Histone modifying enzymes play critical roles in many key cellular processes and are appealing proteins for targeting by small molecules in disease. However, while the functions of histone modifying enzymes are often linked to epigenetic regulation of the genome, an emerging theme is that these enzymes often also act by non-catalytic and/or non-epigenetic mechanisms. SETD2 (Set2 in yeast) is best known for associating with the transcription machinery and methylating histone H3 on lysine 36 (H3K36) during transcription. This well-characterized molecular function of SETD2 plays a role in fine-tuning transcription, maintaining chromatin integrity, and mRNA processing. Here we give an overview of the various molecular functions and mechanisms of regulation of H3K36 methylation by Set2/SETD2. These fundamental insights are important to understand SETD2’s role in disease, most notably in cancer in which SETD2 is frequently inactivated. SETD2 also methylates non-histone substrates such as α-tubulin which may promote genome stability and contribute to the tumor-suppressor function of SETD2. Thus, to understand its role in disease, it is important to understand and dissect the multiple roles of SETD2 within the cell. In this review we discuss how histone methylation by Set2/SETD2 has led the way in connecting histone modifications in active regions of the genome to chromatin functions and how SETD2 is leading the way to showing that we also have to look beyond histones to truly understand the physiological role of an ‘epigenetic’ writer enzyme in normal cells and in disease. All molecular processes in the eukaryotic cell that involve transactions with genomic DNA have to deal with chromatin—the complex of DNA and histone proteins. In general, wrapping DNA around histone octamers to form nucleosomes acts as a barrier for processes that require DNA as a template such as transcription, replication and DNA repair. Indeed, histones need to be removed at least temporarily to allow access to the DNA for these processes . ASaccharomyces cerevisiae, the SET domain-containing protein Set2 methylates histone H3 on lysine 36 (H3K36). This site is located at the base of the N-terminal tail of H3 and can be either mono-, di-, or trimethylated by Set2 [A large part of our understanding of chromatin modifying (‘writer’) enzymes comes from research on histone methyltransferases in budding yeast. In  by Set2 . H3K36 m by Set2 , 204, bu by Set2 . This ap by Set2 , 95, 120 by Set2 , 217. ThSince Set2’s first function was described in budding yeast, our understanding of Set2 and its homologs in other organisms has greatly expanded. At the molecular level, we are now beginning to get a decent picture of how Set2’s activity towards H3K36 is regulated during transcription. At the same time, the number of functions attributed to Set2 and H3K36 methylation has been steadily growing to include roles in processes such as mRNA splicing and DNA repair. Like with many other chromatin modifiers, Set2 is conserved from yeast to humans, in the form of SETD2 . The app1S2P3T4S5P6S7. Following the recruitment of RNAPII to the transcription start site, phosphorylation of the serine residues in this repeat stimulates the recruitment of elongation factors and chromatin writers [In both yeast and mammalian cells, the most well-characterized function of Set2/SETD2 is to methylate H3K36 during transcription. This function of Set2/SETD2 is regulated by RNA polymerase II (RNAPII) post-translational modifications. The C-terminal domain of the largest subunit of RNAPII consists of a heptad repeat with the consensus Y writers , 80. In  writers ). During writers , 147). A writers , 204. SeS. cerevisiae, Set2 is the only H3K36 methyltransferase and is therefore responsible for all H3K36 methylation states [In n states . In mammn states ). H3K36mn states  was shown states . In vitrn states  but lossn states . All H3Kn states . These fn states .Fig. 1OvCryo-EM studies show that the AWS-SETD-post-SET domains of yeast Set2 interact with DNA, H3 and the H2A C-terminal tail to help position Set2 on its H3K36 substrate , 125. Hutrans histone cross-talk [A positive regulator of Set2 activity is histone