The genome is now known to be folded into a complex 3D arrangement, created and maintained in part by the interplay of the Cohesin complex and the DNA-binding protein CTCF. In the last few years, however, doubt over the relationship between looping and transcriptional activation has emerged, driven by studies finding that only a modest number of genes are perturbed with acute degradation of looping machinery components. In parallel, newer models describing distal enhancer action have also come to prominence. In this article, we explore the emergence and development of the looping model as a means for enhancer–promoter communication and review the contrasting evidence between historical gene-specific and current global data for the role of chromatin looping in transcriptional regulation. We also discuss evidence for alternative models to chromatin looping and their support in the literature. We suggest that, while there is abundant evidence for chromatin looping as a major mechanism for enhancer function, enhancer–promoter communication is likely mediated by more than one mechanism in an enhancer- and context-dependent manner.In mammalian organisms, enhancers can regulate transcription from great genomic distances. How enhancers affect distal gene expression has been a major question in the field of gene regulation. One model to explain how enhancers communicate with their target promoters, the chromatin looping model, posits that enhancers and promoters come in close spatial proximity to mediate communication. Chromatin looping has been broadly accepted as a means for enhancer–promoter communication, driven by accumulating  Cloin loops , 14. Desin loops , 14. Thiin loops , 16. Howin loops . Even wiin vivo and its causal role in transcription regulation. Subsequently, we review more recent data on the mechanisms that give rise to chromatin loops and discuss the results of experiments perturbing the looping machinery and their effects on gene regulation that have raised potential questions regarding the role of looping in gene regulation. Throughout this review, we try to synthesize both historical data and more recent observations to obtain a more holistic view of the role of chromatin looping in enhancer function. Indeed, we address here the possibility that 3D architecture is primarily important in mediating changes in transcriptional state and how we may utilize this concept to more appropriately interpret past data and guide the collection of future data.In this review, we attempt to synthesize classical observations of chromatin looping and enhancer function with more recent studies on the mechanisms that govern chromosomal looping and the effects of perturbing the chromatin looping machinery on gene expression. We start by reviewing the historical evidence that contributed to the rise of the looping model and discuss other early alternative models for E-P communication. We also discuss prior gene specific data that established evidence for chromatin looping via a protein bridge to enable transfer of protein from one region to the other. With the identification of distal, cis-acting regulatory elements in both prokaryotes (A). At the same time, several alternative non-looping models were suggested to explain enhancer function. “Sliding”  was proposed as a potential mechanism by which the SV40 enhancer affects transcription by serving as a “bidirectional entry site” for RNA polymerase II to track or scan along chromatin until it reaches an initiation site . In addition, “conformation” models proposed that conformational changes in DNA initiating at the enhancer could transmit a signal to the promoter region, possibly involving propagation of decompacted chromatin   . The SV4araBAD operon in Escherichia coli . The co . Beyondive gene , 53. Simive gene , 55. Takvia passive mechanisms such as diffusion, this has often been discounted as a means of loop formation, particularly over long genomic distances. This is in part due to the fact that DNA undergoes subdiffusive motion, meaning that two genomic loci if initially separated by large 3D space are very unlikely to come in contact, in particular at short timescales  and was initially identified for its role in sister chromatid cohesion (C) . T. TB) andsion (C) . The glosion (C) , allowinsion (C) , TADs (1sion (C) , 12, 71,sion (C) . These s , suggesRad21 conditional KO systems  system for acute degradation led to the finding that loss of Cohesin causes a near-complete disruption of chromatin loops and domains , 74. Sim systems . Applica systems , 77, 78.A). With regards to the formation of chromatin loops, the loop extrusion model posits that Cohesin will “extrude” a loop of chromatin until it is blocked by an appropriately oriented CTCF-binding site (C). The loop extrusion model, albeit without regard to enhancer function, was proposed by Nasmyth , increased (Fbn2), and decreased (Pcdhα)  , 80. Thepression , 117. Foxpressed . These rShh) is controlled by different enhancers under different contexts, primarily the limb bud-specific enhancer ZRS and the series of brain-specific enhancers SBE. On a correlation basis, Shh and ZRS have been found to have close spatial proximity during periods of Shh expression  is accompanied by a reduction in the spatial proximity between Shh and the contextually speaking most active enhancer SBE6 , which found that close spatial proximity between enhancer and promoter is not a necessary requisite for a transcriptional burst  than what a stable looping model would predict . Indeed, this idea aligns well with much of the early evidence supporting a causal relationship between E-P looping and transcriptional regulation, such as the forced looping experiments for transcriptional activation , but a subset of CohesinSTAG1 remains on chromatin for around 5 h. Based on their contrasting residence times, one would expect the long-lived CohesinSTAG1 subset to modulate stable loops, whereas the more transient CohesinSTAG1 and CohesinSTAG2 would both contribute to dynamic loops. In general, STAG2-containing Cohesin is preferentially distributed at enhancers and promoters that lack CTCF-binding sites, whereas STAG1-containing Cohesin tends to be associated with TAD boundaries, which are enriched for CTCF-binding sites . This may suggest that loop formation initiates a self-perpetuating transcriptional cycle whereby relaxing of the loop, through either innate processes or artificial disruption of looping machinery, has only minimal consequences on recruitment of the transcriptional machinery to the promoter and ongoing gene expression . This process is unlikely to be feasible for inducible genes and implies looping remains necessary for initiating transcription of at least a subset of constitutive genes. In the former context, a stimulus may promote loop formation  or enable transcriptional activation following loop formation , with both of these requiring functional looping machinery. The dynamicity of the majority of Cohesin complexes therefore could create an environment in which transcriptional change is easily accessible.Why does perturbation of 3D architecture have such a limited effect on transcription at steady state but a more pronounced effect on gene expression transitions? One possibility that has been raised in response to the minimal effect of Cohesin/CTCF depletion on transcriptional output is that while the dominant structures are eliminated by CTCF or RAD21 degradation, less prominent interactions between enhancers and promoters may retain connectivity, at least at short timescales. Specifically, RAD21 depletion appears to have a more limited impact on E-P communication, especially at early time points, and that a large number, albeit the minority, of promoter interactions are maintained in the absence of Rad21 , 133. ThFig. 3Much attention has been paid in recent years to the role of Cohesin and CTCF in the formation of chromatin loops in the genome. In light of the minimal effects of CTCF or Cohesin depletion on steady-state transcription, it is important to consider that several non-CTCF/Cohesin proteins have also been implicated as contributing to loop formation either independently or coordinately with Cohesin and CTCF. As the list of non-CTCF/Cohesin looping factors could be quite long with varying levels of support in the literature, we will specifically discuss here factors that we believe have the strongest evidence for a role in E-P looping in mammalian organisms.Drosophila homolog of Ldb1, Chip, was identified in screens of proteins that are required for enhancer function, offering early suggestions that Ldb1 could be required for specific instances of E-P looping . Like CTCF, YY1 is a DNA-binding zinc-finger protein that was originally reported as having either activator or repressor functions depending on promoter context , 142. YYet al.  showed b . Interestingly, when specific loci are tethered to the nuclear lamina, not all nearby genes are repressed . Repressive chromatin environments, such as H3K27me3 Polycomb sites or H3K9me3/HP1 heterochromatin regions, are in a relatively compacted state . If such a phase separated body encompassed both an enhancer and a promoter, then this could lead to gene activation without the gene and enhancer actually having to directly physically contact each other in a loop. Genome-wide evidence for multiway chromatin interactions at potentially phase-separated bodies such as nuclear speckles has recently been observed using the split-pool recognition of interactions by tag extension (SPRITE) assay  assay . If phasOther potential non-looping models for enhancer function have also been proposed over the years, particularly in the early history of the study of enhancers. The aforementioned discussion is not meant to be an exhaustive list of non-looping models for enhancer function but is meant to highlight specific models that have clear or growing evidence in the literature. We would suggest that such nonlooping-based models warrant further investigation, particularly how such events may act in combination, or in contrast, with more traditional looping-based models.in vivo, including chromatin loops, identification of the major looping machinery, and to demonstrable transcriptional regulation induced by loop formation. The relationship between chromatin looping and the control of gene expression is, however, becoming increasingly complex. Indeed, in studies assessing the functional contribution of chromatin looping to transcriptional regulation, there are distinct classes of evidence; initial in vivo challenges found that looping was often sufficient for transcriptional activation, while later global challenges suggested that extensive perturbations in looping had minimal effect on steady-state transcription. We posit that these classes are not incompatible and instead reflect differential contributions to transcriptional initiation and maintenance, with chromatin looping particularly relevant for mediating E-P communication to enable changes in transcriptional state. The terms of this relationship are likely context dependent, but we currently lack the appropriate global studies to understand the specifics of looping-mediated transcriptional activation. Regardless, the path forward must take into account that multiple mechanisms likely bridge enhancers and promoters to enable their communication, with chromatin looping one of many.Chromatin looping was among the earliest models to account for the observed influence of enhancers over their associated promoters and has persisted as the predominant model through decades of accumulated evidence. This evidence extends from observations of 3D genome structures AID is a system for rapid degradation of a target protein. It involves fusion of a protein of interest to a miniAID and introduction of the E3 ligase complex member OsTIR1 to the cell line (ell line . Additioβ-globin locus is region of the genome that encodes the embryonic (ε), fetal (ɣ), and adult (δ and β) β-globin genes. These genes are encoded and expressed sequentially, with the upstream LCR regulating their expression via loop formation.Chromatin conformation capture (3C) is a family of techniques based on crosslinking, restriction digest, and proximity ligation to identify regions of DNA that are spatially proximal (circular chromosome conformation capture (4C) for the unbiased identification of interacting loci with a single locus of interest (chromosome conformation capture carbon copy (5C) for unbiased identification of interacting loci with multiple loci  and B (inactive) compartments.Condensin I and II are protein complexes consisting of SMC2 and SMC4 and additional complex-dependent subunits. These complexes are primarily known for their role in chromosome condensation.CRISPR-GO is a technique developed by Wang et al. (g et al.  that useCTCF is a zinc-finger transcription factor that is able to stall the translocation of Cohesin along chromatin and functions to insulate regions of the genome.eRNAs are noncoding RNAs transcribed from enhancers by RNA PolII.E-P loop is a structure formed by bringing an enhancer in close spatial proximity to its promoter. Loops are proposed to be formed through the process of loop extrusion and mediated by the Cohesin complex and accessory proteins.LPS is a molecule found on the outer membrane of bacteria and can be used to initiate an immune response in cells.Loop extrusion is the proposed mechanism of E-P loop formation by the Cohesin complex. It involves threading a region of chromatin through Cohesin and a progressive increase in loop size as Cohesin translocates along chromatin.Mediator is a protein complex containing up to 26 subunits and primarily functions as a channel of communication between transcription factors and RNA PolII.NIPBL/MAU2 heterodimer is involved in the loading of Cohesin onto chromatin.Nuclear lamina is a network of filamentous lamin proteins and lamin-binding proteins that sits inside of the inner nuclear membrane. In addition to playing a structural role for the nucleus, it has multiple functional contributions, including to the control of transcription, DNA replication, apoptosis, cell cycle, and cell differentiation.Phase separation is the concept behind the formation of membraneless organelles, which form from the aggregation of proteins and other macromolecules to give liquid-like structures known as “biomolecular condensates.”SPRITE is a technique used to map genome-wide interactions (ractions . Unlike Shh is a developmentally regulated gene that is involved in the patterning of a number of tissues during embryonic development. Tissue-specific enhancers control the expression of Shh, including the limb bud enhancer ZRS and the brain enhancer SBE. While not mentioned here, additional enhancers of Shh expression are the lung and gut enhancers MACS1 and SLGE (and SLGE , brain/fand SLGE , and theand SLGE .TADs are a feature of the 3D genome in which self-interaction is disproportionately favored. TAD boundaries are often defined by CTCF binding.TALEs are DNA-binding proteins derived from Xanthomonas that can be customized to bind specific regions of DNA.VP64 and VP128 are derivatives of the herpes simplex virus-1 transcription factor VP16. VP64 and VP128 contain 4X and 8X, respectively, tandem repeats of the minimal activation domain of VP16, creating a powerful transcriptional activator.WAPL is responsible for the removal of Cohesin from chromatin.The authors declare that they have no conflicts of interest with the contents of this article.


