.Silencing the tomato DNA demethylase using RNAi can prevent fruit ripening, and it has been later confirmed by CRISPR/Cas9 mutationACS2 and CNR are already expressed in the developing tomato fruit before their promoter regions are demethylated. In addition, there are many down-regulated genes associated with promoter demethylation during tomato fruit ripening29. Again, we could conveniently expand the function of DNA methylation and suggests that it can activate transcription. This is indeed supported by recent experimental evidence30. However, it is meaningless to discuss DNA methylation change on its own as it affects thousands of genes either directly or indirectly, and can both activate and repress transcription.However, it is often overlooked that there are many genes whose expression profiles are not associated well with DNA methylation changes. For example, some key ripening genes such as In addition, if we knockout key tomato DNA methylase genes, which will surely affect the expression of thousands of genes and perhaps also disrupt many biological processes such as ripening, should we start to claim that DNA hypermethylation, not just demethylation, also regulate fruit ripening? The same logical fallacy could be applied to any “global” factors, such as protein post-translational modification and small RNA biogenesis, or even transcription itself, as we can easily disrupt any complex developmental processes by altering these core machineries. But what is the significance of claiming that these core machinery is necessary for ripening? The key question is not whether global regulators such as DNA methylation are involved in ripening. Instead, we should ask which gene(s) necessary for ripening is directly regulated by DNA methylation.31. They found a strong anti-correlation between DNA methylation and chromatin accessibility. Hence, it was proposed that the majority of the DNA methylation changes are passive and only a small number of them could be actively controlling TF binding. DNA methylation is likely to be automatically deposited onto DNA when a TF vacates the binding sites and the promoter region become inaccessible.This might be new to the horticulture field, but it has been well recognized in the mammalian research. The human ENCODE project has systematically profiled gene expression, DNA methylation, and chromatin accessibility in multiple cell lines and tissues5. These observations suggest that the majority of the promoter DNA methylation changes in fruits are passive marks of promoter chromatin remodeling, just like the mammalian ones. Hence, it is futile and often misleading to discuss the function of DNA methylation when it is taken out of the context of the chromatin environment.In the fruitENCODE project, we did not observe conserved DNA methylation changes targeting the ripening genes in different plant species, neither did other fruits experience a tomato-like whole-genome demethylation. In fact, all of them have local DNA methylation changes, and we found the same inverse-correlation between their methylation change and chromatin accessibility change5. We propose that evolving this strategy enable plants to control most if not all ripening genes through a few key TFs, which is efficient and could be of significant evolutionary advantage.Fleshy fruit is a classic example of convergent evolution and as they have developed independently multiple times in the history of the angiosperm, this already suggests that they could have evolved different mechanisms to achieve this. However, some of them, mainly those classified as climacteric fruits, utilize the same plant hormone ethylene to regulate ripening. Their ripening is associated with a burst of respiration, and most importantly, autocatalytic ethylene synthesis, which is necessary for ripening to initiate and progress. In all seven cases  that have been examined in the fruitENCODE project, the autocatalytic ethylene is produced by positive feedback loops formed between ethylene and the so-called “ripening TFs” Fig. . Many ge5. However, further investigations in more species are necessary in order to show whether other regulatory systems do exist.Interestingly, the lack of more diverse ripening circuits appears to suggest that the evolution of fruit ripening was constrained by some unknown factors, which could be the limited optimal signaling molecules and genetic and epigenetic materials in the carpel tissue. What we have observed might be the most optimized path(s) for plants to evolve a ripening fruit, which is to repurpose an existing carpel senescence gene, or use the duplicated floral organ identify genesAnother important lesson from the fruitENCODE project and these CRISPR/Cas9 mutants is that ripening is controlled by a complex and highly redundant transcriptional regulatory network. Many biological processes are robust to change, and such redundant system can tolerate multiple perturbations. The evolutionary benefit of a robust system is the increase of neutral mutations, meaning the population could carry more genetic variations that might be useful in the future. Analysis of such a complex system at a single level could easily lead to an incomplete picture such as the traditional genetic approaches or examining a single epigenetic factor while ignoring others. Therefore, if we want to fully understand a complex plant developmental process such as fruit ripening, we might need to adopt a more holistic approach considering regulatory inputs from multiple dimensions such as 3D chromatin organization, DNA methylation, chromatin accessibility, histone modification, TF network and transcriptome. How to combine the different advantages of traditional genetic experiment and the big data driven approaches is a new challenge for plant biologists.Solanum lycopersicum cv. Ailsa Craig), nor and Cnr mutants, and transgenic lines were grown in a greenhouse under controlled condition with natural light. Fruits samples of WT, nor and Cnr mutants, and transgenic lines were harvested at the red-ripe stage. Pericarp tissues were collected, immediately frozen in liquid nitrogen and stored at −80 °C for RNA isolation.Wild-type (WT) tomato plants (http://cbi.hzau.edu.cn/crispr/) was used to select four specific sgRNAs that targeted the tomato NOR or CNR, respectively . Three biological replicates were used for TrueSeq library preparation. Illumina sequencing was performed on HiSeq2500 using the PE150 mode. The raw data were mapped to the tomato reference genome v3 using tophat, and differentially expressed genes were called using DEGseq2.Total RNA was extracted from the pericarp tissues of WT, Dataset 1


Cricetulus griseous (Chinese hamster), associated to the Cytomegalovirus (CMV) promoter, to increase recombinant antibodies production in Chinese Hamster Ovary (CHO) cells.The production of recombinant proteins in mammalian cell lines is one of the most important areas in biopharmaceutical industry. Viral transcriptional promoters are widely used to express recombinant proteins in mammalian cell lines. However, these promoters are susceptible to silencing, thus limiting protein productivity. Some CpG islands can avoid the silencing of housekeeping genes; for that reason, they have been used to increase the production of recombinant genes in cells of animal origin. In this study, we evaluated the CpG island of the promoter region of the β-actin gene of dihydrofolate reductase gene.We focused on the non-coding region of CpG island, which we called RegCG. RegCG behaved as a promoter, whose transcriptional activity was mainly commanded by the CAAT and CArG boxes of the proximal promoter. However, the transcription started mainly at the intronic region before the proximal transcription start site. While the CMV promoter was initially more powerful than RegCG, the latter promoter was more resistant to silencing than the CMV promoter in stable cell lines, and its activity was improved when combined with the CMV promoter. Thereby, the chimeric promoter was able to maintain the expression of recombinant antibodies in stable clones for 40 days at an average level 4 times higher than the CMV promoter. Finally, the chimeric promoter showed compatibility with a genetic amplification system by induction with methotrexate in cells deficient in the We have generated an efficient synthetic hybrid transcription promoter through the combination of RegCG with CMV, which, in stable cell lines, shows greater activity than when both promoters are used separately. Our chimeric promoter is compatible with a genetic amplification system in CHO DG44 cells and makes possible the generation of stable cell lines with high production of recombinant antibodies. We propose that this promoter can be a good alternative for the generation of clones expressing high amount of recombinant proteins, essential for industrial applications.The online version of this article (10.1186/s13036-019-0187-y) contains supplementary material, which is available to authorized users. The production of recombinant proteins is one of the most important areas in biopharmaceutical industry. Among these proteins, recombinant monoclonal antibodies are of great interest because of their diversity and high specificity ; moreoveChinese Hamster Ovary (CHO) cells have been used as the main platform for the industrial production of antibodies and other complex biopharmaceuticals, as they generate recombinant proteins with correct assembly and glycosylation patterns compatible with their clinical use. Viral transcriptional promoters, such as Cytomegalovirus (CMV) and simian virus 40 (SV40), among others, are strong promoters that are widely used