H2B mono-ubiquitination. During transcription elongation, H2B is ubiquitinated at lysine 123 in budding yeast (lysine 120 in humans) by the E2 ubiquitin conjugase Rad6 (UBE2A in humans) and E3 ligase Bre1 (RNF20/40 heterodimer in humans) in a process that also depends on Paf1C , 220. H2oss-talk , 192. H2oss-talk , 191. Sioss-talk . In the oss-talk . Consistoss-talk , 199. H2oss-talk , 168.Set2 contains additional domains C-terminally of the catalytic core which control Set2 activity and nucleosomal substrate recognition. These domains are conserved from yeast Set2 to human SETD2. The Set2-Rpb1 interacting (SRI) domain specifically interacts with RNAPII-pCTD . Both th, deletion of the SRI domain (Set2-ΔSRI) abolishes H3K36me3 and reduces H3K36me2 levels [In yeast2 levels , 213. Ho2 levels , 213, in2 levels . Interes2 levels . Similar2 levels . The SRI2 levels  suggestitrans-acting factors, including the elongation factors Spn1 (IWS1 in humans) and Spt6 (SUPT6H in humans). IWS1/Spn1 has several roles in co-transcriptional processes including pre-mRNA processing and mRNA export from the nucleus [The control of SET domain activity by the AID and SRI domains is influenced by multiple  nucleus , 212. IW nucleus , 138, wh nucleus , 211. Im nucleus . Initial nucleus , 160. Th nucleus . It is t nucleus . The emeThe Set2 AID appears to be conserved in SETD2 based on the similarity of the predicted structure of the AID in yeast Set2 and human SETD2 using AlphaFold . The preTwo other domains in Set2 are the WW domain and the coiled-coil (CC) domain, both of which mediate protein–protein interactions. The WW domain is named for its two conserved tryptophan residues and generally binds to proline-rich proteins. In yeast Set2, deletion of the WW domain does not affect the interaction with RNAPII or H3K36me3 levels . In humaThe CC domain of SETD2/Set2 was found through in silico prediction as CC domains have a typical amino acid composition. Not much is currently known about the function of the yeast Set2 CC domain. Deletion of the CC domain does not affect yeast Set2 RNAPII binding  suggestiSETD2 has a large N-terminal region (~ 1400 amino acids) that is not present in yeast Set2. This N-terminal region is predicted to be mostly unstructured . RecentlUnstructured domains can also contribute to the liquid–liquid phase separation of proteins. In line with this, SETD2 was recently reported to phase separate in cells , 14. Not. Following recruitment, activation of Rpd3S requires an interaction between either H3K36me2 or me3 and the Rpd3S subunits Eaf3 and Rco1 [Unlike acetylation, methylation does not change the electric charge of the modified lysine residue, and lysine methylation is therefore believed to mainly function via reader and/or effector proteins. In budding yeast, the most well-characterized function of H3K36me2/3 is to promote chromatin restoration in the wake of transcription by directing the Rpd3S histone deacetylase complex to active gene bodies  in addition to histone deacetylation [In mammals, SETD2 has also been reported to repress cryptic transcription from within active gene bodies , 146. Notylation , 190. Intylation . Importatylation , which stylation , 206. DNtylation , 206. SEtylation ,reviewedtylation . In summDrosophila. In this landmark study on the function of H3K36 methylation in higher eukaryotes, all the canonical H3 copies were mutated to H3K36R in Drosophila, but no evidence could be found for cryptic transcription [Drosophila and mammalian cells? Unlike most other insects, Diptera (flies) have very low levels of DNA cytosine methylation and lack DNMT1 and DNMT3 [Drosophila. So far, it has also not been shown that SETD2 represses cryptic transcription in Drosophila , so determining this could be a first step in unraveling this apparent discrepancy. In addition, a challenge for the future is to establish a mammalian model in which all  H3 copies can be mutated to H3K36R to confirm that SETD2 indeed represses cryptic transcription via H3K36me in mammals, as suggested by Neri et al. [Drosophila. Interestingly, H4 acetylation is increased in both H3K36R and SETD2-depleted Drosophila cells [Drosophila  directly leads to