Salvia miltiorrhiza synthesises tanshinones with multidirectional therapeutic effects. These compounds have a complex biosynthetic pathway, whose first rate limiting enzyme is 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR). In the present study, a new 1646 bp fragment of the S. miltiorrhiza HMGR4 gene consisting of a promoter, 5′ untranslated region and part of a coding sequence was isolated and characterised in silico using bioinformatics tools. The results indicate the presence of a TATA box, tandem repeat and pyrimidine-rich sequence, and the absence of CpG islands. The sequence was rich in motifs recognised by specific transcription factors sensitive mainly to light, salicylic acid, bacterial infection and auxins; it also demonstrated many binding sites for microRNAs. Moreover, our results suggest that HMGR4 expression is possibly regulated during flowering, embryogenesis, organogenesis and the circadian rhythm. The obtained data were verified by comparison with microarray co-expression results obtained for Arabidopsis thaliana. Alignment of the isolated HMGR4 sequence with other plant HMGRs indicated the presence of many common binding sites for transcription factors, including conserved ones. Our findings provide valuable information for understanding the mechanisms that direct transcription of the S. miltiorrhiza HMGR4 gene. Salvia miltiorrhiza Bunge, also known as Red sage, or Chinese sage, is an important species used in traditional Chinese medicine. The dried root is used alone or in combination with other herbs to treat various ailments including cardiovascular diseases, menstrual disorders and insomnia [HMGR in plant metabolism is emphasised by the precise regulation of its function at the level of transcription, post-transcription, translation and post-translation [S. miltiorrhiza HMGR gene sequences (HMGR—HMGR4) have been identified and deposited in the GenBank database [HMGR (EU680958.1) and HMGR4 (JN831103.1), and HMGR2 (FJ747636.1) and HMGR3 (JN831102.1) are probably two pairs of duplicated genes, respectively [S. miltiorrhiza HMGR genes have been identified and described, some of their promoter sequences remain unknown. These include the HMGR4 promoter.insomnia ,2. In adinsomnia , Parkinsinsomnia  and Alzhinsomnia , as wellinsomnia , hepatocinsomnia , acute linsomnia , fibrosiinsomnia , neuropainsomnia , diabeteinsomnia , or alcoinsomnia . The maiinsomnia . The keyinsomnia . The pivnslation ,16. To ddatabase ,18,19. AArabidopsis thaliana indicate that miRNAs can also regulate gene expression during transcription by binding to promoter sequences [Promoter sequence analysis provides much valuable information for understanding the regulation of gene expression. Motifs such as the TATA box, CpG islands, tandem repeats or transcription factor binding sites (TFBSs) deserve special attention. Genome-wide analyses indicate that most in vivo functional TFBSs are located in the proximal promoter region ,21. Thesequences ,28,29,30A. thaliana is the most widely-studied plant in modern biology. Its wide appeal for scientists results from its fast growth rate, easy maintenance and small space requirements [A. thaliana has become the starting point for studying numerous aspects of plant cell-, molecular- and system-biology [A. thaliana has led to the creation of numerous clones, cloning vectors, mutant lines, seeds, databases and online tools containing genomic, epigenomic, transcriptomic and proteomic data [irements . Moreoveirements . Geneticirements . Due to -biology . The abumic data .S. miltiorrhiza HMGR4 promoter and 5′ untranslated region (5′UTR) sequences, and their in silico characterisation via specialised databases such as PlantPan 2.0, TSSP and miRBase. Furthermore, the comprehensive in silico analysis presented herein presents valuable new information about the regulatory functions of HMGR promoters. The data can be used to create modified or synthetic promoters which could be active under certain controlled conditions [This work describes the isolation of a new nditions . In thisHMGR4 gene sequence JN831103.1 identified by Ma et al. [The 1646 bp sequence obtained by the DNA walking method was deposited in GenBank under accession number KT921337.1 . The seqa et al. . The TATA. thaliana were revealed using the PlantPan 2.0 tool ; seven salicylic acid (SA)-responsive elements, including W box and TCA-elements; two brassinosteroid-responsive elements (BRREs); two ethylene-responsive elements (EREs); two abscisic acid-responsive elements (ABREs); four methyl jasmonate-responsive elements (MJREs); one pathogen-responsive element Eli box 3; three wounding- and pathogen-responsive elements WRE3; three anaerobic-responsive elements (AREs); and twenty-four light-responsive elements (LREs), including ATCT sequences, Box 4, GT1 motif, TCT motifs, GA motifs, AE box, Box I  (14 binding sites), BHLH74 (2 sites), BZIP20 (32 sites), WRKY2 (8 sites) and WRKY61 (9 sites) (HMGR4 may play a role in the biosynthesis of tanshinones.As 9 sites)  [36,37,3HMGR4 promoter and 5′UTR revealed potentially interacting miRNAs  was more similar to A. thaliana HMGR1 (NP_177775.2), with an identity of 73.76%, than to A. thaliana HMGR2 (NP_179329.1), with one of 69.48%. Additionally, a phylogenetic study of coding sequences indicated that HMGR4 from S. miltiorrhiza (JN831103.1) and HMGR1 from A. thaliana (NM_106299.4) were more closely related to each other  was used for further co-expression research. As a result of the conducted microarray analysis, 166 TF genes co-expressed with A. thaliana HMGR1 in the r range of 0.5–1.0 were found: 41 in AtGenExpress Hormone and Chemical Compendium, 37 in AtGenExpress Abiotic Stress Compendium, 34 in AtGenExpress Pathogen Compendium, 25 in AtGenExpress Tissue Compendium, and 29 in AtGenExpress Plus—Extended Tissue Compendium , L1 box (P$L1BX), MYB IIG-type binding sites (P$MIIG), DNA binding with one finger (P$DOFF), GT box elements (P$GTBX), MADS box proteins (P$MADS), MYB-like proteins (P$MYBL), MYB proteins with single DNA binding repeat (P$MYBS), NAC factors with transmembrane motif (P$NTMF), plant specific NAC proteins (P$NACF), transcription repressor KANADI (P$KAN1), W box family (P$WBXF), time-of-day-specific regulatory elements (P$TODS), nodulin consensus sequence 1 (P$NCS1), sweet potato DNA-binding factor with two WRKY domains (P$SPF1), zinc finger proteins (P$ZFAT), light-responsive elements (P$LREM), protein secretory pathway elements (P$PSPE), CGCG box binding proteins (P$CGCG), proteins involved in programmed cell death response (P$PCDR), plant nitrate-responsive elements (P$PNRE) and finally, stomatal carpenter (P$SCAP). As can be seen in HMGR1 and HMGR2 promoter sequences was strikingly similar. The above TF family analysis found that the S. miltiorrhiza HMGR1, HMGR2 and HMGR4 genes can be co-regulated in response to abiotic factors , biotic factors  and during root, stem, leaf and flower organogenesis  and HMGR2 (KF297286.1) promoters to demonstrate the greatest similarity (97%). In contrast, only 17% similarity was found between HMGR4 (KT921337.1) and HMGR2, and 14% between HMGR4 and HMGR1. The greatest number of identical areas was revealed in the proximal fragments of the analysed promoters, as well as in the beginning and the middle of the distal parts. Common overlapping TFBSs were identified in locations where all three tested sequences showed high local similarity; these included two binding sites for Arabidopsis homeobox proteins (P$AHBP), one site for SBP domain proteins (P$SBPD), one site for W box family proteins (P$WBXF), one site for DNA binding with one finger factors (P$DOFF), one GT box element (P$GTBX) and, finally, one L1 box (P$L1BX). The PlantPan 2.0 and MatInspector (Genomatix) databases indicated that P$AHBP proteins are mainly involved in the response to hormones  and the initiation and development of shoot, root and flower meristems. P$SBPD TFs are associated with inflorescence development, flowering and leaf epidermis differentiation. In turn, P$WBXF matrix family responds to hormonal stimulation , other abiotic factors  and biotic agents , and also participate in leaf