to express recombinant proteins in mammalian cell lines . Howeverβ-actin (ACTB) gene is a well-studied HK gene. It has a promoter with a CpG island that extends the proximal transcription start site (TSS), the intron I, and part of the exon II [ACTB gene promoters from human [The CpG islands, which correspond to regions of DNA rich in CpG dinucleotides, provide the maintenance of extensively demethylated regions of the genome. The demethylated state of these islands allows the nucleosome to acquire a less compact configuration, which makes it more accessible to the transcriptional machinery –12. More exon II . Such chom human , chickenom human , shrimp om human , fish [1om human , 20, andom human  origin fACTB gene promoter of Cricetulus griseus (Chinese hamster) to increase the expression of recombinant antibodies in CHO cells. We studied the promoter characteristics of the ACTB gene upon incorporation of a CpG island (RegCG). In addition, we generated chimeric promoters that combined RegCG with the CMV promoter and other regulation elements, such as glucocorticoid response elements (GRE) and their promoter activities were compared with those of CMV promoter alone. Finally, we tested the ability of one chimeric promoter to produce antibodies in CHO cell lines.The general aim of this work was to evaluate the use of the CpG island of the E. coli DH5α cells using the Wizard® Plus SV Minipreps DNA Purification System Kit , and then cut with restriction enzymes from Invitrogen (USA) or New England Biolabs (USA). The DNA fragments were purified with the Wizard® SV Gel and PCR Clean-Up System Kit  from agarose gels between 1 to 1.2% (w/v) in TBE buffer. Genomic DNA obtained from CHO-K1 (ATCC® CCL-61™) cells was purified using the PureLink™ Genomic DNA Mini Kit . The PCR reactions for DNA fragments analysis were carried out with the GoTaq® Green Master Mix Kit . The PCR reactions to generate cloning fragments were performed with KAPA HiFi HotStart high-fidelity DNA polymerase .The plasmids were purified from transformed CHO-K1 cells were cultured in adherent conditions in the chemically defined medium DMEM/F12 , supplemented with 5% (v/v) of fetal bovine serum (FBS)  and 100 mM of L-glutamine . Suspension-adapted, dihydrofolate reductase (DHFR)-deficient CHO DG44 cells were obtained from ThermoFisher Scientific (USA) and initially cultured in a chemically defined CD-DG44 medium  with a mixture of sodium hypoxanthine and thymidine (HT).TNF-L and dhfr genes under the control of the same promoter (pRegCG/GRE/CMV-TNF-L)  Fig. a.Fig. 1R2. When reaching 80 to 90% of confluence, the cells were transfected; for this purpose, the culture medium was removed and a mixture containing 500 ng of expression vector was added with 20 μL of Lipofectamine 2000 CD  in 100 μL of OptiMEM medium , and cells were incubated for 4 h at 37 °C and 5% CO2. Next, the transfection mixture was removed, cells were fed with 500 μL of DMEM-F12 medium supplemented with 10% FBS and then cells were incubated for 48 h at 37 °C and 5% CO2.For transient assays to generate stable cells lines, the following transfection protocol was carried out: the parental CHO-K1 cells were incubated at 300,000 cells/mL in 500 μL of DMEM-F12 medium  supplemented with 10% FBS and 100 mM L-glutamine at 37 °C and 5% (v/v) COTo measure the transient activity of promoters, CHO-K1 cells were co-transfected with the transcription reporter vector derived from the pGL4.17 vector , and the normalizing pGL4.73 vector , following the protocol described above. The transfected cells were collected and lysed after 48 h of culture, according to the instructions of the “Dual-Glo® Luciferase Assay System” , and luminescence was measured in a Luminoskan ASCENT luminometer (Thermo Electronic Corporation). The promoter activity was calculated by the quotient between the luciferase activities of firefly and renilla (FIR/REN) and expressed as relative light units (RLU).2, in the presence of the selection antibiotic G418. Reporter activity from the cell lysate was determined using the Bright-Glo™ luciferase assay system , and normalized by the total protein concentration determined by the Bradford assay. Reporter activity was expressed as RLU, equivalent to the quotient between the luciferase activity of firefly and the total protein concentration (FIR/Prot).To measure the activity of promoters in stable cell lines, CHO-K1 cells were transfected with pre-linearized expression reporter vectors followed by clone isolation through limiting dilution in the presence of the selection antibiotic G418 ; dilutions were performed in 96-well culture plates with DMEM-F12 medium supplemented with 10% FBS, and 100 mM L-glutamine at 37 °C and 5% COCHO-K1 cells were transfected with a pre-linearized bicistronic expression vector (pRegCG/GRE/CMV-aCD20LH) coding for the heavy and light chains of the anti-human CD20 antibody in one vector, according to the protocol described above.6 cells/mL, and ≥ 95% viability. All cell cultures were incubated in a Mco-17Aic CO2 Incubator  at a 5% CO2 enriched atmosphere.The parental CHO DG44 cells were co-transfected with the linearized plasmids pRegCG/GRE/CMV-aTNF-H and pRegCG/GRE/CMV-aTNF-L, which code for the heavy and light chains of the chimeric anti-human TNF antibody, respectively, as described in Torres et al., 2018 . The celThe production levels of anti-human CD20 and anti-human TNF antibodies in CHO-K1 and CHO DG44 cells were determined by measuring the concentration of the antibodies in the culture supernatants, which was quantified by an antigen capture ELISA prepared in our laboratory, and described in .RNA was extracted from cells using E.Z.N.A.® Total RNA Kit I , according to the manufacturer’s instructions. RNA concentration was quantified using a Synergy 2 Spectrophotometer . RNA extracts (2 μg) were treated with DNase I  to remove any trace of genomic DNA contamination. Reverse transcriptase production of cDNA from the RNA was performed using an Affinity Script enzyme  and oligo dT. The amplification of the specific cDNA was carried out by PCR with the GoTaq® Green Master Mix Kit , and the amplification products were analyzed by electrophoresis on 1% (w/v) agarose gels.ACTB gene of Cricetulus griceus, we analyzed the ACTB gene promoter sequence obtained from a contig of the genome of Cricetulus griseus using nBLAST of National Center for Biotechnology Information (NCBI). The sequence of this contig was compared with its orthologous human, mouse, and rat genomic DNA sequences, using Clustal Omega , to identify the promoter and coding regions  into the transcription reporter vector pGL4.17  through transient expression assays performed on the CHO-K1 cell line after 48 h of culture. We observed that the RegCG promoter possessed approximately 40% activity from the CMV promoter and that its activity was 4-fold greater than that of the CMV Core promoter . This mutant showed a 5-fold decrease in activity compared to the RegCG promoter . Although promoters with the disrupted CMV promoter had less transcriptional activity than RegCG in transient assays Fig. d, surpriACTB gene with an expected amplification size of 711 bp from genomic DNA and 256 bp from mRNA. The intensity of amplification from recombinant mRNA was comparable to amplification from plasmid DNA vector  as the selection systems, and clones were treated with methotrexate. At the end of the selection process, 6 clones were obtained that showed a productivity equal to or greater than 2 pg/cell/day  la methylation free CgG island upstream the CMV promoter decreased silencing of the recombinant gene with the consequent increase in the production of recombinant proteins . This efACTB gene of embryonic expression, whose transcription begins in the intron I, which is commanded by its own CAAT, CArG and TATA elements that are present inside the intron I [ACTB gene also contains a CArG box [Feng et al. reported an alternative isoform of an amphiouxus intron I . In mammCArG box , which mACTB gene may facilitate a nucleosomal conformation that makes it more accessible to the transcription machinery [Recent evidence shows that there is coordination between the transcription machinery, the splicing machinery and the state of chromatin , 32. It achinery . Interesachinery . TherefoThe UCOE has been widely studied for their capacity to improve CMV promoter activity in stably transfected CHO cells , 35. TheACTB gene from Cricetulus griseus (RegCG) with the CMV promoter. RegCG behaves as a promoter that sustains the expression of recombinant proteins in stable cell lines, which is more resistant to gene silencing than the CMV promoter. The generation of promoters by the combination of RegCG with CMV results in promoters with greater activity in stable cell lines as compared to both promoters separately. We showed that these chimeric promoters are compatible with the genetic amplification system in CHO DG44 cells and allow the generation of stable cell lines with high production of recombinant antibodies. We propose that this promoter can be used for the generation of industrial cell lines for the production of recombinant antibodies.We have generated an efficient transcription promoter by combining a CpG island fragment of the Additional file 1:Table S1. List of primers. (DOCX 18 kb)Additional file 2:Figure S1. Pairing of the primers for the synthesis of a glucocorticoid response element (GRE) tandem. The alignment of the hybridized primers is shown, prior to their ligation with T4 Ligase through the cohesive ends generated. a) Synthesis of short portion of CMV promoter (Core). b) Synthesis of a tandem of five Glucocorticoid Response Element (GRE). In celestial the palindromic elements of the GRE site are shown, and in yellow the cohesive ends compatible with restriction enzymes are shown. (DOCX 13 kb)Additional file 3:Figure S2. Alignment of promoter sequences of the gene for the beta actin of Gallus , Human (Hum), Chinese Hamster (CHO), mouse (Mus) and rat (Rat). In Amarillo, the CAAT, TATA and CArG boxes stand out. In green, the XhoI restriction site and the start codon of beta-actin are highlighted. The arrows> and < indicate the start and end, respectively of the CpG Island, the start of transcription and the intron I for the sequence of the CHO cell genome. (DOCX 16 kb)