a chromatin state in which nucleosomes are constantly lost and replaced, this remains to be functionally assessed.In addition to histone hyperacetylation, Set2 deletion leads to increased replication-independent (RI) histone deposition in active gene bodies in budding yeast . Normalltivators , 207. Hotivators , 93. Howtivators , and as tivators , 210. HoSet2 might also suppress histone turnover independently of histone acetylation and cryptic transcription initiation. For example, H3K36me3 recruits the chromatin remodeler complex Isw1b , which mLens epithelium-derived growth factor  and hepatoma-derived growth factor 2 (HDGF2) are two H3K36me2/3 reader proteins that have been reported to have FACT-like functions in mammalian cells, namely promoting transcription through a chromatin template . LEDGF bTo summarize, there is evidence in both yeast and human cells that H3K36me3 directly recruits transcription elongation factors that stimulate histone recycling during transcription. This could provide a mechanistic explanation for the observed increase in histone turnover in active genes in absence of Set2 in budding yeast . HoweverSETD2 deficient and proficient primary clear cell renal cell carcinoma (ccRCC) tumors revealed that splicing defects or alternative splicing events such as intron inclusion and differential exon usage are widespread in SETD2 deficient tumors [SETD2 deficient cells display increased chromatin accessibility in active genes, which might be due to impaired nucleosome recycling during transcription, as discussed above. The increase in chromatin accessibility may also contribute to the splicing defect observed in SETD2 deficient cells as nucleosome occupancy is also important for proper mRNA splicing [In both yeast and human cells, Set2/SETD2 has been reported to regulate pre-mRNA splicing. In yeast, proper co-transcriptional splicing of several genes has been shown to be dependent on Set2-mediated H3K36me (predominantly H3K36me2) by promoting the recruitment of spliceosome components . This met tumors . Interessplicing .What evidence is there that SETD2 regulates pre-mRNA splicing via H3K36me3 in human cells? As mentioned previously, the human Eaf3 orthologue Mortality Factor 4 Like 1  recognizes H3K36me2 and H3K36me3 in vitro . SimilarAnother factor that influences splicing through SETD2 activity is Zinc Finger MYND-Type Containing 11 (ZMYND11), which is a chromatin reader protein that has been reported to associate with spliceosome components and regulate intron retention . ZMYND11There is strong evidence that SETD2 not only regulates splicing but that splicing in turn regulates SETD2. In mammalian cells, H3K36me3 is predominantly found on genes containing introns . TogetheAs mentioned above, it was recently demonstrated that SETD2 interacts with multiple factors that regulate splicing including hnRNP L through its SHI domain , 14. Do Drosophila as no evidence for splicing defects could be found in H3K36R cells [Drosophila? In the Drosophila study by Meers et al. [Drosophila (but not yeast) based on comparative genomics . It is therefore possible that recruitment of this potential ZMYND11 homologue by H3.3K36me3 in H3K36R Drosophila cells is sufficient to maintain normal splicing, although this will require further studies. In addition, it will be interesting to determine the role of the Drosophila orthologue of Eaf3/MORF4L1 in regulating splicing and if it is independent of H3K36. Furthermore, similar as for cryptic transcription, a mammalian model system in which all H3 copies can be replaced by H3K36R would be a useful tool to directly probe the role of H3K36me in pre-mRNA splicing.In contrast to yeast and human cells, H3K36me is dispensable for normal splicing in 6R cells . How cans et al. , all canDrosophila. However, H3K36R Drosophila cells did display a defect in mRNA polyadenylation suggesting a role for H3K36me in mRNA maturation [As discussed above, H3K36me is dispensable for repression of cryptic transcription and pre-mRNA splicing in turation . Messengturation . NotablyAs with other processes that involve DNA transactions, the response to DNA damage is extensively controlled by histones and histone