senescence. P$DOFF proteins are primarily involved in the regulation of flowering, circadian rhythm and in response to hormones (auxins and SA). P$GTBX factors participate in the organogenesis of flowers and shoots. In addition, P$L1BX proteins are needed for epidermis development and seed germination. These data are available in The DiAlign TF tool analysis found the HMGR genes revealed the presence of conserved regions; these are marked in blue in Arabidopsis homeobox protein) and P$GCCF (GCC box family) families were detected in 27.8% of cases. Sites for P$TDTF (transposase-derived proteins), P$MYBL (MYB-like proteins) and P$L1BX (L1 box) proteins were identified in 25% of the sequences. Binding sites for P$DREB (dehydration responsive element binding factors) and P$ROOT (root hair-specific cis-elements in angiosperms) families were found in 22.2%. These TFBSs are highlighted in red in MEGA X software alignment of 36 sequences spanning the proximal promoters and 5′UTRs of the plant S. miltiorrhiza HMGR4 promoter was found to contain several other common binding sites for TFs, belonging to P$AHBP (Arabidopsis homeobox protein), P$GTBX (GT box elements), P$DOFF (DNA binding with one finger) and P$L1BX (L1 box); these were shared by 8–13% of the tested sequences. Nucleotide pairwise alignment of S. miltiorrhiza HMGR4 with the remaining tested sequences found 13 to 31% identity (mean 24.7%).Apart from the conserved TATA box and PRS motifs, the investigated S. miltiorrhiza HMGR4 promoter and 5′UTR and compares these sequences with other plant HMGRs.Our study presents new data regarding the isolated A. thaliana promoters [S. miltiorrhiza HMGR2 promoter [Initially, the sequences were examined for the presence of certain distinctive motifs . One sucromoters . It was promoter . Previoupromoter ,46,47,48promoter ,46,47,48promoter .HMGR4 promoter is also characterised by the presence of a single tandem repeat. This motif is estimated to be present in only 25% of promoters [S. miltiorrhiza HMGR2 promoter [The romoters , and is promoter . As tandpromoter .HMGR2 promoter and the studied HMGR4 promoter lack CpG islands [Both the  islands . The cyt islands ,52. Howe islands .S. miltiorrhiza HMGR2 gene [A PRS is also detected in the 5′UTR of the described sequence. This is a rather rare observation, but not an unprecedented one, as a PRS has also been found in the 5′UTR of the GR2 gene . It is bGR2 gene .S. miltiorrhiza HMGR4 promoter sequence turned out to be rich in TFBSs recognised by specific TFs  had an inhibitory effect [HMGR1 in roots almost 5-fold compared to an untreated control [HMGR transcript level, peaking at three-times higher than baseline after 36 h [HMGR2 promoter in leaf material [HMGR expression. The addition of Pseudomonas brassicacearum subsp. neoaurantiaca and Pseudomonas thivervalensis to S. miltiorrhiza root culture resulted in 2.1- and 1.5-fold enhancements in HMGR enzyme activity, respectively [Streptomyces pactum Act12 increased HMGR1 expression by more than a factor of 35 on day 14 relative to the calibrator [HMGR2 promoter, resulting in manifold higher expression compared to the calibrator in 96 h [HMGR1 and HMGR2, respectively [HMGR in leaves and roots over 48 h of exposure [HMGR1 transcript in leaves and roots as compared to the calibrator [Furthermore, the examined ific TFs . The cont stress , suggestor roots . It is iy effect . In cont control . Incubatter 36 h . In turnmaterial . A decreectively . In addilibrator . Exposur in 96 h . In turnectively ,61. Saltexposure . It was librator .As non-coding regions are generally not highly conserved, from an evolutionary perspective, finding such motifs in the promoter or 5′UTR sequences suggest they have functional importance .HMGR sequences, the most frequently-identified conserved motif was the TATA box  or multimers. Dimers and multimers are often preferred by nature because they allow specific interactions with the promoter sequence and bind with high affinity [S. miltiorrhiza HMGR4 proximal promoter: HD-ZIP , WRKY  and Dof (DOF5.4)  . The HD-thaliana , and to thaliana . It has thaliana ,73,74, W vulgare , while Wa sativa . The Dofa sativa  and to eHMGR4 sequence was searched for miRNA binding sites and interacting miRNAs [HMGR4 promoter and 5′UTR sequences  according to the manufacturer’s instructions. A 5′-terminal fragment of the HMGR4 gene, deposited in GenBank under accession number JN831103.1, was used as a target for designing GSP1 and GSP2 specific primers .Genomic DNA used for isolation of the n et al. . The DNA primers . The PCRS. miltiorrhiza HMGR4 promoter took approximately one month.The isolation and sequencing of the HMGR4 promoter sequence was characterised in silico using available tools and databases [The obtained atabases . The proatabases . Tandem atabases . The proatabases .A. thaliana HMGR genes is a homologue of the S. miltiorrhiza HMGR4 gene. Analyses were performed on coding sequences. Expression Angler  [Arabidopsis RNA-seq Database [A. thaliana HMGR gene. The Expression Angler tool has access to the expression results for approximately 22,000 Arabidopsis genes, while the Arabidopsis RNA-seq Database integrates 28,164 publicly available Arabidopsis RNA-seq libraries. The following microarray dataset compendiums were used during the study: AtGenExpress Hormone and Chemical, AtGenExpress Abiotic Stress, AtGenExpress Pathogen, AtGenExpress Tissue, and AtGenExpress Plus—Extended Tissue. The Pearson’s correlation coefficient (r) ranging from 0.50 to 1.00 (moderate to strong positive correlation) was applied to identify co-regulated genes. Information on the detected TFs was obtained from the UniProt database [Protein BLAST  and MEGA X version 10.2.6   were emp Canada)  and ArabDatabase  were utidatabase . The coldatabase .S. miltiorrhiza HMGR promoter sequences, i.e., HMGR1 (GU367911.1), HMGR2 (KF297286.1), and HMGR4 (KT921337.1) were analysed with Common TFs, FrameWorker and DiAlign TF tools from Genomatix, Munich, Germany. Common TFs was used for preliminary analysis of the common TFBSs and interacting TFs located anywhere in the investigated promoters. The search only included sites that were common to all three sequences. The similarity of the matrix to the tested sequences was set to the highest possible value, i.e., 0.05. The FrameWorker tool permitted the common, most complex framework of TFBSs to be extracted from the input promoters. Frameworks are defined as TFBSs that occur in the same order and in a specificed space range in all of the sequences. The DiAlign TF allowed for multiple alignment of the studied HMGR promoters, and revealed conserved regions and TFBSs located therein. The analyses using the Genomatix tools were based on matrix library version 11.3 and default search criteria.The entire available HMGR promoters derived from plants such as A. thaliana, Arabidopsis lyrata, Glycine max, Gossypium hirsutum, Oryza sativa, Solanum lycopersicum, Zea mays and S. miltiorrhiza was assessed by aligning their sequences. Proximal promoters and 5′UTR sequences (each sequence 500 bases long) were obtained from PlantPan 3.0 [The conservation of the 36 available tPan 3.0  and NCBItPan 3.0 . AlignmetPan 3.0 . TFBSs lS. miltiorrhiza HMGR4 gene expression can occur during flowering, embryogenesis, organogenesis and circadian rhythm, and are influenced mainly by factors such as light, SA, bacterial infection and auxins.Regulation of S. miltiorrhiza HMGR4 gene plays an important role in the production of these metabolites.The presence of binding sites for TFs that promote the biosynthesis of tanshinones may indicate that the S. miltiorrhiza HMGR1, HMGR2, and HMGR4 promoter sequences indicates that these genes can be co-regulated in response to abiotic and biotic factors, and during organogenesis.A comparison of TFBSs and TFs in the S. miltiorrhiza HMGR4 promoter is not highly conserved.The S. miltiorrhiza HMGR4 promoter could be developed towards preparing promoter deletion mutants, and studying their transcriptional activity [Future research on the activity . Moreoveactivity . The TFsactivity . Isolateactivity ,92. Thesactivity . Finallyactivity ,95.