Long non-coding (lnc) RNAs are non-coding RNAs longer than 200 nt. lncRNAs primarily interact with mRNA, DNA, protein, and miRNA and consequently regulate gene expression at the epigenetic, transcriptional, post-transcriptional, translational, and post-translational levels in a variety of ways. They play important roles in biological processes such as chromatin remodeling, transcriptional activation, transcriptional interference, RNA processing, and mRNA translation. lncRNAs have important functions in plant growth and development; biotic and abiotic stress responses; and in regulation of cell differentiation, the cell cycle, and the occurrence of many diseases in humans and animals. In this review, we summarize the functions and mechanisms of lncRNAs in plants, humans, and animals at different regulatory levels. Long non-coding (lnc) RNA transcripts are longer than 200 nt in length and have no protein-coding ability; however, they may have short open reading frames (ORFs). Most annotated lncRNAs are RNA polymerase II (Pol II) transcribed; hence, they are similar in structure to mRNA and may have cap structures and poly A tails. In addition, the plant-specific RNA polymerases, Pol IV and V, can produce lncRNAs ,2. LncRNAccording to the current understanding of lncRNA identification and function, these transcripts are broadly classified into those mediating chromatin modifications and DNA methylation involved in epigenetic regulation, proteins (primarily transcription factor) and DNA interactions involved in transcriptional level regulation, and in mRNA processing at the post-transcriptional level, as well as direct interactions with proteins to regulate protein translation and post-translation modification . In thisEpigenetics refers to heritable alterations in gene expression without alterations in DNA sequence. The molecular mechanisms involved primarily include DNA methylation, histone modification, chromatin remodeling, and, in a broad sense, non-coding RNA. Histone modifications are a major class of chromatin modifications responsible for the epigenetic regulation of gene expression. Histones undergo various modification processes such as methylation, acetylation, phosphorylation, and ubiquitination under the action of related enzymes, and different modifications are associated with activation or inhibition of gene expression. Histone methylation and acetylation are the most important histone modifications, and they occur primarily at histone H3 lysine residues. In response to a variety of diseases and plant flowering, histone modification can induce changes in chromatin structure, thus affecting transcriptional regulation ,21,22. lFLC), encoding an important flowering inhibitor protein, thus regulating the vernalization in Arabidopsis. The lncRNA transcribed from FLC intron 1, consistent with the direction of FLC transcription, has been named COLD ASSISTED INTRONIC NONCODING RNA (COLDAIR) ,29FLC),  of MAF4 . MAS is omplexes . Chromatpression B 30]..FLC), enFEZF1-AS1 has been identified in gastric cancer. High expression of FEZF1-AS1 increases the sizes of gastric cancer tumors in patients and also promotes the proliferation of gastric cancer cells by inducing cell cycle progression and by decreasing apoptosis. The lincRNA FEZF1-AS1 inhibits P21 expression in the cell cycle. RNA pull-down and RIP experiments have confirmed that the lincRNA FEZF1-AS1 specifically binds lysine-specific demethylase 1(LSD1), thereby regulating the expression of P21 at the transcriptional level. ChIP assays have confirmed that LSD1 directly binds the promoter of P21, thereby mediating H3K4me2 demethylation modification; knocking out FEZF1-AS1 affects the binding of LSD1 to the P21 promoter and H3K4me2 modification. The transcription factor SP1 upregulates FEZF1-AS1 expression, and ChIP assays have indicated that SP1 binds the promoter of FEZF1-AS1. The lincRNA FEZF1-AS1 recruits LSD1 to the promoter region of P21, thereby inducing demethylation of H3K4me2 and inhibiting the transcription of P21  is transcribed from the promoter region of ST3Gal6 in the opposite direction from transcription. The lncRNAs ST3Gal6-AS1 and ST3Gal6 are down-regulated in colorectal cancer (CRC), and their expressions levels are positively correlated—lncRNA ST3Gal6-AS1 activates the expression of ST3Gal6 in CRC cells. ChIP has been used to detect the H3K4me3 signal in the promoter region of ST3Gal6, and knockout of ST3Gal6-AS1 affects the accumulation of H3K4me3. Bioinformatics analysis and RIP experiments have indicated that lncRNA ST3Gal6-AS1 binds histone methyltransferase MLL1, recruits it to the promoter region of ST3Gal6, and induces H3K4me3 modification, which in turn activates ST3Gal6 transcription C 31]. F. F31]. Fn of P21 D 32]..FEZF1-ASHistone acetylation is a reversible dynamic equilibrium process catalyzed by histone acetyltransferase (HAT) and histone deacetylase (HDAC). Histone acetylation refers to the activation of gene transcription by HAT through acetylation of histone lysine residues, whereas HDAC catalyzes histone deacetylation and inhibits gene transcription. The acetylation sites of histones are primarily H3K4Ac, H3K9Ac, H3K27Ac, H3K56Ac, and H4K16Ac. The mechanism through which lncRNAs regulate histone acetylation modification is similar to the mechanism of regulation of histone methylation modification mentioned above. lncRNAs regulate histone acetyltransferases or deacetylases , males absent on the first (MOF), K acetyltransferases 2A (KAT2A), and sirtuin 6 (SIRT6) ) and thulncPRESS. lncPRESS1 is one of 40 multi-exonic intergenic lncRNAs, and is 756 nt in length. By using gene silencing technology to interfere with p53, lncPRESS1 has been found to regulate the transcription of p53, which keeps the pluripotency of hESC consistent with the core pluripotency factor. RNA-seq techniques have been used to sequence hESCs lacking lncPRESS1, and lncPRESS1 has been found to control the gene network promoting pluripotency. RNA pull-down assays in vitro have indicated that lncPRESS1 binds SIRT6, an H3K56ac/K9ac histone deacetylase ..lncRNA ADNA methylation in genomes is catalyzed by DNA methyltransferases and is regarded as a key player in epigenetic silencing of transcription. The members of the DNA methyltransferase (DNMT) family are conventionally classified as de novo (DNMT3a-b) and maintenance (DNMT1) methyltransferases. lncRNAs can recruit DNA methyltransferases to regulate target gene transcription C. lncRNAAdditionally, researchers have found that a class of lncRNAs recruit multiple DNMTs  to target genes, thereby leading to methylation of target genes and regulating their expression. LINC00313 is highly expressed in thyroid cancer and negatively regulates aristaless-like homeobox 4 (ALX4) expression. Dual-luciferase reporter gene assays have indicated that LINC00313 binds the ALX4 promoter, which has many CpG islands and is easily methylated. RIP and ChIP assays have revealed that LINC00313 binds DNMT1 and DNMT3B, which in turn bind the ALX4 promoter. These results indicate that LINC00313 recruit DNMT1 and DNMT3b and consequently facilitate the methylation of the ALX4 promoter and inhibit ALX4 expression, thereby regulating cell proliferation, migration, and invasion in thyroid cancer C 45]..45].lncRNAs also directly participate in the regulation of transcription itself, a process that primarily occurs in the nucleus. lncRNAs participate in regulation of transcription in many ways. lncRNAs directly bind DNA sequences and inhibit gene transcription; in addition, they directly interact with proteins (primarily transcription factors) and inhibit or activate the expression of downstream genes E,F. HereEvf2 is transcribed from an ultraconserved distal enhancer, which specifically binds the transcription factor DLX2, forming a transcriptional complex, and binds an enhancer, thereby inducing expression of the adjacent protein-coding gene DLX5/6 ..PXN mRNAHnRNP E1 is an RNA binding protein and a known alternative splicing (AS) inhibitor . AffymetmiRNAs are a class of non-coding short-sequence RNAs that are 18 to 25 nt in length, do not have an ORF, and are widely expressed in eukaryotes. MiRNAs play important roles in animals and plants by targeting mRNA for cleavage or directing translational inhibition to negatively regulate mRNA expression . miRNAs IPS1, which belongs to the TPSI1/Mt4 family, was first identified in 2007 as having a ceRNA regulatory mechanism. IPS1 accumulates in leaves and roots under high phosphate starvation. The 23 nt conserved region of the IPS1 sequence is complementary to the partial sequence of miR399. IPS1 is not cleaved by miR399, as a bulge is formed by a mismatch at the 10–11 base position of the miRNA; consequently, IPS1 can mimic miR399 target genes and bind miR399 without being degraded, thereby inhibiting the activity of miR399, increasing the expression of the target gene PHO2, and maintaining the normal growth of Arabidopsis under phosphate starvation conditions ..Cyp7a1 aThe stability of mRNA is a balance