PTMs. Set2/SETD2 has been reported to play multiple roles in the DNA damage response (DDR) in different organisms (for a detailed review see . In budd2 (for review see [SETD2 deficient cells display microsatellite instability (MSI) indicating that the interaction between MSH6 and H3K36me3 is important for functional MMR [Similar as in yeast, human SETD2 is also involved in the early steps of DDR signaling induced by DNA DSBs. In contrast to Set2 in yeast, however, it appears that in mammalian cells SETD2 promotes DNA repair mainly via HR. SETD2 is required for efficient phosphorylation of ATM as well as p53 (which is an ATM target) but not for γH2A.X formation . SETD2 fview see . SETD2 mview see . SETD2 hview see . SETD2 donal MMR . It is nonal MMR .1 and peaking in G2/M), and Set2 also promotes cell cycle progression by repressing cryptic antisense transcription of cell cycle control genes [RRM2 [. Consistent with this, SETD2 depletion reduces replication fork speed in renal carcinoma cells [SETD2 deficient cells are sensitive to inhibition of WEE1 which also depletes dNTP levels, leading to S-phase arrest in SETD2 deficient cells treated with WEE1 inhibitor [SETD2 deficient cells, making it a potential much-needed avenue to develop a targeted therapy for cancers in which SETD2 activity is perturbed. Interestingly, it was recently reported that SETD2 also prevents replication stress by methylating histone H3 on lysine 14 (H3K14) [In both budding yeast and human cells, Set2/SETD2 protein levels vary across the cell cycle (being low in Gol genes . Other sol genes . Mechaniol genes . Intereses [RRM2 . Consistma cells  and delema cells . Consequnhibitor . This im (H3K14) . H3K14me (H3K14) . Thus, SMost of the functions of Set2/SETD2 discussed above have been axiomatically linked to Set2/SETD2’s catalytic function towards H3K36. However, an emerging theme in chromatin biology is that many writer enzymes can have functions independent of their catalytic activity, modify non-canonical histone residues (as discussed above), or even modify proteins other than histones , 219. NoThe SRI domain is an important regulator of Set2/SETD2 activity towards H3K36. Recent studies indicate that the SRI domain can also regulate SETD2’s activity towards non-histone substrates. The binding of SETD2 to α-tubulin depends on both the acidic unstructured C-terminal tail of α-tubulin as well as on the SRI domain of SETD2 , 152. InAs discussed above, the SRI domain is positively charged at cellular pH and critical positively charged residues facilitate the interaction with the negatively charged RNAPII-pCTD . It is tIs SRI-dependent methylation of non-H3K36 substrates by SETD2 also conditionally stimulated by post-translational modification of these substrates? The acidic C-terminal tails of both α- and β-tubulin are extensively modified (for review see Janke ). AnalogSETD2  have been found in many cancer types, including clear cell renal cell carcinoma (ccRCC), gliomas, and in several types of leukemia [SETD2 is also mutated in other cancer types such as (among others) lung adenocarcinoma, endometrial carcinoma, colon adenocarcinoma, and melanoma [SETD2 mutations were described in ccRCC [SETD2 mutations in ccRCC. A frequent mutational event in ccRCC is the (heterozygous) loss of the short arm of chromosome 3  will be important to uncover the relative contribution of splicing regulation by SETD2 to tumor suppression. In addition to pre-mRNA splicing, SETD2’s function in mRNA polyadenylation and transcription termination has also been suggested to have tumor suppressive effects [SETD2 deficient ccRCC cell lines, read-through transcription affects the expression levels of downstream genes and leads to the formation of chimeric transcripts [SETD2, e.g. through its reported ability to stimulate DNA methylation (as opposed to Rpd3S activation in yeast). Notably, if read-through induced silencing indeed depends on H3K36me3/SETD2 in human cells, then it will be interesting to consider how exactly read-through transcription in SETD2 deficient cancer cells interferes with downstream genes. Alternative models could include RNAPII collisions or DNA topological stress, for