Although significant advancement has been made in the study of adipogenesis, knowledge about how chromatin accessibility regulates yak adipogenesis is lacking. We here described genome-wide dynamic chromatin accessibility in preadipocytes and adipocytes by using the assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq), and thus revealed the unique characteristics of open chromatin during yak adipocyte differentiation. The chromatin accessibility of preadipocytes and adipocytes exhibited a similar genomic distribution, displaying a preferential location within the intergenic region, intron, and promoter. The pathway enrichment analysis identified that genes with differential chromatin accessibility were involved in adipogenic metabolism regulation pathways, such as the peroxisome proliferator activated receptor-γ (PPAR) signaling pathway, wingless-type MMTV integration site (Wnt) signaling pathway, and extracellular matrix-receptor (ECM–receptor) interaction. Integration of ATAC-seq and mRNA-seq revealed that genes with a high expression were associated with high levels of chromatin accessibility, especially within 1 kb upstream and downstream of the transcription start site. In addition, we identified a series of transcription factors (TFs) related to adipogenesis and created the TF regulatory network, providing the possible interactions between TFs during yak adipogenesis. This study is crucial for advancing the understanding of transcriptional regulatory mechanisms of adipogenesis and provides valuable information for understanding the adaptation of plateau species to high-altitude environments by maintaining whole body homeostasis through fat metabolism. Adipose tissue is a vital endocrine organ for energy storage and metabolism in animals, and the growth of this tissue refers to the increase in adipocyte number and size . Mature The yak is a unique and rare ruminant living in the Qinghai–Tibet Plateau and adjacent regions. As an important livestock species for animal husbandry in this area, yaks provide local residents with agricultural products such as meat, milk, and hair while adapting to environmental conditions including cold, low oxygen, and high altitude and ultraviolet rays ,17. BecaTo determine whether preadipocytes fully differentiated into adipocytes, Oil Red O staining and mRNA detection of adipogenic marker genes were performed. The results of Oil Red O staining revealed that preadipocytes were fully differentiated into mature lipid-filled adipocytes after induction with adipogenic agents for 12 days . At the To explore epigenetic regulation during yak adipocyte differentiation, we used ATAC-seq to identify open chromatin regions in preadipocytes and adipocytes . An average of 167,249,845 and 159,616,847 high-quality reads were obtained from Pread (preadipocytes) and Ad (adipocytes) groups, respectively. The reads information of each ATAC-seq library before and after filtering is shown in p < 0.05). The results revealed 1293 differential peaks, of which 1265 were downregulated and 28 were upregulated  were identified, of which 381 genes were downregulated and 286 upregulated . The mRNTFs are a class of protein molecules that bind to promoters and cooperate with RNA polymerase II to initiate gene transcription. We used AME from the MEME suite  to identify the enriched TFs based on conservative motifs of differential peaks. A total of 26 TFs were enriched in these differential peaks, among which FOS (FBJ osteosarcoma oncogene), JUNB (jun B proto-cologene) and KLF5 (Kruppel-like factor 5) were confirmed to be related to the adipogenic metabolism regulation . The mecTo our knowledge, this study is the first to use ATAC-seq technology to detect the landscape of chromatin accessibility during yak adipocyte differentiation. This will provide a new perspective on the molecular mechanism of adipogenesis in plateau species. Transcriptional regulatory elements of genes include enhancers, upstream activators, and proximal promoters. When the chromatin region containing transcriptional regulatory elements is in an open state, TFs can bind to the region and recruit RNA polymerase to the core promoter to initiate transcription. By contrast, condensed chromatin prevents TFs from binding to regulatory elements to silence gene expression . ATAC-seWe noted that the number of Pread peaks  was much higher than that of Ad peaks (7683), suggesting that ATAC-seq worked better in preadipocytes, or that differentiation would lead to reduced chromatin accessibility in yak adipocytes. The peaks of Pread and Ad groups exhibited a similar genomic distribution, demonstrating the preferential location within the distal intergenic region, intron and promoter. This distribution is consistent with the chromatin accessibility features reported in bovine myogenic differentiation, suggesting the vital role of non-promoter cis-regulatory elements in the regulation of cell differentiation . AlthougGO terms of differential chromatin accessibility were mainly implicated with binding, transcription regulator activity, molecular transducer activity, metabolic process, regulation of biological processes, developmental processes, signaling, cell part and protein-containing complex being enriched, further illustrating that adipocyte differentiation was a complex transcriptional regulatory activity mediated by a cascade of multiple TFs. The KEGG pathway enrichment analysis found that the signaling pathways of differential chromatin accessibility was significantly associated with adipogenesis, such as the PPAR signaling pathway, Wnt signaling pathway, regulation of actin cytoskeleton, and ECM–receptor interaction. Among them, the PPAR signaling pathway, Wnt signaling pathway, and ECM–receptor interaction are the classical pathways of fat metabolism. The actin cytoskeleton is a highly dynamic structure involved in the maintenance of cell morphology and structural stability . InsulinOpen chromatin regions can provide binding sites for TFs, thereby making initiation of target gene transcription possible. Therefore, integration of genome-wide chromatin accessibility and gene expression in specific biological processes can reveal the role that open chromatin plays in gene expression regulation. Chromatin accessibility has a significant correlation with gene expression during adipogenesis of human adipose-derived stem cells , bovine In the present study, differential chromatin accessibility was enriched in some crucial TFs, including FOSL2 (FOS-like antigen 2), JUND, FOS, and JUNB. FOSL2 and FOS are members of the FOS gene family. A study on the adipogenesis of deep intra-abdominal preadipocytes found that the induction of FOS protein was essential for preadipocyte differentiation into adipocytes . StudiesAll experimental procedures involved in this study were reviewed and confirmed by the Animal Administration and Ethics Committee of Lanzhou Institute of Husbandry and Pharmaceutical Sciences, Chinese Academy of Agricultural Sciences (SYXK-2014–0002).3 pieces in a sterile environment. Cells were isolated through type I collagenase (1 mg/mL) digestion with constant stirring for 60 min in a 37 °C in water bath. Subsequently, the digested tissue was sequentially filtered through 100 µm and 70 µm nylon mesh films, and the filtrate was centrifuged at 1400× g for 5 min. Cell pellets obtained through centrifugation were incubated with red blood cell lysis buffer  for 10 min. After washing with PBS, the mixture was centrifuged at 1400× g for 5 min to obtain the final cell pellets. Finally, the pellets were dissolved with DME-F12  containing 10% fetal bovine serum , inoculated into flasks, and cultured under conditions at 37 °C with 5% CO2.Three healthy 3-day-old infant Datong yaks from Datong Yak Breeding Center  were selected as experimental animals for this study. The collected subcutaneous adipose tissue samples were first rinsed with 0.9% NaCl and then with phosphate buffered saline (PBS) containing penicillin (200 U/mL) and streptomycin (200 U/mL). The adipose tissue was then cut into approximately 1 mmWhen the density of preadipocytes reached 70–80%, the cells were induced using adipogenic agents containing 3-isobutyl-methylxanthine (0.5 mM), dexamethasone (1 μM), and insulin (10 μg/mL)  for 2 days. The cells were then cultured with adipogenic agents including only insulin (10 μg/mL) until day 12. Finally, the adipocytes were washed three times with PBS, fixed with 4% formaldehyde crosslink for 1 h, and reacted with Oil Red O for 30 min. The stained samples were observed through light microscopy.® 96 Instrument  with the SYBR Green dye. Primer sequences used to determine the credibility of RNA-seq are listed in RNA extraction and reverse transcription of the cells were performed according to the manual of TriZol reagent  and PrimeScript™ 1st Strand cDNA Synthesis Kit , respectively. Quantitative RT-PCR was conducted on the LightCyclerWe performed ATAC-seq using preadipocytes (n = 3) and adipocytes (n = 3). Nuclear suspensions of samples were incubated in the transposition reaction mix containing Tn5 transposase at 37 °C for 30 min. When Tn5 entered the nuclei, open chromatin regions were preferentially fragmented. Meanwhile, an adapter was added to the ends of the fragments. Immediately following transposition, the sample was purified using the QIAGEN MinElute PCR purification kit  . The libTotal RNA of preadipocytes and adipocytes was isolated using the TriZol reagent kit in accordance with the manufacturer’s manual. RNA quality was verified through RNase-free agarose gel electrophoresis and evaluated on an Agilent 2100 Bioanalyzer . Then, mRNA was enriched using oligo(dT) beads. Subsequently, the enriched mRNA was cut into short fragments using fragmentation buffer and reverse transcribed into cDNA by utilizing NEBNext Ultra RNA Library Prep Kit for Illumina . The purified double-stranded cDNA fragments were end fixed, and a base was added. Then, the fragments were connected to Illumina sequencing adapters. The connection reaction was purified using AMPure XP beads (1.0×). Ligated fragments were subjected to size selection through polymerase chain reaction (PCR) amplification and agarose gel electrophoresis. The resulting cDNA library was sequenced using Illumina Novaseq6000 by Gene Denovo Biotechnology Co. .p < 0.05 and |log2FC| > 1. The MEME-ChIP and MEME-AME suite was used to identify the motifs. The ChIP and AME were selected to examine motifs with high credibility through peak regions and confirm the existences of any specific known motifs, respectively. Gene Ontology (GO) enrichment analysis was performed to recognize the main biological functions of peak-related genes. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was performed to identify metabolic pathways or signal transduction pathways associated with peak-related genes. GO terms and pathways were considered to be significantly enriched if they met the threshold of p < 0.05.To ensure the high quality of ATAC-seq reads, we conducted three stringent filtering standard procedures including removing reads with adapters, more than unknown nucleotides (N), and more than 50% of low-quality  bases. Filtered reads of each sample were mapped to LU_Bosgru_v3.0 (Ensembl_release104) by using Bowtie2  . We split all genes into three groups  by using a threshold value determined by dividing the gene activity value (RPKM) in three quantile groups based on their means (Hmisc::cut2 R function ). Then, t-test in SPSS  was used to evaluate statistics. The results were displayed as mean ± SEM, and p < 0.05 was defined as statistically significant.Student’s In summary, our study described genome-wide dynamic chromatin accessibility and gene expression during adipocyte differentiation in yaks, which are an ideal model for studying the adipogenesis of plateau species. Integration of ATAC-seq and mRNA-seq revealed that genes expressed at high levels were associated with high levels of chromatin accessibility, especially within 1 kb upstream and downstream of the TSS. Additionally, we identified a series of TFs and created the TF regulatory network, clarifying the possible interactions between TFs during yak adipogenesis. Taken together, our study sheds light on the mechanism by which open chromatin regulates gene expression during adipogenesis and provides the theoretical and material bases for research on its epigenetic roles in fat metabolism.