between the synthesis and degradation of the required RNA in the processes of gene transcription and translation. The regulation of mRNA stability occurs primarily through the degradation of various recognition proteins and nucleases. In vivo, the stability of mRNA is associated with the existence of specific binding proteins. Recent studies have reported that lncRNAs can also participate in regulating the stability of mRNA post-transcriptionally L. The lnSirt1 antisense transcript (Sirt1 AS) transcribed from the Sirt1 antisense strand in myogenesis. Sirt1 AS lncRNA overexpression promotes myoblast proliferation but inhibits differentiation. miR-34a targets Sirt1 mRNA, and the expression of miR-34a is negatively correlated with the expression of Sirt1 and Sirt1 AS. The lncRNA Sirt1 AS alleviates the inhibition of Sirt1 expression and rescues the expression of proliferating genes by inhibiting the expression of miRNA-34a. Srit1 AS lncRNA interacts with Sirt1 mRNA, forming an RNA duplex, thus improving the stability of Sirt1 through competition with miRNA-34a in the 3’-UTR region of the Sirt1 gene and thereby promoting the proliferation and inhibiting the differentiation of C2C12 cells  encoded by the lncRNA LINC00961, which is highly expressed in skeletal muscle in both humans and mice. Knockout of LINC00961 in HeLa cells results in increased activation of mechanistic target of rapamycin complex 1 (mTORC1) under amino acid starvation and stimulation. The polypeptide, named ‘small regulatory polypeptide of amino acid response’ (SPAR) is conserved in both humans and mice, and localizes to the late endosome/lysosome. Further experiments have revealed that SPAR interacts with subunits of the v-ATPase proton pump, but it has no effect on v-ATPase proton pump activity. The v-ATPase complex interacts with the Ragulator complex at the lysosome, thereby regulating the activation of mechanistic target of rapamycin 1 (mTORC1) under amino acid stimulation . SPAR afGmENOD40 is an lncRNA that is specifically expressed in legumes, is associated with symbiotic nitrogen-fixation nodule formation, and can encode short peptides. This lncRNA has a complex expression pattern during the development of soybean nodules, and studies have shown that ENOD40 peptide may have a transport function and participate in the control of sucrose use in nitrogen-fixing nodules ..114].Post-translational modification of proteins is an important part of proteomics. Post-translational modification of proteins changes their spatial conformation, activity, stability and interaction with other molecules, and consequently participates in regulating diverse life activities in the body. Post-translational modification of proteins is a complex process. Currently, more than 20 modification types are known in eukaryotes, including phosphorylation, acetylation, ubiquitination, glycosylation, and SUMOylation . lncRNAsPhosphorylation, the most extensive covalent modification of protein after translation, plays an important role in cell signal transduction. Protein phosphorylation occurs mainly on serine and tyrosine residues, and is regulated by some protein kinases. lncRNAs have been found to regulate protein phosphorylation. lncRNA lnc-DC is solely expressed in human conventional dendritic cells (DCs) and mediates DC differentiation. Histone H3K4me3 and H3K27ac modifications and the transcription factor PU.1 directly activate lnc-DC transcription. RNA fluorescence in situ hybridization (RNA FISH) has indicated that lnc-DC is localized in the cytoplasm. RNA pull-down and RIP assays have confirmed that lnc-DC interacts with signal transducer and activator of transcription 3 (STAT3) protein; RNA FISH has further verified that lnc-DC colocalizes with STAT3 in the cytoplasm. lnc-DC enhances STAT3 phosphorylation on Tyr-705 and participates in its post-translational modification. SHP1 is a protein tyrosine phosphatase that promotes dephosphorylation of tyrosine. Knockout of lnc-DC promotes the binding of SHP1 and STAT3, thereby enhancing the dephosphorylation of STAT3, whereas overexpression of lnc-DC attenuates their binding, preventing dephosphorylation of STAT3 Tyr705, and activating downstream transcriptional activity C 116]. . 116]. AlncRNA HULC, the first lncRNA identified in liver cancer, enhances the cell malignant phenotype and cell proliferation by promoting G1/S transformation. RNA pull-down, RIP, deletion-mapping, and colocalization analyses have indicated that HULC specifically interacts with YB-1 (Y-box protein-1). Extracellular signal-regulated kinase (ERK) protein kinase has been shown to phosphorylate YB-1, and RNA pull-down experiments have shown that HULC interacts with ERK. Elevated expression of HULC promotes phosphorylation of YB-1 by activating the ERK pathway, thereby reducing the interaction of certain oncogenic mRNAs with YB-1, such as cyclin D1, cyclin E1, and MMP11, and accelerating the translation of these mRNAs during tumorigenesis and promoting cell proliferation D 118]..118].Protein ubiquitination is a common form of post-translational modification, which regulates various protein substrates in different cellular pathways. Ubiquitin labels proteins to be degraded in proteasomes, a process requiring three enzymes: ubiquitin-activating enzyme (E1), ubiquitin-binding enzyme (E2), and ubiquitin protein ligase (E3). lncRNAs have a variety of functions, such as regulating protein ubiquitination and affecting protein activity. lncRNA HOTAIR (HOX antisense intergenic RNA) is involved in various regulatory pathways, including regulating the ubiquitination of proteins. HuR interacts with HOTAIR, thereby promoting its degradation by recruiting the let7–Ago2 complex. RIP experiments have indicated that HOTAIR specifically binds to two E3 ubiquitin ligases Mex3b and Dzip3 and their respective substrates Snurportin-1 and Ataxin-1. HOTAIR is mainly used as a scaffold molecule that, by bringing together ubiquitin ligases with their substrates, increases substrate degradation. Accordingly, overexpression of HOTAIR promotes ubiquitination and protein degradation of Snurportin-1 and Ataxin-1 E 119]..119].lncRNA uc.134 is an ultraconserved lncRNA, and its expression is significantly decreased in highly aggressive HCC cells. Gain-of-function and loss-of-function studies in vitro and in vivo have indicated that the lncRNA uc.134 inhibits HCC cell proliferation, invasion, and metastasis. RNA pull-down, RIP, and western blot (WB) assays have confirmed that lncRNA uc.134 directly interacts with the E3 ubiquitin ligase CUL4A. lncRNA uc.134 affects the localization of CUL4A, and overexpression of uc.134 promotes the accumulation of CUL4A protein in the nucleus, whereas knockout of uc.134 increases CUL4A protein levels in the cytoplasm. LATS1 protein contains an N-terminal ubiquitin-binding domain, and CUL4A activates ubiquitination and degradation of LATS1. Ubiquitination of LATS1 blocks its kinase activity in phosphorylating YAP, primarily at Ser127 . By direWe have previously mentioned the regulation of histone acetylation by lncRNA. In addition to histone acetylation, non-histone acetylation occurs; dynamic changes in protein acetylation and deacetylation are regulated by acetyltransferase and deacetylase. Non-histone acetylation also plays an important role in life processes . SimilarlncRNAs account for 80% of ncRNA, have no protein-coding function, and play roles as RNA molecules. lncRNAs participate in regulation at the epigenetic, transcriptional, post-transcriptional, translational, and post-translational levels in the form of RNA. In addition, recent studies have found that lncRNAs can encode polypeptides and regulate gene expression in the form of polypeptides. Studies have demonstrated multiple mechanisms of lncRNAs in animals, such as lncRNA-encoded peptides and interaction with DNA or RNA methylases and demethylases; however, the mechanism of action of lncRNAs to regulate RNA methylation of mRNA and protein expression have not been reported in plants. Although plant lncRNAs involved in these functions have not been reported yet, we have reason to believe that many of the unknown functions of lncRNAs will be realized through these modes. These mechanisms of lncRNA action in plants can provide directions for future research, and more functions of plant lncRNAs can be determined.lncRNAs do not function in a single manner or function alone; instead, they interact with other genes and proteins, and their functions are complex. On the basis of the summary of the above mechanisms, lncRNAs primarily interact with other molecules and directly or indirectly regulate gene transcription and translation, not only through simple single regulatory pathways but also through participating at multiple regulatory levels. The field of lncRNAs research is also expanding rapidly thanks to developing technologies such as CRISPR/Cas9, RNA pull-down, RIP, ChIP, and ChIRP that have helped in understanding the functions and mechanisms of action of lncRNAs. Recently, these experimental techniques have been applied in plants, thus greatly aiding in the study of the functions and mechanisms of lncRNAs, as well as in promoting the rapid development of plant lncRNA research. By combining these technologies with other future technologies, more attractive and unique features and functions of lncRNAs will certainly be found. Obviously, there is no universal function of lncRNAs, and only through specific case studies can meaningful understanding be obtained. When we study lncRNAs, we cannot classify them as independent actors, as this would limit our thinking and understanding of their functions and mechanisms.