example. A more fundamental insight in the role of SETD2 in transcriptional interference in mammalian cells would greatly contribute to understanding any potential role of this process in tumor progression.So far, SETD2’s tumor-suppressor function has mostly been attributed to its chromatin-associated roles in transcription regulation, mRNA processing and maintaining genome stability Fig. . In ccRCor genes . In this effects . In SETDnscripts . In buddnscripts , 76, 96.SETD2 allele), with subsequent mutations in the remaining SETD2 allele  occurring in more advanced stages of the disease [. Interestingly, mono-allelic inactivation of SETD2 does not affect global H3K36me3 levels in ccRCC tumors [SETD2 loss early-on in ccRCC development does not contribute to tumorigenesis via processes that depend on H3K36me3. However, mono-allelic SETD2 loss does impact α-TubK40me3 levels, leading to mitotic defects and the formation of micronuclei [SETD2 allele (compared to H3K36me3) and that mitotic defects caused by impaired tubulin methylation are likely early drivers in ccRCC tumor development [SETD2 loss due to mutations could further contribute to tumor progression and metastasis by impacting H3K36me3-dependent processes such as MMR (via MSH6) and possibly mRNA processing.In addition to mRNA processing, SETD2 has tumor suppressor functions through its role in DNA repair (HR and MMR) and promoting chromosome segregation during mitosis . As mentioned previously, the majority of primary ccRCC tumors are characterized by heterozygous 3p loss . Structural studies have shown that H3.3K36M rearranges the SET domain, increasing the affinity for the H3 tail and trapping SETD2 on its nucleosomal substrate, thereby explaining how H3.3K36M inhibits SETD2 [Besides direct stone H3 , 99, 126stone H3 , 65. A f3 [H3F3B . H3.3K363 [H3F3B  and in h3 [H3F3B . Even th3 [H3F3B , 128, suts SETD2 , 221. H3ts SETD2 .. Interestingly, H3.3K36M also leads to a redistribution of H3K27me3 away from developmentally silenced genes to regions normally enriched in H3K36me3, which may contribute to the derepression of PRC2 target genes that prevent differentiation [, H3K36me3 nucleosomes are a poor substrate for PRC2 [Drosophila H3K36R cells [In chondroblastoma, H3.3K36M has been reported to contribute to tumor development by promoting colony formation, and inhibiting apoptosis and chondrocyte differentiation , 128. Infor PRC2 , providi6R cells  so futurH3F3A) are H3.3G34R/V in osteosarcoma [in cis [SETD2 deficient cells.Other mutations that have been found in H3.3 (predominantly in osarcoma  and glioosarcoma , 202, [1osarcoma , and H3.osarcoma . Unlike  [in cis . H3.3G34 [in cis , 221. It [in cis , which i [in cis . A commo [in cis . Express [in cis , 208 indSETD2 are found in distinct cancer types and both affect SETD2 activity but can do so in distinct manners (Fig. in-trans inhibition of other writers such as NSD2.To summarize, mutations in H3.3 genes and ers Fig. . BesidesSETD2 deficient versus KDM4A overexpressing tumors.SETD2’s function can also be affected in cancer through misregulated expression of the demethylase KDM4A , which dThe SETD2/Set2 enzymes have been a prime example of how different approaches in different model systems have led the way in unravelling the molecular role and regulation of a chromatin modifying system associated with RNA polymerase moving along genes. SETD2/Set2 is a key enzyme in the cell involved in a broad range of genome-associated processes. The studies on SETD2/Set2 and H3K36 methylation showcase that teasing apart the various functions requires perturbing not only the writer itself, but also the other domains of the enzymes, the opposing demethylation activity, and the substrate lysine. Moving beyond chromatin, the story of SETD2 emphasizes the importance of knowledge about non-histone substrates of so called ‘epigenetic writers’. An emerging theme in chromatin biology is that non-catalytic functions or activities towards non-histone substrates of epigenetic enzymes need to be considered to fully understand the physiological role of these enzymes. Looking forward, it will be important to develop tools to identify substrates of SETD2 in different cellular contexts in an unbiased way and to perturb SETD2 functions in a substrate-specific manner, e.g. by mutation of a substrate lysine, or by isolation of separation-of-function mutations. With the current advances in genome engineering and proteomics it can be expected that more SETD2 surprises will be discovered and that the function of SETD2 in normal cells and in disease will be further unraveled at a molecular level.