Nkx3.2 that is deeply conserved in most gnathostomes but undetectable in the jawless hagfish and lamprey. This enhancer is active in the developing jaw joint region of the zebrafish Danio rerio, and was thus designated as jaw joint regulatory sequence 1 (JRS1). We further show that JRS1 enhancer sequences from a range of gnathostome species, including a chondrichthyan and mammals, have the same activity in the jaw joint as the native zebrafish enhancer, indicating a high degree of functional conservation despite the divergence of cartilaginous and bony fish lineages or the transition of the primary jaw joint into the middle ear of mammals. Finally, we show that deletion of JRS1 from the zebrafish genome using CRISPR/Cas9 results in a significant reduction of early gene expression of nkx3.2 and leads to a transient jaw joint deformation and partial fusion. Emergence of this Nkx3.2 enhancer in early gnathostomes may have contributed to the origin and shaping of the articulating surfaces of vertebrate jaws.The acquisition of movable jaws was a major event during vertebrate evolution. The role of NK3 homeobox 2 (Nkx3.2) transcription factor in patterning the primary jaw joint of gnathostomes (jawed vertebrates) is well known, however knowledge about its regulatory mechanism is lacking. In this study, we report a proximal enhancer element of  The establishment of jaw joints was one of the major events that enabled the evolutionary transition from jawless to jawed vertebrates. The earliest known articulated jaws are found in fossil placoderms from the late Silurian period 423 MYA (million years ago)  The primNkx3.2 displays focal expression in the first pharyngeal arch, between Meckel’s cartilage/anguloarticular and the palatoquadrate/quadrate in non-mammalian vertebrates, as shown in zebrafish , which acts as a chondrocyte maturation inhibitor , is thouebrafish , Xenopus, proximal to the Nkx3.2 gene. We show that JRS1 has a highly conserved sequence and demonstrate that JRS1 sequences from multiple gnathostomes drive fluorescent reporter expression in the primary jaw joint of zebrafish, implying functional conservation between diverse clades. To test if JRS1 is essential for the jaw joint development, we generated a CRISPR/Cas9-induced enhancer deletion zebrafish line and show that homozygous mutants display transient dysmorphology and partial fusion of the jaw joint-articulating cartilages.In this study, we identified a novel gnathostome-specific cis-regulatory element, Nkx3.2, reasoning that if we find homologous genes upstream and downstream of Nkx3.2 in different vertebrate species, the locus is unlikely to have undergone major rearrangements that would have disrupted the intergenic non-coding sequences. Our synteny analysis showed that in almost all examined jawed vertebrate genomes, Nkx3.2 is located in a highly conserved syntenic region between Bod1l1 (Biorientation of chromosomes in cell division protein 1-like 1) and Rab28 (Ras-related protein Rab-28) genes (Bod1l1 and downstream gene Rab28 have the same orientation (located on the same DNA strand) as Nkx3.2. However, we could not identify Bod1l1 in the coelacanth genome or Rab28 in the zebrafish genome. On sea lamprey chromosome 11, Nkx3.2  is located upstream from Rab28 and next to Nkx2.6 (LOC116941469), which is located on the opposite strand. Bod1l1 (LOC116941243) is located more than 8.5 Mb upstream on chromosome 11 (Nkx3.2-like sequence (LOC116940711) located on sea lamprey chromosome 9. However, this region did not share synteny with the Nkx3.2-containing genomic region. In the hagfish genome, Nkx3.2 (ENSEBUG00000015739) is located on the contig FYBX02009947.1 next to Nkx2.5 (ENSEBUG00000006582), but on the opposite DNA strand. Rab28 (ENSEBUG00000007671) is located downstream from Nkx3.2, but there is an additional gene, annotated as Spondin 2  between them  but on the opposite strand. This region, however, also did not share synteny with the Nkx3.2-containing genomic region. These results indicated that the intergenic sequences flanking Nkx3.2 were likely to be homologous in examined vertebrates, and therefore were appropriate to align in search of CNEs. In jawless sea lamprey and hagfish we searched for CNEs around both Nkx3.2 and Nkx3.2-like sequences.In order to search for proximal conserved non-coding elements (CNEs), we first performed an analysis of the gene synteny around 8) genes . The upsosome 11 . BLAST seen them . BLAST sNkx3.2, between Nkx3.2 and Rab28, for all examined gnathostome species: human, mouse, koala, painted turtle, chicken, frog, coelacanth, zebrafish, spotted gar, bichir, and elephant shark  were extracted and a search for conserved motifs within these peak sequences was performed with MEME, identifying a core ~245 bp sequence , electric eel (Electrophorus electricus), Atlantic salmon , Atlantic cod (Gadus morhua), slimehead (Gephyroberyx darwinii), pineconefsh (Monocentris japonica), soldierfish (Myripristis murdjan), alfonsino (Beryx splendens), cusk-eel (Lucifuga dentata), toadfish , mudskipper , seahorse (Hippocampus comes), tilapia (Oreochromis niloticus), amazon molly (Poecilia formosa), stickleback (Gasterosteus aculeatus), and pufferfish (Tetraodon nigroviridis).In addition to the broad sampling of gnathostome species, we performed a more detailed analysis of teleost fish species beyond just the zebrafish. These additional teleost species included the arowana , and all non-acanthopterygian actinopterygians appears to be well conserved.Using a combination of mVISTA alignments and genome-wide BLASTN searches using the spotted gar and bichir as query sequences, we were able to identify JRS1 in all teleost species with the exception of most members of the clade Percomorpha: toadfish, mudskipper, soldierfish, seahorse, tilapia, amazon molly, stickleback, and pufferfish . The onlnkx3.2(JRS1):mCherry transgenic lines, which allowed further characterization of enhancer activity.To test JRS1 for enhancer activity in vivo, we generated Tol2 reporter constructs with species-specific JRS1 sequences from human, mouse, frog, zebrafish, bichir, and elephant shark upstream of a membrane-tagged mCherry coding sequence. Embryos injected with JRS1 reporter constructs displayed mosaic reporter gene expression within the first pharyngeal arch elements Meckel’s cartilage and palatoquadrate. At least two positive founders were identified for each injected construct and used for generation of stable nkx3.2 enhancer within the jaw joint-forming region of the first pharyngeal arch (nkx3.2(JRS1):mCherry line furthermore displayed mCherry-expressing cells in the notochord, which could be detected up to 3 days post fertilization (dpf)  we detected persistent mCherry expression driven by the eal arch , Video 1nkx3.2(JRS1):mCherry/sox10:egfp double transgenic fish were used for the characterization of joint progenitor cells from 2 dpf. The sox10:egfp line labels neural crest cell-derived populations comprising pharyngeal arch cartilages (nkx3.2(JRS1):mCherry and sox10:egfp in condensed mesenchyme cells in the jaw joint-establishing zone :mCherry-expressing cells were densely packed in the joint-forming region, overlapping with sox10:egfp expressing cells (nkx3.2(JRS1):mCherry-expressing cells were lined up from the posterior Meckel’s cartilage up to the anterior palatoquadrate.By 3 dpf ng cells  and dispng cells . Double-ng cells . At bothnkx3.2(JRS1):mCherry-expressing cells :mCherry-expressing cells in nkx3.2 gene mutants we crossed previously reported +/uu2803nkx3.2 mutant alleles (nkx3.2(JRS1):mCherry/sox10:egfp fish and incrossed these heterozygotes to generate homozygous uu2803/uu2803nkx3.2 mutants (abbreviated to nkx3.2−/−). Notably, nkx3.2(JRS1):mCherry-expressing cells were detected lining the outside of the cartilage in the fused jaw joint region of nkx3.2−/− mutants at 3–5 dpf :mCherry lines  and used Alcian blue and alizarin red staining to characterize the craniofacial morphology in comparison to heterozygotes and wild-type siblings at 5, 9, 14, and 30 dpf.As JRS1 appears functionally conserved and active specifically in the jaw joint, we next investigated its function more closely by using CRISPR/Cas9 genome editing to generate a zebrafish line with the entire JRS1 enhancer sequence deleted from the genome. Two sgRNAs were targeted to sequences either side of JRS1 resulting in a 445 bp deletion spanning the conserved core of the JRS1 and ~100 bp flanking sequences . The resnkx3.2ΔJRS1/ΔJRS1 mutants tended to display a reduced RAP on Meckel’s cartilage and the cartilage of the palatoquadrate often failed to form a pronounced convex joint process  and 47% (p = 0.0002) reduction in urements . The relat 6 dpf .nkx3.2+/+ (N = 12), nkx3.2+/ΔJRS1 (N = 10), and nkx3.2ΔJRS1/ΔJRS1 (N = 8), were imaged using optical projection tomography (OPT), and the whole heads  in a number of gnathostome species. Next, we applied Tol2-mediated transgenesis to test JRS1 sequences from human, mouse, frog, zebrafish, bichir, and elephant shark for enhancer activity in zebrafish. The zebrafish nkx3.2(JRS1):mCherry line labelled areas corresponding to the endogenous expression of nkx3.2 in both the first pharyngeal arch and the early notochord (nkx3.2(JRS1):mCherry expression overlapped with sox10:egfp-positive neural crest derived cells at early stages, consistent with the neural crest origin of jaw joint-establishing cells :mCherry- and sox10:egfp-positive cells in nkx3.2−/− mutants that displayed the fused jaw cartilages as early as 3 dpf, in accordance with previous report by nkx3.2(JRS1):mCherry-expressing cells lined the outside of the fused jaw joint cartilages, indicating that some joint or intermediate domain markers may still be present in the fusion region. This is reminiscent of the jaw joint fusion caused by the dlx3b;4b;5a-MO injection described by trps1, normally expressed in the articular cartilage of the wild-type jaw joint, was detected in cells surrounding the fusion in the morphants. Despite these cell patterning changes, nkx3.2(JRS1):mCherry-positive cells at 5 dpf displayed a similar accumulation of fluorescence reporter protein in the cell membrane of nkx3.2−/− mutants as in wild types, indicating the absence of cell identity changes.Notably, there was no apparent overlap of nkx3.2(JRS1):mCherry expression at 3 dpf was found in cells displaying this characteristic interzone cell morphology. nkx3.2(JRS1):mCherry expression in the mandibular arch was consistent over time, displaying a strong fluorescence signal even at 14 dpf. The scattered membranous mCherry expression we could observe from 5 dpf onwards could be a consequence of the increasing extracellular matrix production by the interzone cells, necessary to facilitate the joint cavitation process :mCherry transgenic line. These data suggest that the general landscape of transcription factors binding to JRS1 is most probably conserved between gnathostomes, suggesting a highly conserved role for this cis-regulatory element. This is also supported by our prediction of transcription factor-binding motifs broadly shared between species that are associated with functions in the pharyngeal arch patterning and skeletogenesis  joint cartilage  pharyngeal arch, consistent with other gnathostomes. In the related elasmobranch chondrichthyans, gene expression of l arches . If we achthyans . On the chthyans . In thisJRS1 appears to be absent in all analysed percomorph (PERC) teleost fish with the exception of the cusk-eel, and only a small conserved sequence fragment is present in the cusk-eel, alfonsino, and soldierfish – acanthopterygian fish from the orders Ophidiiformes (O) and Beryciformes (B) that narrowly post- and pre-date the phylogenetic origin of percomorph fish, respectively . This sunkx3.2 loci, we were unable to find a JRS1-like conserved sequence within this clade, which is more consistent with a total deletion or degeneration of JRS1 in the last common ancestor of PERC+B. We cannot rule out the presence of novel or pre-existing shadow enhancers in this clade that may have taken over the role of