Endothelial cells (ECs) make up the innermost layer throughout the entire vasculature. Their phenotypes and physiological functions are initially regulated by developmental signals and extracellular stimuli. The underlying molecular mechanisms responsible for the diverse phenotypes of ECs from different organs are not well understood.To characterize the transcriptomic and epigenomic landscape in the vascular system, we cataloged gene expression and active histone marks in nine types of human ECs (generating 148 genome-wide datasets) and carried out a comprehensive analysis with chromatin interaction data. We developed a robust procedure for comparative epigenome analysis that circumvents variations at the level of the individual and technical noise derived from sample preparation under various conditions. Through this approach, we identified 3765 EC-specific enhancers, some of which were associated with disease-associated genetic variations. We also identified various candidate marker genes for each EC type. We found that the nine EC types can be divided into two subgroups, corresponding to those with upper-body origins and lower-body origins, based on their epigenomic landscape. Epigenomic variations were highly correlated with gene expression patterns, but also provided unique information. Most of the deferentially expressed genes and enhancers were cooperatively enriched in more than one EC type, suggesting that the distinct combinations of multiple genes play key roles in the diverse phenotypes across EC types. Notably, many homeobox genes were differentially expressed across EC types, and their expression was correlated with the relative position of each organ in the body. This reflects the developmental origins of ECs and their roles in angiogenesis, vasculogenesis and wound healing.This comprehensive analysis of epigenome characterization of EC types reveals diverse transcriptional regulation across human vascular systems. These datasets provide a valuable resource for understanding the vascular system and associated diseases. The vasculature pervades almost all body tissues. It consists of arteries, veins and interconnecting capillaries and has numerous essential roles in physiology and disease . EndotheIt is widely recognized that certain specific vessels are susceptible to pathological changes, which include those related to atherosclerosis and inflammation . AtherosGATA6, in human umbilical vein endothelial cells (HUVECs) and human dermal microvascular endothelial cells (HMVECs) [Several animal models have been developed to reveal the enhancer elements that function during the differentiation of various tissues, including ECs , 9. With(HMVECs) , 11. Deshttps://rnakato.github.io/HumanEndothelialEpigenome/).As a part of the International Human Epigenome Consortium (IHEC) project , we collTo establish an epigenetic catalog for different EC types, we generated a total of 491 genome-wide datasets, consisting of 424 histone modification ChIP-seq and 67 paired-end RNA sequencing (RNA-seq) datasets, encompassing a total of 22.3 billion sequenced reads. ECs were maintained as primary cultures with a physiological concentration of vascular endothelial growth factor (VEGF) and a minimal number of passages (fewer than six). We generated genome-wide normalized coverage tracks and peaks for ChIP-seq data and estimated normalized gene expression values for RNA-seq data.Human aortic endothelial cells (HAoECs),Human coronary artery endothelial cells (HCoAECs),Human endocardial cells (HENDCs),Human pulmonary artery endothelial cells (HPAECs),Human umbilical vein endothelial cells (HUVECs),Human umbilical artery endothelial cells (HUAECs),Human common carotid artery endothelial cells (HCCaECs),Human renal artery endothelial cells (HRAECs), andHuman great saphenous vein endothelial cells (HGSVECs).In this study, we selected a subset of 33 EC samples (131 datasets) as a representative set comprising nine types of vessels from the human body ” and “enhancer (H3K27ac only)” sites for each sample Fig. b, left. We compared the distribution of the reference sites with gene annotation information. As expected, active promoter sites were enriched in the transcription start sites (TSSs) of genes, whereas enhancer sites were more frequently dispersed in introns and intergenic regions  based on the H3K27ac read densities in the integrated EC enhancer sites with the 117 cell lines from the Roadmap Epigenomics Project . We founWe sought to identify the corresponding gene for the reference enhancer sites and used chromatin loop data obtained from the Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET) data using RNA Polymerase II (Pol II) in HUVECs. We identified 292 significant chromatin loops , 49.3% (144 loops) of which connected promoter and enhancer sites. Even when we used all chromatin loops (at least one read pair), 27.4%  of them linked to enhancer–promoter sites. Remarkably, 48.1% (4228 of 8782) of loops connected the distal enhancer sites. In total, we identified 2686 distal enhancer sites that are connected by chromatin loops. We also detected enhancer–enhancer  and promoter–promoter  loops, suggesting physically aggregated chromatin hubs in which multiple promoters and enhancers interact . As the KDR; Fig. KDR was marked as an active promoter (enriched for both H3K4me3 and H3K27ac) and physically interacted with the EC-specific enhancer sites indicated by H3K27ac, ~ 50 kbp upstream and downstream of the TSS. Another example is intercellular adhesion molecule 2 . As a result, we obtained 3765 EC-specific enhancer sites  from the GWAS catalog  and idenbcd, oc, Gsc and PITX1-3  assigned in the JASPAR database  classifications under Biological Process for the enhancer sites using GREAT  and founOne important issue of this study is to clarify the epigenomic/transcriptomic diversity across EC cell types. To circumvent variances at the level of the individual in each cell type observed Fig. c and difTo further investigate this tendency, we implemented a multiple-group differential analysis with respect to H3K4me3, H3K27ac and gene expression data to obtain sites and genes whose values varied significantly between any of the nine cell types. With the threshold FDR < 1e−5, we identified 753 differential H3K4me3 sites , 2979 differential H3K27ac sites  and 879 differentially expressed genes . As expected, DPs and DEs were more enriched around DEGs, as compared with all genes. DPs were enriched within ~ 10 kbp from TSSs, whereas DEs were more broadly distributed (~ 100 kbp)  to characterize the overall variability of DEGs, DEs Fig. b and DPs to charapes Fig. b, even tHAND2) and GATA-binding protein 4 (GATA4) were expressed in HAoECs, HENDCs and HPAECs (cluster 3). HAND2 physically interacts with GATA4 and the histone acetyltransferase p300 to form the enhanceosome complex, which regulates tissue-specific gene expression in the heart [HEY2, also called Hrt2), a positive marker for arterial EC specification [CDH4); the protein product of this gene mediates cell–cell adhesion, and mutation of this gene is significantly associated with chronic kidney disease in the Japanese population [CDH4 and HEY2 loci, H3K4me3 was also enriched in some EC types in which the genes were not expressed, whereas the