ClHDZs were systemically identified in the watermelon genome, which were subsequently divided into four distinctive subfamilies (I–IV) based on the phylogenetic topology. HD-ZIP members in the same subfamily generally shared similar gene structures and conserved motifs. Syntenic analyses revealed that segmental duplications mainly contributed to the expansion of the watermelon HD-ZIP family, especially in subfamilies I and IV. HD-ZIP III was considered the most conserved subfamily during the evolutionary history. Moreover, expression profiling together with stress-related cis-elements in the promoter region unfolded the divergent transcriptional accumulation patterns under abiotic stresses. The majority (13/23) of ClHDZs in subfamilies I and II were downregulated under the drought condition, e.g., ClHDZ4, ClHDZ13, ClHDZ18, ClHDZ19, ClHDZ20, and ClHDZ35. On the contrary, most HD-ZIP genes were induced by cold and salt stimuli with few exceptions, such as ClHDZ3 and ClHDZ23 under cold stress and ClHDZ14 and ClHDZ15 under the salt condition. Notably, the gene ClHDZ14 was predominantly downregulated by three stresses whereas ClHDZ1 was upregulated, suggesting their possible core roles in response to these abiotic stimuli. Collectively, our findings provide promising candidates for the further genetic improvement of abiotic stress tolerance in watermelon.Homeodomain-leucine zipper (HD-ZIP) transcription factors are one of the plant-specific gene families involved in plant growth and response to adverse environmental conditions. However, little information is available on the HD-ZIP gene family in watermelon. In this study, forty  HD-ZIP transcription factors are a type of protein containing the homeodomain (HD)-leucine zipper (LZ) motif, which are prevalent in the plant kingdom and play important roles in growth and developmental processes. According to the structural characteristics, sequence conservations, and functional properties, the HD-ZIP gene family can be further divided into four subfamilies, i.e., HD-ZIP I, HD-ZIP II, HD-ZIP III, and HD-ZIP IV ,2. In adAtHB7 and AtHB12 in Arabidopsis can repress the expression of two ABA receptors, PYL5 and PYL8, thereby functioning as mediators of the negative feedback effect in ABA signaling in response to water deficit [AtHB2 and AtHB4 act together to control early exit from proliferation during leaf development under canopy shade [AtHB8 can promote vascular cell differentiation, while the decreased transcriptional level of the miR166-mediated gene AtHB15 can alter the vascular system with expanded xylem tissues and inter-fascicular regions [ANL2 affecting anthocyanin accumulation in subepidermal cells and root development [atml1 pdf2 double mutants exhibiting severe defects in shoot epidermal cell differentiation [Numerous evidence has confirmed that HD-ZIP TFs participate in a wide variety of developmental processes ,2. To da deficit . For inspy shade . HD-ZIP  regions ,11. In antiation .Citrullus lanatus L.) is cultivated worldwide because of its important economic and horticultural performance. However, the production and quality of watermelon is threatened by numerous environmental stresses, including heavy metal toxicity, drought, salinity, cold, and pathogen and/or virus invasion. To adapt to and resist these adverse conditions, plants have evolved a series of defense mechanisms. Transcription factors (TFs) have been well-known to play vital roles in plant responses to biotic and abiotic stimuli. Furthermore, with the availability of a high-quality watermelon reference genome [cis-elements, and phylogenetic relationship. Moreover, the expression patterns of members in subfamilies I and II under abiotic stresses were also investigated. Our results provide