JRS1 in regulating nkx3.2, as our search for conserved sequences was limited to the bod1l1-nkx3.2-rab28 locus while other enhancers may be present in more distant intergenic regions . Synteny data including upstream and downstream genes from Nkx3.2 were extracted from the Ensembl and NCBI databases for several genomes: human, Homo sapiens (GRCh38.p12); mouse, Mus musculus (GRCm38.p6); koala, Phascolarctos cinereus (phaCin_unsw_v4.1); painted turtle, Chrysemys picta (Chrysemys_picta_bellii-3.0.3); chicken, Gallus gallus (GRCg6a); tropical clawed frog, Xenopus tropicalis ; coelacanth, Latimeria chalumnae (LatCha1); zebrafish, Danio rerio (GRCz11); spotted gar, Lepisosteus oculatus (LepOcu1); elephant shark, Callorhinchus milii ; inshore hagfish, Eptatretus burgeri (Eburgeri_3.2); and sea lamprey, Petromyzon marinus (kPetMar1). Nkx3.2 sequences were identified in hagfish and lamprey genomes with TBLASTN searches using the protein sequence of the spotted gar Nkx3.2 (ENSLOCG00000009892) as a query. A genomic scaffold containing the Nkx3.2 locus was obtained from genomic databases of the bichir, Polypterus senegalus  were collected for a number of vertebrate species and submitted to mVISTA ; electric eel, Electrophorus electricus (Ee_SOAP_WITH_SSPACE); Atlantic salmon, Salmo salar (ICSASG_v2); Atlantic cod, Gadus morhua (gadMor3.0); Darwin’s slimehead, Gephyroberyx darwinii (GCA_900660455.1); pineconefish, Monocentris japonica (ASM90032336v1); pinecone soldierfish, Myripristis murdjan (fMyrMur1.1); splendid alfonsino, Beryx splendens (ASM90031256v1); toothed Cuban cusk-eel, Lucifuga dentata (Ldentata1.0); Amazon toadfish, Thalassophryne amazonica (fThaAma1.1); Korean mudskipper, Periophthalmus magnuspinnatus (fPerMag.1.pri); tiger-tail seahorse, Hippocampus comes (H_comes_QL1_v1); Nile tilapia, Oreochromis niloticus (O_niloticus_UMD_NMBU); Amazon molly, Poecilia formosa (Poecilia_formosa-5.1.2); three-spined stickleback, Gasterosteus aculeatus (BROAD S1); and green spotted pufferfish, Tetraodon nigroviridis (TETRAODON 8.0). For Darwin’s slimehead, the nkx3.2 coding sequence was not included in the relevant contig, as it terminated at the 5′ boundary of the 3′ UTR. mVISTA analysis was performed as described above, and additional BLASTN searches were performed with the following parameters: match/mismatch scores: 1, −1; gap costs: 3, 2; seed word size: 11.To search for JRS1 in a range of teleost species, additional sequences were extracted from the Ensembl and NCBI Genome databases, representing the from human H. sapiens, mouse M. musculus, frog X. tropicalis, zebrafish D. rerio, bichir P. senegalus, and elephant shark C. milii was used to amplify identified JRS1 sequences. Forward primers contained four guanine residues at the 5′ end followed by attB4 (5′-ACAACTTTGTATAGAAAAGTT-3′) attachment sites and finally the species-specific template sequence: human 5′-GTCACACAGCTTGGAATTGGTG-3′, mouse 5′-AGTTTTACAGGTTCCTAGCCCATAC-3′, frog 5′-TCTGAACTGTTTTGCCCACATT-3′, zebrafish 5′-AGACGTGATGCTGTGACACGCTAACTGCTG-3′, bichir 5′-GAACCGAGTGCTTTACAATTAGGTA-3′, elephant shark 5′-GAATGGAGTCACACGATAGTAATCC-3′. Reverse primers contained four guanine residues at the 5′ end, attB1r (5′-ACTGCTTTTTTGTACAAACTTG-3′) attachment sites, and the adenovirus E1b minimal promoter sequence followed by species-specific template sequences: human 5′- AAGTGGTTCAAAGGCTAAAGTT-3′, mouse 5′-CCTCATTGCTCCACCTCTCT-3′, frog 5′-ACATTGGCACTGACAGGTAAAC-3′, zebrafish 5′-GATTTACATTTTGACGTCAAT-3′, bichir 5′- TTTCGAAATATTTGATACCGACAGT-3′, elephant shark 5′-AAAGTGCATTGTGAACAAATAGACA-3′. PCR products were subsequently recombined into pDONR P4-P1R donor vectors by the BP reaction according to the MultiSite Gateway cloning protocol (Genomic DNA protocol . The LR D. rerio) embryos were microinjected with freshly mixed injection solution containing the expression clone plasmid (175 ng/μl) and transposase mRNA (125 ng/μl) according to nkx3.2(JRS1):mCherry expression. F0 embryos displaying the strongest mosaic nkx3.2(JRS1):mCherry expression were selected and raised to sexual maturity (90 dpf). Mature adults were outcrossed with AB fish. Positive Tg(nkx3.2(JRS1):mCherry) F1 embryos were raised to establish stable transgenic lines. For generating double transgenic zebrafish, the Tg(nkx3.2(JRS1):mCherry) line was crossed with previously published zebrafish lines Tg(fli1a:egfp) (sox10:egfp) (One-cell stage zebrafish (1a:egfp)  or Tg(so10:egfp) .nkx3.2 gene mutant zebrafish, the previously reported uu2803 null allele (nkx3.2(JRS1):mCherry/sox10:egfp and further incrossed to produce −/−nkx3.2 fish.For comparison between wild-type and homozygous l allele  was crosConfocal microscopy was performed on an inverted Leica TCS SP5 microscope using Leica Microsystem LAS-AF software. Embryos were sedated with 0.16% MS-222 and embedded in 0.8% low-melting agarose on the glass bottom of the 35 mm dishes. To prevent drying, embedded embryos were covered with system water containing 0.16% MS-222. Images are presented as single images or maximum projections, as specified.nkx3.2 JRS1 enhancer were selected with the use of CRISPOR online design tool  fish  using the same PCR primers.Injected F0 fish were raised to adulthood and individually outcrossed with AB zebrafish. Clutches of 5 dpf larvae were genotyped to determine the presence of the enhancer deletion in the germline of F0 parents. Three batches of 8–12 randomly selected larvae were sacrificed and lysed in a solution of 150 µl 50 µM NaOH for 20 min at 95°C. The lysis solution was subsequently stabilized by adding 100 µl 0.1 mM Tris. 1 µl of the resulting lysis solution was added into a 25 µl PCR reaction containing Platinum Taq DNA Polymerase and the two primers flanking the JRS1 deletion site, and was run for 35 cycles. Resulting PCR products were run on 1% agarose gels to determine their size. The PCR product representing the JRS1 deletion allele was 352 bp in length, and easily distinguishable from 790 bp wild-type allele. Four deletion-positive F0 zebrafish were outcrossed with Tg(FP) fish  to produZebrafish larvae and juveniles were stained with Alcian blue and alizarin red following a protocol modified from A custom-built OPT  system wN = 12), nkx3.2+/ΔJRS1 (N = 10), and nkx3.2ΔJRS1/ΔJRS1 (N = 8) zebrafish were then aligned to the reference.The registration workflow was similar to the methods described by nkx3.2 gene sequence forward 5′-CTTCAACCACCAGCGTTATCTC-3′ and reverse 5′-ACATGTCTAGTAAACGGGCGA-3′. Fragments were cloned from zebrafish cDNA into the pCR II TOPO vector and antisense RNA probes were synthesized with either SP6 or T7 RNA polymerase and digoxigenin labelling mix (Roche). In situ hybridization on zebrafish whole-mount 48 and 56 hpf embryos was performed as previously described  versus nkx3.2ΔJRS1/ΔJRS1  groups was analysed with unpaired t-tests.Primers were designed for the zebrafish escribed . Genotyprpl13a forward 5′-TCTGGAGGACTGTAAGAGGTATGC-3′; rpl13a reverse 5′-AGACGCACAATCTTGAGAGCAG-3′; nkx3.2 forward 5′-ACTGCGTGTCCGACTGTAACAC-3′; nkx3.2 reverse 5′-GTCTCGGTGAGTTTGAGGGA-3′. Amplicon sizes for these target genes were 148 and 187 bp, respectively. A dissociation step was performed at the end of the analysis to verify the specificity of the products, and standard curves were generated from pooled cDNA from all samples in triplicate for each target gene to verify the efficiency of the primers (R2 > 0.99). nkx3.2 expression was normalized to rpl13a levels and relative quantification of gene expression was calculated using the 6 dpf larvae produced from incrossing heterozygous parents were euthanized with an overdose of MS-222 (300 mg/l) and the tails were removed for genotyping while the head and remaining body were stored in RNALater (Invitrogen). Total RNA was extracted with Trizol reagent (Fisher) from a pool of seven larvae of each genotype, with three biological replicates per genotype. To prevent genomic DNA contamination, extractions were DNase-treated using the Turbo DNA-free kit (Ambion). cDNA was synthesized from 200 ng total RNA from each sample using the SuperScript IV First-Strand cDNA Synthesis kit (Invitrogen) with random hexamers in a 20 µl reaction. qPCR was performed on all samples in technical triplicates with PowerUp SYBR Green Master Mix using a 7500 Real Time PCR System (Applied Biosystems) and the following primers: We employed 2D geometric morphometric analysis using 100 landmarks defined along the edge of Meckel’s cartilage at the jaw joint interface. From maximum projection images generated by OPT, standardized with a lateral orientation, the shapes of Meckel’s cartilage were traced in Adobe Illustrator 2020. These shapes were then aligned by orientation and the joint interfacing surface, but not resized, before most of the anterior portion of the shapes were cut away to a standardized extent, leaving shapes representing just the posterior head of Meckel’s cartilage. These shapes were digitized using 100 equidistant landmarks using tpsDig2 (v2.3) , and impGeneralized procrustes analysis was used to align coordinates of the landmarks for subsequent morphospace analysis using the geomorph package (v3.3.2)  and biva In this elegant and important study, Leyhr et al. identify the first potent nkx3.2 jaw joint enhancer, which they show to be deeply conserved across gnathostomes and absent from jawless fishes. The data are convincing and beautifully presented, supporting the hypothesis that this enhancer arose with the origin of hinged jaws during vertebrate evolution and is required for some aspects of early joint development in zebrafish. The work has important implications both for our basic understanding of enhancer function and evolution as well as potential genetic causes of craniofacial defects in humans. public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: (i) Decision letter after peer review:Nkx3.2 contributes to early jaw joint morphology in zebrafish" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Marianne Bronner as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Chrissy Hammond (Reviewer #1); Thomas F Schilling (Reviewer #3).Thank you for submitting your article "An evolutionarily conserved cis-regulatory element of The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:(1) Determine changes in nkx3.2 expression in the enhancer-less mutants, including characterisation of any tissue-specific expression changes using in situ hybridisation.(2) Check the available lamprey genome assemblies for presence of a JRS-like sequence. This is essential before making strong conclusions about its absence in agnathans.