H3K27ac enrichment pattern at TSSs was correlated with the expression level of these genes  were expressed in ECs, and their expression pattern varied across EC types  genes were differentially expressed , each of which contains 9–11 genes essential for determining the body axes during embryonic development, as well as regulating cell proliferation and migration in diverse organisms [HOX clusters A, B and D were highly expressed in all EC types, except HENDCs, possibly because HENDCs are derived from cardiac neural crest, whereas the other EC types are derived from mesoderm [HOXC genes were moderately expressed in HRAECs, HUVECs and HUAECs, but not in the upper-body ECs. More interestingly, perhaps, HOXD genes were not expressed in HPAECs, despite their similar expression pattern relative to other EC types around the heart , and thus human ECs that overexpress HOXD10 fail to form new blood vessels [HOXD10 was highly expressed in HGSVECs, which leads to the inhibition of the angiogenic phenotype as regulated by HOXD10 in this cell type.It has been suggested that the more 3′ henotype . For exas in ECs . In cont vessels . Figure HOX genes, multiple non-HOX homeobox genes were also differentially regulated across ECs. For example, cluster 3 in the DEGs contained NK2 homeobox 5 (Nkx-2.5), which is essential for maintenance of ventricular identity [PITX2) and paired related homeobox 1 (PRRX1), which are both associated with the atrial fibrillation and cardioembolic ischemic stroke variants loci [MEIS1), which is required for heart development in mice [PITX3 was mainly expressed in HENDCs (cluster 6), unlike PITX2. Another example is Mesenchyme Homeobox 2 , which regulates senescence and proliferation in HUVECs [PITX were identified among the EC-specific enhancer sites , the centromeric domain (C-DOM) and the telomeric domain (T-DOM), which are activated during limb and digit development, respectively [LINC01117 (Hotdog) and LINC01116 (Twin of Hotdog), which physically contact the expressed HOXD genes and are activated during cecum budding [HOXD genes in ECs , sex, race  and medical history, all features that may influence the epigenomic profiles of these isolated cells. Although it is difficult to completely avoid effects from these factors in a human study because of the limited number of donors , we did The integrative ChIP-seq analysis described here, which was based on samples from the human tissues of multiple donors, is potentially hampered by both individual variation and technical noise derived from sample preparation under various conditions, relative to the smaller differences among EC types. To overcome this issue, at least in part, we developed a robust procedure for comparative epigenome analysis. We focused on two histone modifications, H3K4me3 and H3K27ac, which exhibit robust, sharp peaks and therefore are suitable for identifying shared and/or unique features across EC cell types, in contrast to more broad histone marks . To ensure the robustness of this study, we used a rather conservative procedure for detecting EC-specific sites, which considered only the common sites among all samples that did not overlap with peaks in all 116 cell lines from the Roadmap Epigenomics Project. Therefore, there may be “true” enhancer sites among the filtered ones. In the small comparative analysis using a subset of our data , more enhancer sites may be included for the downstream analysis. In addition, for motif analysis, the information concerning the precise position of TF binding might have been lost because multiple nearby sites were merged into a single large reference site. A more specialized scheme for each EC type may increase the power to identify additional candidates of binding TFs.In the future, we aim to expand this analysis to other core histone marks including suppressive markers  and apply semi-automated genome annotation methods . BecauseWe successfully identified 3765 EC-specific enhancer sites, 67 of which were highly significantly overlapping with GWAS SNPs. We also found variation in H3K4me3 enrichment at TSSs that may reflect the competence of gene expression, which cannot be fully captured by gene expression analysis , a PCR-based sterility test  and immunostaining-based characterization for von Willebrand factor (vWF) (> 95% cells are positively stained ) and alp2 incubator, and the medium was changed every 3 days. The cells used in the experiments were from passage 6 or less. The cryopreservation solution used consisted of VascuLife VEGF Endothelial medium, containing 250 pg/mL VEGF, 12% fetal bovine serum and 10% dimethylsulfoxide.Purified ECs were cultured in VascuLife VEGF Endothelial Medium (Lifeline Cell Technology) with 250 pg/mL VEGF. Cells were maintained at 37 °C in a humidified 5% COwww.ncbi.nlm.nih.gov/sra) under accession number SRR2952390. The full list of gene expression data is available at the NCBI Gene Expression Omnibus (GEO) under the accession number GSE131953.Poly(A)-containing mRNA molecules were isolated from total RNA and then converted to cDNA with oligo(dT) primers using a TruSeq RNA Sample Preparation kit v2 (Illumina) and were sequenced with a HiSeq 2500 system (Illumina). We applied sequenced paired-end reads to kallisto version 0.43.1  with theFor each EC sample, two million ECs were plated on a 15-cm culture plate and cultured until confluency. The cells were crosslinked for 10 min using 1% paraformaldehyde. After quenching using 0.2 M glycine, cells were collected using a scraper, resuspended in SDS lysis buffer  and fragmented by sonication . Samples were stored at − 80 °C before use. To perform ChIP, antibodies against histone modifications   were useSequencing libraries were made using the NEBNext ChIP-Seq Library Prep Master Mix Set of Illumina (New England Biolabs). Sequenced reads were mapped to the human genome using Bowtie version 1.2  allowinghttps://github.com/arq5x/bedtools2); and the pairwise correlations of read coverage by deepTools version 2.5.0 [We checked the quality of each sample based on the peak number, library complexity and GC content bias by DROMPA; the normalized strand coefficient and background uniformity by SSP ; inter-son 2.5.0   or H3K27ac peaks (enhancer sites) of all cells except for E122 (HUVECs) from the Roadmap Epigenomics Project. Similarly, we further excluded the sites that overlapped H3K27ac peaks of IMR90 cells generated by this study, to avoid the protocol-dependent false-positive peaks. The resulting sites were used as EC-specific sites. We also defined “distal enhancer sites” as those that are > 10 kbp from the nearest TSS. These sites are summarized in Additional file We implemented GWAS enrichment analysis using a strategy similar to that of Lake et al. . We obtaWe applied the ANOVA-like test in edgeR  based onWe used ChIA-PET data mediated by RNA Pol II for HUVECs . We acquWe used MEME-ChIP version 5.0.1  with theAdditional file 1: Table S1. EC sample list.Additional file 2: Table S2. Quality values of ChIP-seq and RNA-seq data.Additional file 3. Supplementary figures.Additional file 4: Table S3. EC enhancer sites.Additional file 5: Table S4. GWAS and enhancer sites.Additional file 6: Table S5. DEG clusters.Additional file 7: Table S6. DE clusters.Additional file 8: Table S7. DP clusters.