new insights for further functional studies of stress-responsive HD-ZIPs in watermelon.Watermelon . Additionally, the related protein sequences of the HD-ZIP genes were retrieved for Arabidopsis (48 AtHDZs) and cucumber (40 CsHDZs) according to recently published research [AtHDZs and CsHDZs were used as queries to identify HD-ZIP homologs in watermelon. The reliability of the candidate genes was verified through searching against the Pfam and SMART databases, and that with both the conserved HD and LZ domains were retained for further analyses.For the genome-wide identification of HD-ZIP genes in watermelon, the protein sequence of reference genome 97103 (V2) were downloaded from Cucurbit Genomics Database . The genomic distribution of ClHDZs on the chromosome was drawn with the software TBtools (v 1.100) [The theoretical molecular weights (MW), isoelectric points (pI), and grand average of hydropathicity (GRAVY) of v 1.100) .http://circos.ca/, accessed on 25 October 2021).Full-length protein sequences of the HD-ZIP genes from watermelon, cucumber, and Arabidopsis were aligned using the software Muscle under default parameters , which wClHDZs was displayed with the online tool GSDS2.0 (http://gsds.gao-lab.org/ accessed on 25 October 2021) based on their corresponding genomic and cDNA sequences. The conserved motifs in ClHDZs were predicted by the online program MEME (http://meme-suite.org/tools/meme accessed on 25 October 2021), setting the maximum number of motifs to 20 and motif width of 6–50. To obtain the cis-regulatory elements of ClHDZs, approximately 2.0 Kb of sequence upstream of the ATG start codon was retrieved and submitted to the PlantCARE server for scanning.The exon-intron organization of The watermelon inbred line ‘YL’ was used for the materials, which was provided by the Cucurbits Germplasm Resource Research Group at Northwest A&F University, Yangling, Shaanxi, China. The germinated seeds were sown in plastic pots filled with commercial peat-based compost (one seed for each pot) and cultivated under natural conditions in a greenhouse at Northwest A&F University. Finally, seedlings at the four-leaf stage were subjected to treatment under drought, low-temperature, and salt stimuli.To simulate a natural drought treatment ,20, the Following the manufacturer’s instructions, the total RNA was extracted from the samples using the RNASimple Total RNA Kit  and the cDNA was synthesized using the FastKing RT Kit with gDNase .ClACT (Cla007792) as the internal reference, the relative expression level for each selected gene was calculated using the 2−∆∆Ct method, as described in our previous study [ClHDZs are listed in The qRT-PCR amplification was performed on a StepOnePlus Real-Time PCR system . Using the housekeeping gene us study ,22. All ClHDZ1 to ClHDZ40 based on their chromosome locations  values being below zero , Arabidopsis (48 AtHDZs), and rice (48 OsHDZs)  and cucumber (~1.36 Mb), the size between Arabidopsis and two cucurbit crops were much smaller, i.e., the average size for Arabidopsis (~0.25 Mb), watermelon (~1.01 Mb), and cucumber (~0.52 Mb)  . Without OsHDZs) . Notably0.52 Mb) .ClHDZs, gene structures and intron numbers were comparatively depicted based on their gene annotations and genomic sequences  was only discovered in subfamily III. Consistent with the literature [Similar to the exon-intron organization, the distribution of the motifs were also conserved within a particular subfamily but diverse among subfamilies C. DetailClHDZs, approximately 2.0 kb of genomic sequences upstream of the translation site were retrieved to predict the cis-regulatory elements. As shown in cis-elements were identified and divided into four categories, including plant growth and development, phytohormones, various stresses, and possible binding sites for transcription factors (TFs). Among them, four types of cis-elements were related to plant growth and development, and eight were responsive to phytohormones, such as ABRE (abscisic acid-responsive), CGTCA motif (gibberellin-responsive), ERE (ethylene-responsive), GARE-motif (gibberellin-responsive), and P-box (gibberellin-responsive). The largest category was predicted to be involved