(3) Validate one or more of the candidate transcription factors binding to JRS1 using ChIP, or, if ChIP-type tools are not available, move the speculative section on transcription factor binding sites and associated literature on their function from the Results to the Discussion.The full reviews are appended below for your information.Reviewer #1 (Recommendations for the authors):The work throughout is well performed and the imaging is beautiful. The main points are well made and well evidenced.The section on matching of the JRS1 to transcription factors doesn't flow as well as the rest of the manuscript. It would have been nice to see validation of that one or more of the TFs discussed binding to the JRS using CHiP.Sox9/10 positivity to Col2+ and potentially to Col10 in the fish in which the JRS1 is deleted. While there are transgenics for these, there are also decent antibodies that work in zebrafish (see for example https://faseb.onlinelibrary.wiley.com/doi/full/10.1096/fj.202101167R) which would allow relatively rapid assessment.As the authors introduce the idea of OA (in relation to the factors that bind the JRS1)and as nkx3.2 is known to inhibit chondrocyte maturation in addition to the Alcian blue/AR and joint shape ideally one might show maturation state of chondrocytes using progression from Reviewer #2 (Recommendations for the authors):While this study is clear and well designed, it could be meaningfully strengthened by making several modifications to the text and performing a few additional analyses, none of which would require additional models to be generated.1. The authors note that they were unable to detect the JRS1 sequence in the hagfish genome, but do not comment about lamprey. This is worth checking, as there are several builds of the lamprey genome available. I can find an annotated "nkx3-2" gene in the SIMRbase genome  though nothing very promising on the UCSC genome browser.2. The authors should further probe and discuss the role of Hox genes in regulating Nkx3.2 expression. Notably, a previous study in zebrafish established that nkx3.2 is suppressed in the second pharyngeal arch by Hox activity . Specifically, moz mutants lacking Hox expression in this arch and hoxa2b/b2a morphants both showed ectopic second arch nkx3.2 expression. I was thus intrigued to note that the authors identified a predicted Meis2 – though not a Hox – binding motif in the second conserved motif of the JRS1 enhancer . Meis proteins are of course known for being key Hox cofactors, along with Pbx proteins. To check whether a stronger connection between the JRS1 enhancer and Hox regulation could be made using existing data, I quickly consulted ChIP-seq datasets for Hoxa2, Meis, and Pbx in mouse pharyngeal arch tissue that were published by Donaldson et al. (2012) and Amin et al. (2015). Distinct peaks and summits for all three  were present directly over the mouse JRS1 sequence. This suggests that the same element that activates nkx3.2 expression in the first arch may be directly repressed in the second arch by a Hox-Pbx-Meis complex.3. This notion may also be relevant for the authors' discussion of the chondrichthyan element (lines 468-476) – is the Meis motif intact in the elephant shark sequence?4. It is intriguing that the JRS1 enhancer was not detected in Acanthopterygii species. The authors note that it may have been translocated to a different genomic location rather than completely lost – to test this, they should perform unbiased Ensembl BLAST searches of these fish genomes using, e.g., the spotted gar sequence as bait, outside the original locus. If they do not find any evidence in favor of translocation, they could comment on whether there are any potential conserved shadow enhancers that could have taken over regulation of nkx3.2 expression in these species. I appreciate that they note that fish in this group show unusual upper jaw protrusability, which may be a consequence of altered selective pressures on the jaw joint.5. The authors detected a conserved Hey1 binding motif in the JRS1 enhancer and noted that hey1 was reported to be expressed downstream of Notch activity in the dorsal domain of the zebrafish pharyngeal arches . They should rephrase this comment to note that those previous authors did indeed find that nkx3.2 expression was dorsally expanded in Notch loss-of-function mutants, supporting the notion that Notch activity, possibly via Hey1, is suppressing its expression dorsally.6. The finding that JRS1:mCherry expression was still expressed in the fused jaw joint of nkx3.2-/- fish argues against Nkx3.2 playing a significant role in autoregulation of its own expression through the JRS1 enhancer (as proposed in lines 176-177). They should consider noting this in the discussion.7. Deletion of the enhancer caused a mild phenotype that resolved with age. This finding, together with the fact that they saw no difference in nkx3.2 transcript levels at 6 dpf, suggests that shadow enhancers may activate later and compensate for early loss of expression. However, the qPCR experiment appears to have been done on whole larvae, meaning that normal levels of nkx3.2 in other parts of the body could have masked an effect specific to the jaw joint. To resolve this, the authors should assess nkx3.2 expression in the jaw region of the JRS1 mutants by in situ hybridization, which, although not quantitative, is sensitive enough to reveal differences in local expression levels. In addition , the authors should perform the qPCR at an earlier stage of development to test for diminished expression at the stage when the joint is first being specified. Tail biopsy-based genotyping of zebrafish larvae can be done as early as 24 hpf.Reviewer #3 (Recommendations for the authors):(1) Look further into changes in Nkx3.2 expression with deletion of the JRS in zebrafish. While there appear to be no statistically significant changes at 6 days postfertilization (6 dpf) homozygotes show slightly reduced expression. Has this experiment been done at 2-5 dpf when jaw joint development is initiated? If so and no changes were detected this might suggest that either loss of the JRS disrupts spatial patterns of Nkx3.2 expression, which the authors should test with in situ hybridization, or the enhancer may act on other nearby genes.(2) Quantify variation in the jaw joint phenotype in the homozygous JRS mutants.(3) Check the available lamprey genomes for presence of a JRS-like sequence. If absent this would bolster the argument for an involvement of the JRS in the origin of jaws during vertebrate evolution.(4) Move the long list of transcription factor binding sites in the JRS and literature describing their functions that is currently included in the Results into the Discussion. Essential revisions:(1) Determine changes in nkx3.2 expression in the enhancer-less mutants, including characterisation of any tissue-specific expression changes using in situ hybridisation.nkx3.2 expression changes at 48 and 56 hpf to the manuscript, demonstrating that the loss of JRS1 severely reduces the early expression of nkx3.2 We have added an analysis of jaw joint-specific (2) Check the available lamprey genome assemblies for presence of a JRS-like sequence. This is essential before making strong conclusions about its absence in agnathans.We have repeated our searches for a JRS1-like sequence in both hagfish and the sea lamprey genomes and can now make more strong claims about its absence in agnathans .(3) Validate one or more of the candidate transcription factors binding to JRS1 using ChIP, or, if ChIP-type tools are not available, move the speculative section on transcription factor binding sites and associated literature on their function from the Results to the Discussion.We have been able to validate Meis binding at JRS1 by accessing the publicly available datasets generated as part of two previous publications, in addition to moving the detailed description of all of the predicted binding sites and their associated literature to the Discussion section as suggested .The full reviews are appended below for your information.Reviewer #1 (Recommendations for the authors):The work throughout is well performed and the imaging is beautiful. The main points are well made and well evidenced.We thank the reviewer for the comments.The section on matching of the JRS1 to transcription factors doesn't flow as well as the rest of the manuscript. It would have been nice to see validation of that one or more of the TFs discussed binding to the JRS using CHiP.We have moved the text describing the predicted transcription factor binding sites to the discussion, and providing a briefer description in the Results section to improve the flow. Reviewer 3 directed us to existing ChIP-seq datasets in E11.5 mouse embryonic branchial arches showing the strong presence of Meis bound to JRS1, validating the predicted Meis2 binding. This validation is described in the Results section (lines 185-192) and has been added to the discussion (lines 626-655).As the authors introduce the idea of OA (in relation to the factors that bind the JRS1)and as nkx3.2 is known to inhibit chondrocyte maturation in addition to the Alcian blue/AR and joint shape ideally one might show maturation state of chondrocytes using progression from Sox9/10 positivity to Col2+ and potentially to Col10 in the fish in which the JRS1 is deleted. While there are transgenics for these, there are also decent antibodies that work in zebrafish (see for example https://faseb.onlinelibrary.wiley.com/doi/full/10.1096/fj.202101167R) which would allow relatively rapid assessment.nkx3.2 expression with phenotypic effects. Future studies may address precisely how different nkx3.2 expression levels influence jaw joint morphology.We thank the reviewer for raising this suggestion and we agree that it would certainly be interesting to determine how the chondrocyte maturation state relates to the observed phenotypes, but feel this goes beyond the scope of the current paper which is primarily aimed at characterising JRS1 and demonstrating that it influences Reviewer #2 (Recommendations for the authors):While this study is clear and well designed, it could be meaningfully strengthened by making several modifications to the text and performing a few additional analyses, none of which would require additional models to be generated.1. The authors note that they were unable to detect the JRS1 sequence in the hagfish genome, but do not comment about lamprey. This is worth checking, as there are several builds of the lamprey genome available. I can find an annotated "nkx3-2" gene in the SIMRbase genome  though nothing very promising on the UCSC genome browser.Nkx3.2 gene loci, and identified in lamprey a Nkx3.2 locus with some conservation in synteny to hagfish and the gnathostomes, in addition to an Nkx3.2-like gene in both lamprey and hagfish with different syntenic surroundings. We performed the mVISTA analysis on all four Nkx3.2/Nkx3.2-like loci from hagfish and lamprey, and were unable to identify any JRS1-like sequences proximal to any of them. This analysis is described in the updated Results section (lines 123-146 and 161-163), and we have updated Figure 1 and Figure 2A to show the results from the syntenic Nkx3.2 loci only. We decided not to focus on the nonsyntenic Nkx3.2-like loci as they likely represent Nkx-family paralogues, which are challenging to robustly annotate or describe in the absence of a comprehensive study of the diversity of the gene family in vertebrates.We agree, and thank the reviewer for raising this point. To be as rigorous as possible, we have repeated the synteny analysis of both the hagfish and sea lamprey genomes to search for 2. The authors should further probe and discuss the role of Hox genes in regulating Nkx3.2 expression. Notably, a previous study in zebrafish established that nkx3.2 is suppressed in the second pharyngeal arch by Hox activity . Specifically, moz mutants lacking Hox expression in this arch and hoxa2b/b2a morphants both showed ectopic second arch nkx3.2 expression. I was thus intrigued to note that the authors identified a predicted Meis2 – though not a Hox – binding motif in the second conserved motif of the JRS1 enhancer . Meis proteins are of course known for being key Hox cofactors, along with Pbx proteins. To check whether a stronger connection between the JRS1 enhancer and Hox regulation could be made using existing data, I quickly consulted ChIP-seq datasets for Hoxa2, Meis, and Pbx in mouse pharyngeal arch tissue that were published by Donaldson et al. (2012) and Amin et al. (2015). Distinct peaks and summits for all three  were present directly over the mouse JRS1 sequence. This suggests that the same element that activates nkx3.2 expression in the first arch may be directly repressed in the second arch by a Hox-Pbx-Meis complex.We thank the reviewer for this suggestion and for checking the datasets themselves for the relevant binding. We have added a summary of the Meis/Hoxa2/Pbx binding data from Donaldson et al. (2012) and Amin et al. (2015) as new figure supplement: Figure 2 —figure supplement 2. These data and their implications are now described in lines 185-192 of the results and in lines 626-655 of the discussion.3. This notion may also be relevant for the authors' discussion of the chondrichthyan element (lines 468-476) – is the Meis motif intact in the elephant shark sequence?The predicted Meis2 binding motif is indeed intact in the elephant shark sequence (and in other chondrichthyans), and we have added this fact to the discussion (lines 738-743).4. It is