STM pluripotency gene promotes initiation of flowers and uncover the mechanism for STM silencing. The ARFs ETTIN (ETT) and ARF4 promote organogenesis at the reproductive shoot apex in parallel with MP via histone-deacetylation mediated transcriptional silencing of STM. ETT and ARF4 directly repress STM, while MP acts indirectly, through its target FILAMENTOUS FLOWER (FIL). Our data suggest that – as in animals- downregulation of the pluripotency program is important for organogenesis in plants.Specification of new organs from transit amplifying cells is critical for higher eukaryote development. In plants, a central stem cell pool maintained by the pluripotency factor SHOOTMERISTEMLESS (STM), is surrounded by transit amplifying cells competent to respond to auxin hormone maxima by giving rise to new organs. Auxin triggers flower initiation through Auxin Response Factor (ARF) MONOPTEROS (MP) and recruitment of chromatin remodelers to activate genes promoting floral fate. The contribution of gene repression to reproductive primordium initiation is poorly understood. Here we show that downregulation of the  STM via chromatin modifications and allow initiation of primordia that lead to formation of flowers.The pluripotency factor SHOOTMERISTEMLESS (STM) maintains stem cell identity in the centre of the shoot apical meristem. Here Chung et al. show that Auxin Response Factors work in concert with the YABBY transcription factor FIL to silence  Flowers are plant organs important for reproductive success and yield3. Perception of a local auxin maximum triggers transcriptional responses that enable specification of reproductive primordium founder cells5. When auxin levels are low, small nuclear Aux/IAA proteins bind to MONOPTEROS/Auxin Response Factor 5  and recruit co-repressor complexes to prevent activation of auxin responsive genes7. Increased auxin levels lead to ubiquitin-mediated degradation of Aux/IAA proteins8, releasing the co-repressors and allowing MP to recruit the SWI/SNF family chromatin remodelers BRAHMA and SPLAYED (SYD)9. In this fashion, MP directly upregulates genes important for flower development, such as those encoding the transcription factors FILAMENTOUS FLOWER (FIL) and LEAFY (LFY)10. MP also directly modulates expression of genes that control hormone accumulation and response13.Plants give rise to new organs continuously throughout their life. During reproduction, primordia that give rise to flowers and cryptic bracts (henceforth termed reproductive primordia), arise from a population of transit amplifying cells at the flanks of the inflorescence shoot apex14. Class A ARFs are classified as transcriptional activators, while class B and C ARFs are classified as transcriptional repressors16. MP is a class A ARF with an unstructured glutamine-rich central domain that can interact with SWI/SNF family chromatin remodelers9. Besides MP, only two other ARFs are strongly expressed in founder cells of the reproductive primordia: ETTIN  and ARF417. Both ETT and ARF4 are ‘repressive’ class B ARFs16. ETT and ARF4 have partly redundant roles in several developmental processes in Arabidopsis, including lateral root initiation and leaf polarity20.Auxin Response Factors are divided into three evolutionarily conserved classes, A, B and CSHOOTMERISTEMLESS (STM)21. STM promotes meristematic fate in part by upregulating biosynthesis of the hormone cytokinin, which acts in a positive feedback loop to promote stem cell fate23. STM frequently acts in concert with other class I KNOX genes such as BREVIPEDICELLUS (BP) and the activity of the class I KNOX proteins is modulated by a different class of homeodomain proteins, the BEL-like proteins (see24 for review). STM expression is high throughout the shoot apical meristem, but is downregulated at sites of auxin maxima in transit amplifying cells26. Neither the functional significance nor the mechanism for STM downregulation in primordium initials are currently understood.A key pluripotency gene in plants is the class I KNOX homeobox transcription factor STM repression promotes initiation of reproductive primordia. We further demonstrate that the class B ARFs ETT and ARF4, which are expressed in incipient reproductive primordia, act in parallel with MP to downregulate STM as well as BP. We implicate the MP target FIL in direct repression of the class I KNOX genes together with ETT and ARF4. Finally, we reveal that silencing of the pluripotency gene STM and BP by FIL and ETT/ARF4 is mediated by histone deacetylation.We show here that mp allele mp-S31927. mp-S319 has a T-DNA insertion in the second to last exon of MP28 and displays weaker phenotypes than mp null mutants, including partially compromised flower initiation  was not dramatically increased, suggesting that the expansion of the STM expression domain is not accompanied by an increase in the size of the CLV3-expressing stem cell pool.  in entire mp-S319 ett arf4 or syd-5 ett arf4 inflorescences  are formed during embryo development33. mp-12 ett arf4 mutants, like mp-12 mutants, did not form roots  likewise enhanced the flower initiation defects of the hypomorph mp-S319 mutant , whose expression is also elevated in mp-S319 ett arf5 and mp-12 ett arf4 mutants . On the basis of prior studies, BP downregulation also contributes to reproductive primordium initiation51.Organogenesis is critical to establish the body plan of higher eukaryotes. Here we investigate the initiation of reproductive primordia that give rise to flowers, plant organs critical for reproductive success. Prior studies uncovered the gene expression programs activated by MP and the mechanism for this activation in response to auxin accumulation in the founder cells of reproductive primordiaion Fig. . We furtion Fig. , 5. FinaSTM Fig. . RemovalSTM is known to be a target of Polycomb Repressive Complex 2 (PRC2)54. In our genetic enhancer tests, we did not observe enhanced flower initiation defects when we combined mp-S319 with a PRC2 mutant . Evidence is accumulating for unique DNA sequence binding preferences of ETT and MP59. It is likely that multiple different types of interactions between the evolutionarily conserved classes of ARFs61 enable unique transcriptional and developmental outcomes in response to a single hormonal cue, auxin. Additional specificity is provided by ARF interactions with other classes of transcription factors, such as the YABBY transcription factor FIL we identify here.It has been proposed that class B ARFs may inhibit activity of class A ARFs by competing for binding sites at the same target genesFIL accumulation in incipient reproductive primordia by triggering ubiquitin-mediated degradation of Aux/IAA proteins complexed with MP, thus enabling MP to recruit chromatin remodelers to activate FIL expression9. How auxin modulates ETT/ARF4 accumulation or activity to promote flower initiation is not clear. In a pathway parallel to MP, auxin may regulate ETT and ARF4 message abundance transcriptionally or post transcriptionally via the transacting siRNAs that target these class B ARFs18. Alternatively, ARF4 activity may be inhibited by Aux/IAA proteins in conditions of low auxin. In this scenario, activity of ETT, which lacks the Aux/IAA protein interacting domain, could be blocked by Aux/IAA proteins as a DNA-binding domain mediated heterodimer with other ARFs, such as ARF458. Since ETT activity is modulated by changes in auxin concentration59, auxin might instead control ETT interaction with FIL and/or HDAC-containing co-repressor complexes.Auxin promotes 62, organogenesis requires both activation of lineage-specific gene expression programs and silencing of pluripotency genes.In summary, our study implicates epigenetic silencing of a pluripotency factor in initiation of reproductive primordia, and elucidates the mechanism for this silencing. Our findings moreover suggest that in plants, like in animals63. All mutations were in the Columbia accession unless otherwise indicated. Mutant alleles described before include ett-743, arf4-2 (SALK_070506)20, mp-S31927, hda19-164, mp-12 (SALK_049553)31, clf-2865, pETT:ETT-GFP ett -336, syd-566 and pHDA19:HDA19-GFP9. fil-867 is in the Ler background and was outcrossed to Columbia more than three times. fil_049 (WiscDsLox367E6_049) was obtained from the Arabidopsis Biological Resource Center.Plants were grown at 22 °C in long-day conditions (16 h day/8 h dark) in soil or on ½ Murashige and Skoog (MS) platesmp-S319, plants were treated with 1 µM dexamethasone  plus 0.015% Silwet from 12 days of age onwards, the stage when the first reproductive primordium initiates68. Treatment was continued for 9 days, because new reproductive primordia initiate continuously and the synthetic steroid rapidly decays in plants69. For expression studies, 9-day-old MS agar-grown seedlings were sprayed with 10 µM Dex and seedlings were harvested after 4 h. Mock treatment consisted of 0.1% DMSO and 0.015% Silwet. For the cytokinin treatment, mp-S319 inflorescences were treated with 1 µM cytokinin  in 0.015% Silwet from 11 days of age onwards every 2 days, for a total of eight treatments. Mock treatment consisted of 0.1% DMSO and 0.015% Silwet. For deacetylase inhibitor treatment, soil-grown plants were sprayed with 5 µM of Trichostatin A  plus 0.015% Silwet or with 50 µM of Suberoylanilide hydroxamic acid  plus 0.015% Silwet every 3 days from 11 days of age onwards for a total of six treatments. After manifestation of pin inflorescence phenotypes, spraying was resumed once weekly for 2 weeks. Mock treatment consisted of 0.1% DMSO plus 0.015% Silwet. For NPA treatment, fil ett arf4 inflorescences were treated with 500 nM of NPA plus 0.015% Silwet every 2 days on from 11 days of age onwards for a total of total eight treatments. Mock treatment consisted of 0.05% DMSO plus 0.015% Silwet. Because treated plants are less vigorous and make fewer flowers then untreated plants  were identified by