intriguing that the JRS1 enhancer was not detected in Acanthopterygii species. The authors note that it may have been translocated to a different genomic location rather than completely lost – to test this, they should perform unbiased Ensembl BLAST searches of these fish genomes using, e.g., the spotted gar sequence as bait, outside the original locus. If they do not find any evidence in favor of translocation, they could comment on whether there are any potential conserved shadow enhancers that could have taken over regulation of nkx3.2 expression in these species. I appreciate that they note that fish in this group show unusual upper jaw protrusability, which may be a consequence of altered selective pressures on the jaw joint.We agree that it would be useful to more rigorously test this conjecture, and we have now performed the suggested BLAST searches. We did this using both the spotted gar and bichir JRS1 sequences as queries against the whole genomes of the teleost species, including additional species from a more complete sampling of clades near the origin of Acanthopterygii. The methods section was updated accordingly, and the results are described in lines 223-250. We supported the text with a new Figure 3 depicting both the BLASTN search results  and the new summary of how JRS1 seems to have been lost. In the first version of the manuscript we proposed that JRS1 had been lost or translocated at the base of Acanthopterygii, but now with the inclusion of additional species and a more robust analysis, we show that JRS1 began to be partially lost/diverged after the divergence of Trachichthyiformes with the remainder of the Acanthopterygian clades, and that JRS1 is only completely lost  near the base of Percomorpha, after Ophidiiformes diverged. The Discussion section has been updated accordingly (lines 745-771).We added a comment on the presence of additional shadow enhancers in lines 771-775.5. The authors detected a conserved Hey1 binding motif in the JRS1 enhancer and noted that hey1 was reported to be expressed downstream of Notch activity in the dorsal domain of the zebrafish pharyngeal arches . They should rephrase this comment to note that those previous authors did indeed find that nkx3.2 expression was dorsally expanded in Notch loss-of-function mutants, supporting the notion that Notch activity, possibly via Hey1, is suppressing its expression dorsally.We have rephrased this comment to add this important note (lines 659-666).6. The finding that JRS1:mCherry expression was still expressed in the fused jaw joint of nkx3.2-/- fish argues against Nkx3.2 playing a significant role in autoregulation of its own expression through the JRS1 enhancer (as proposed in lines 176-177). They should consider noting this in the discussion.We agree, and have added this caveat (lines 700-705).7. Deletion of the enhancer caused a mild phenotype that resolved with age. This finding, together with the fact that they saw no difference in nkx3.2 transcript levels at 6 dpf, suggests that shadow enhancers may activate later and compensate for early loss of expression. However, the qPCR experiment appears to have been done on whole larvae, meaning that normal levels of nkx3.2 in other parts of the body could have masked an effect specific to the jaw joint. To resolve this, the authors should assess nkx3.2 expression in the jaw region of the JRS1 mutants by in situ hybridization, which, although not quantitative, is sensitive enough to reveal differences in local expression levels. In addition , the authors should perform the qPCR at an earlier stage of development to test for diminished expression at the stage when the joint is first being specified. Tail biopsy-based genotyping of zebrafish larvae can be done as early as 24 hpf.nkx3.2 gene expression differences is required to support our conclusions. To this end, we performed in situ hybridisation to assess nkx3.2 gene expression in the jaw joints of 48 and 56 hpf embryos. Following the protocol described by Dobrzycki et al. (2020), we were able to quantify the intensity of ISH staining in the jaw joints and show that homozygous JRS1 deletion mutants displayed a ~45% reduction in staining intensity, demonstrating that nkx3.2 expression was severely reduced in the absence of JRS1. These results have been added to Figure 6 in panels K-O, described in lines 471-473, and discussed in lines 795-804. The methods section at lines 1080-1097 has also be updated accordingly. We are still unable to distinguish whether the absence of nkx3.2 expression differences between genotypes at 6 dpf is the result of other enhancers “taking over”, or that extra-jaw-joint expression swamps the signal, and have added this caveat clearly at lines 815-822.We agree that further quantification of Reviewer #3 (Recommendations for the authors):(1) Look further into changes in Nkx3.2 expression with deletion of the JRS in zebrafish. While there appear to be no statistically significant changes at 6 days postfertilization (6 dpf) homozygotes show slightly reduced expression. Has this experiment been done at 2-5 dpf when jaw joint development is initiated? If so and no changes were detected this might suggest that either loss of the JRS disrupts spatial patterns of Nkx3.2 expression, which the authors should test with in situ hybridization, or the enhancer may act on other nearby genes.nkx3.2 gene expression differences is required to support our conclusions. To this end, we performed in situ hybridisation to assess nkx3.2 gene expression in the jaw joints of 48 and 56 hpf embryos. Following the protocol described by Dobrzycki et al. (2020a), we were able to quantify the intensity of ISH staining in the jaw joints and show that homozygous JRS1 deletion mutants displayed a ~45% reduction in staining intensity, demonstrating that nkx3.2 expression was severely reduced in the absence of JRS1. These results have been added to Figure 6 in panels K-O, described in lines 471-473, and discussed in lines 795-804. The methods section at lines 1080-1097 has also be updated accordingly. We are still unable to distinguish whether the absence of nkx3.2 expression differences between genotypes at 6 dpf is the result of other enhancers “taking over”, or that extra-jaw-joint expression swamps the signal, and have added this caveat clearly at lines 815-822.We agree that further quantification of (2) Quantify variation in the jaw joint phenotype in the homozygous JRS mutants.The most rigorous presentation of the variation in jaw joint phenotype is present in Figure 7A, but we have added numbers to Figure 6C, D, F, G, I, J to quantify how representative the images are of the mutant phenotypes.(3) Check the available lamprey genomes for presence of a JRS-like sequence. If absent this would bolster the argument for an involvement of the JRS in the origin of jaws during vertebrate evolution.Nkx3.2 gene loci, and identified in lamprey a Nkx3.2 locus with some conservation in synteny to hagfish and the gnathostomes, in addition to an Nkx3.2-like gene in both lamprey and hagfish with different syntenic surroundings. We performed the mVISTA analysis on all 4 Nkx3.2/Nkx3.2-like loci from hagfish and lamprey, and were unable to identify any JRS1like sequences proximal to any of them. This analysis is described in the updated Results section (lines 123-146 and 161-163), and we have updated Figure 1 and Figure 2A to show the results from the syntenic Nkx3.2 loci only. We decided not to focus on the non-syntenic Nkx3.2-like loci as they likely represent Nkx-family paralogues which are challenging to robustly annotate or describe in the absence of a comprehensive study of the diversity of the gene family in vertebrates.We agree, and thank the reviewer for raising this point. To be as rigorous as possible, we have repeated the synteny analysis of both the hagfish and sea lamprey genomes to search for (4) Move the long list of transcription factor binding sites in the JRS and literature describing their functions that is currently included in the Results into the Discussion.We have moved this text into the discussion.


Recent studies have shown that different epigenetic factors affect the transcription of auxin biosynthetic genes. In this review, we focus our attention on the molecular mechanisms through which epigenetic modifications regulate auxin biosynthesis.Auxin is one of the most important plant growth regulators of plant morphogenesis and response to environmental stimuli. Although the biosynthesis pathway of auxin has been elucidated, the mechanisms regulating auxin biosynthesis remain poorly understood. The transcription of auxin biosynthetic genes is precisely regulated by complex signaling pathways. When the genes are expressed, epigenetic modifications guide  As an important plant growth regulator, auxin plays a central role in regulating the growth and development of plants  is synthesized through two major pathways: the Tryptophan-Independent (TI) pathway and the Tryptophan-Dependent (TD) pathway /ANTHRANILATE SYNTHASE ALPHA-SUBUNIT 1 (ASA1) and WEI7/ANTHRANILATE SYNTHASE BETA-SUBUNIT 1 (ASB1)  family proteins have overlapping functions and catalyze the conversion of Trp to IPyA  . Surrounding the nucleosome is the junction histone H1, which compresses the nucleosome into higher-order structures to form chromatin fibers , and TAA1/TARs , which is an important epigenetic regulator of developmental processes ], Suppressor of zeste 12 [Su(z)12], Extra sex combs (Esc), and the histone-binding Nurf55 (nucleosome remodeling factor 55 kDa) , Homolog of Su(z)12, CURLY LEAF (CLF), Homolog of E(z), FERTILIZATION INDEPENDENT ENDOSPERM (FIE), Homolog of Esc, and MULTIPLE SUPPRESSOR OF IRA 1 (MSI1), Homolog of Nurf55]  in the Arabidopsis genome  was expressed at the floral meristem-organ boundaries to define flower patterning. The loss function of SUP leads to higher levels of auxin in these boundaries, resulting in flowers with supernumerary stamens. As shown in YUC1 and YUC4 promoters through the C2H2 zinc-finger domain, SUP recruits the PCR2 complex by interacting with the CLF subunit and LHP1 to form the SUP-LHP1-PRC2 complex, which represses YUC1 and YUC4 expression by trimethylation modification of histone H3 at K27 , which is a unique homolog of YUC1, YUC3, YUC7, YUC8, YUC9, and YUC11 via specifically demethylating H3K27me3 and H3K27me2 in Arabidopsis  marks activates YUC8 transcription by inhibiting the expression of some negative regulators  proteins have been identified to function as Histone DeMethylases (HDMs) in mammalian and plant cells  is involved in chromatin silencing by promoting histone demethylation  and EML3 in the Tudor/Agent family repress Since histone acetylation was first reported  cis-element of the YUC8 promoter , have been shown to mediate high-temperature-associated plant growth , which inhibits the transcription of YUC4  and biosynthetic gene YUC6 were regulated by the subunit SWI3B of the chromatin remodeling complex SWI/SNF  is another important epigenetic modification related to gene transcriptional activation  that was linked to the loss of function of the gene HUB1 , indicating that H2Bub1 is required for the transcriptional regulation of these genes  and two homologous HUB2s , which are directed by 24-nt small interfering RNAs (siRNAs), and methylates all three sequence contexts via the RNA-directed DNA methylation (RdDM) pathway   were targeted by RdDM. This means that DNA methylation is a very important yet poorly understood mechanism regulating auxin biosynthesis, marking methylation as an interesting subject that requires further study in plants.It has also been reported that the auxin biosynthetic genes TAA gene family, which participates in the main auxin biosynthesis pathway. Moreover, multiple histone modifications act in a combinatorial fashion to specify distinct chromatin states , the Fundamental Research Funds for the Central Universities (lzujbky-2020-33 and lzujbky-2021-kb05), and the Natural Science Foundation of Gansu Province (20JR5RA270).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.