BASTA treatment and plants heterozygous for mp and ett by genotyping. In the T2 generation, T1 progeny was sprayed with BASTA to identify plants hemi or homozygous for amiRSTM. mp ett arf4 triple mutants were identified visually (they form very few leaves) and confirmed by genotyping. amiRSTM arf4 plants were identified by visual inspection and genotyping from the same T1 progeny. Phenotypic analyses were conducted in the T2 generation on arf4 and mp-S319 ett arf4 siblings. The arf4 single mutant is indistinguishable from the WT20.p35S:amiR STM19 were mutagenised by site-directed mutagenesis using primer sets previously described19. The ETTr fragment was amplified by PCR and cloned into pENTR/D Topo (Thermo Fisher Scientific). To generate ARF4r, the full length ARF4 coding sequence was amplified and cloned into pENTR/D TOPO. 266 base pairs including the sequence with the mutated tasi-RNA target sites19 in ARF4 was synthesized as a gBlock Gene Fragment (Integrated DNA Technologies). The endogenous sequence was replaced by the synthesized gBlock in pENTR/D TOPO using Gibson assembly with the Gibson assembly master mix (New England BioLabs). For BiFC assays, ETTr, ARFr, as well as FIL and HDA19 in pENTR/D TOPO  in an entry Gateway compatible vector. Following LR reaction (Invitrogen), the pFIL:FIL-6xHA-tNOS fragment was recombined into a modified Gateway-compatible version of the pPZP222 vector. To increase the cut-ligate reaction efficiency, site-specific mutagenesis was used to mutate two endogenous BsaI site in the FIL locus. The final destination vectors were transformed into in the fil_049 mutant background. Constructs used for stable plant transformation were transformed into Agrobacterium tumefaciens strain GV3101 by electroporation, followed by plant transformation by floral dip.To generate ETTr, the tasi-RNA target sites of ETTSTM 3′UTR was used as a query for finding sequences of closely related species. The genomic sequences of Brassicaceae species were obtained by running BLASTn discontinuous megablast. The mVISTA program (http://genome.lbl.gov/vista/index.shtml) was employed to identify regions of conservation. Clustal omega  was used to identify conserved cis motifs in the conserved regions of the STM locus.Genomic sequence covering from AT1G62370 to the 72. For inflorescence ChIP, ~0.5 g of young inflorescences from plants with 0~5 cm bolts were harvested. All flowers from stage 6 onwards were removed. Anti-GFP  and Anti-HA  were used for IP. Antibody quality was validated by confirming published ChIP data for LFY-GFP73 and FIE-HA71. In addition, Anti-H3K27ac antibodies , anti-H3K9ac antibodies  and anti-H3 antibodies  were used for IP. The antibodies were validated by the manufacturers. For ChIP in wild-type leaves and ap1 cal inflorescences, ~2 g of fully expanded Columbia leaves and 0.5 g of ap1-1 cal-174 inflorescence tissue was collected and fixed for analysis. Antibody amounts were 5 µg for anti GFP and HA, and 2 µg for anti H3, H3K27ac and H3K9a. The TA3 retrotransposon (At1g37110) served as negative control locus (NC)9. The LFY locus region e (LFYe)10 served as positive control for MP ChIP and as a negative control for ETT ChIP. Throughout, ChIP was also performed using the same antibodies in the WT to control for nonspecific DNA enrichment. To estimate the DNA enrichment, the qPCR value of the ChIP product was normalized over input DNA. Primer sequences are listed in Supplementary Table ChIP was performed as described in our detailed published protocolSTM, CLV3, MP and ETT was performed as described in our published protocol75 with minor modifications. The antisense probes for STM, FIL and ARF4 were generated using in vitro transcription from the full- length STM (1149 base pair), FIL (690 base pairs) and ARF4 (2367 base pairs) coding sequences cloned into pGEM-T (Promega). The full-length (516 base pair) CLV3 coding sequence and 1273 base pair of the ETT coding sequence (551–1824 base pairs) were cloned into pGEM-T easy for probe synthesis. The MP probe has been described10. Inflorescence tissue was harvested at 21–28 days of age for WT and ett arf4 and at 28 days of age for genotypes showing pin phenotypes (mp-S319 and mp-S319 ett arf4). Seedling tissue was grown on MS agar medium and harvested at 10 days of age. Pictures were taken of 8 μ sections using a brightfield microscope (Olympus BX51).In situ hybridization of UBQ10 (AT4G05320).For qRT-PCR expression analysis from 1–5 cm bolt inflorescences and fully expanded adult leaves, RNA was extracted by TRIzol (Thermo Fisher Scientific) and further purified using the RNeasy mini kit (Qiagen). First strand cDNA was synthesized using the superscript III kit (Thermo Fisher Scientific) from 1 µg of RNA. Quantitative real time PCR was performed using Power SYBR Green master mix (Thermo Fisher Scientific). Relative expression value was determined using a cDNA standard curve and normalized over that of the housekeeping gene For test of interaction between ETT, ARF4, FIL and HDA19 in yeast, full length clones for all four proteins, as well as ETT-N (amino acids 1–389) and ETT-C (amino acids 390–608) were cloned into pENTR/D TOPO and recombined into pDEST22 and pDEST32. These constructs were co-transformed into competent AH109 yeast cells using the Frozen-EZ Yeast Transformation II Kit (Zymo research). To examine yeast growth, serially-diluted yeast cells were spotted on media lacking Leucine and Tryptophan. To test for interactions, yeast cells were spotted on media lacking Leucine, Tryptophan and Histidine and containing 3-amino-1,2,4-triazole (3-AT) as competitive inhibitor. 3-AT concentrations used were 1 mM and 0.1 mM. Pictures were taken after 3 days of growth.Arabidopsis leaf protoplasts was performed as previously described76. Only protoplasts prepared and transfected at the same time were used for direct comparison. Three-week-old soil-grown Columbia plants were either sprayed with 10 µM of indole-3-acetic acid  plus 0.015% silwet or with 0.01% DMSO plus 0.015% Silwet nine hours prior to protoplasting. To avoid message depletion by tasiRNAs, tasiRNA resistant versions of ETT (ETTr) and ARF4r version were employed. Protoplasts (70–280) were counted per replicate and sample. Confocal imaging was conducted using a Leica TCS SP8 confocal microscope and a 40X water immersion objective.Bimolecular fluorescence complementation (BiFC) in 77 or FLAG-tag and 4xMyc-tag fusion, respectively. ETT-FLAG was generated using golden gate cloning and a previously described L0 clone for ETT36, combined with the p35S promoter (AddGene #50266), the C-terminal 3xFLAG epitope (AddGene #50308), and the Nos-terminator (AddGene #50339) into the L1 (AddGene #48000)78. The epitope-tagged proteins were transiently expressed in 4-week-old N. benthamiana leaves by means of infiltration with an Agrobacterium suspension (OD600 = 0.5). Two days after infiltration, 1 g of leaf tissue was collected and ground in liquid nitrogen. All subsequent steps were carried out at 4 °C. The ground powder was homogenized for 30 min in two volumes of extraction buffer ). The homogenized samples were cleared by centrifugation at 14,000 × g for 10 min. The supernatant was transferred to 2 ml microcentrifuge tubes and cleared for another 5 min by centrifugation 14,000 × g. Next, the cleared lysate was transferred to fresh 2 ml microcentrifuge tubes and incubated for 2 h with 20 µl anti-FLAG M2 magnetic beads . The beads were subsequently washed five times with IP buffer ). Proteins were eluted by adding 80 µl 1x SDS loading buffer followed by an incubation at 95 °C for 10 min. The eluate was analysed by western blot using an anti-FLAG antibody  or an anti-Myc antibody . Both antibodies were used at a 1:10000 dilution. The antibodies were validated by the manufacturer.For co-immunoprecipitation, ETT-Myc, ARF4-FLAG, ARF4-Myc, HDA19-FLAG and HDA19-Myc were generated using the same entry vectors as for BiFC (above) following recombination into pGWB12 and pGWB18For scanning electron microscopy, 10-day-old seedlings were fixed overnight in FAA  and dehydrated in a serial dilution of ethanol. The samples were critical point dried, mounted and gold coated. Images were taken with a Zeiss Supra 55VP Field emission scanning electron microscope using an acceleration voltage of 3 kV.mp-12/+ett/+ arf4/- plants homozygous for a pSTM:GUS reporter30 were dissected from mature siliques, and stained in restrictive GUS staining solution  for 5 h at 37 °C. Embryos were mounted in concentrated Hoyer’s solution , cleared overnight, and observed with a Leica DMR6000 microscope with Nomarski optics at ×10 magnification. Homozygous mp ett arf4 embryos were identified by comparing segregation ratios of phenotypes between mp-12/+ and mp-12/+ett/+arf4/- plants.Late bent cotyledon stage embryos from 9. For qRT-PCR and ChIP-qPCR, one representative of three experiments is shown. Box and whisker plots: Lower vertical bar = sample minimum. Lower box = lower quartile. Black Line = median. Upper box = upper quartile. Upper vertical bar = sample maximum. A nonparametric Mann Whitney U test was used for phenotypic data, which frequently are not normally distributed. Unpaired t-tests were used for qRT-PCR and ChIP-qPCR data, which were normally distributed based on Kolmogorov–Smirnov tests. We employed one-tailed tests to examine differences between groups in a specific direction, and two-tailed tests to probe differences between groups in any direction. Sample size was determined by expedience and based on prior studies that found significance using similar samples sizes73.Throughout, error bars shown represent the standard error of the mean for all numerical values. For frequencies, the error bars are proportional to the standard error of the pooled percentage computed using binomial distributionFurther information on experimental design is available in the Supplementary InformationReporting SummarySource Data