in stress responses and consisted of 15 different cis-regulatory elements such as ARE (anaerobic-responsive), LTR (low temperature-responsive), WUN motif (wound-responsive element). Moreover, the binding sites for MYB- and MYC-type bHLH TFs were frequently detected in the promoter of ClHDZs. Although the number of cis-regulatory elements varied dramatically, the stress-related and TF-binding elements occupied the major proportion of all ClHDZs  after treatment  were gathered together in subfamily HD-ZIP II  values being below zero  in line with the published classifications for other species such as Arabidopsis, cucumber, tomato, and rice [ClHDZs were detected in the segmental region of the watermelon genome, with the majority contributing to the expansion of HD-ZIP subfamilies I and IV , and salt  was identified as a ortholog of AtHB51/LMI1 in Arabidopsis, which is characterized as a vital regulator in leaf proportionality [Cl018360 was also detected and designated as ClHDZ13 (ClHDZ13 was extremely reduced by drought stress (BnHB1423 (an ortholog of AtHB51/LMI1) in Brassica napus L. was found to be significantly upregulated by salt stimuli [HD-ZIP transcription factors have been reported to be involved in responses to a variety of abiotic stress in plants ,2,4,5, eand salt  stressesnditions . Moreovereatment A. Similaog AtHB6 . Previoutermelon , and theionality . Here, g ClHDZ13 . Accordit stress A, provid stimuli , implyinClHDZs were genome-widely identified in watermelon, which were further divided into four subfamilies. Among them, subfamily III was considered as the most conserved subfamily based on the gene structure and exon-intron organization. Syntenic analyses indicated that the expansion of subfamilies I and IV were possibly due to segmental duplication events. The expression profiling revealed that far more ClHDZs could be induced by cold and NaCl treatments compared with drought stimuli. In addition, some valuable candidates were subsequently identified that are possibly involved in responses to abiotic stresses, e.g., drought, low-temperature, and salinity. For example, the expression of gene ClHDZ23 and ClHDZ32 showed continuous enhancement with the increase in drought stress, similar to that of gene ClHDZ35, ClHDZ38, and ClHDZ39 under the cold condition and ClHDZ1 and ClHDZ18 after salt treatment. Moreover, gene ClHDZ14 was predominantly downregulated by the three stresses, but the expression of gene ClHDZ1 was upregulated by all treatments. Collectively, these findings may expedite the further functional characterization of ClHDZs in response to abiotic stresses.In the present study, a total of 40


Cheilinus undulatus is a protogynous socially hermaphroditic fish. However, the physiological basis of its sex reversal remains largely unknown. cyp19 is a key gender-related gene encoding P450 aromatase, which converts androgens to estrogens. cyp19 transcription regulation is currently unknown in socially sexually reversible fish. We identified NR5A1 by encoding SF-1, and FOXL2 from giant wrasse cDNA and cyp19a1a and cyp19a1b promoter regions were cloned from genomic DNA to determine the function of both genes in cyp19a1 regulation. Structural analysis showed that SF-1 contained a conserved DNA-binding domain (DBD) and a C-terminal ligand-binding domain (LBD). FOXL2 was comprised of an evolutionarily conserved Forkhead domain. In vitro transfection assays showed that SF-1 could upregulate cyp19a1 promoter activities, but FOXL2 could only enhance cyp19a1b promoter transcriptional activity in the HEK293T cell line. Furthermore, HEK293T and COS-7 cell lines showed that co-transfecting the two transcription factors significantly increased cyp19a1 promoter activity. The −120 to −112 bp (5′-CAAGGGCAC-3′) and −890 to −872 bp (5′-AGAGGAGAACAAGGGGAG-3′) regions of the cyp19a1a promoter were the core regulatory elements for SF-1 and FOXL2, respectively, to regulate cyp19a1b promoter transcriptional activity. Collectively, these results suggest that both FOXL2 and SF-1 are involved in giant wrasse sex reversal.The giant wrasse  Interestingly, only radial glial cells (RGC) express aromatase B in adult fish  constitutes the nuclear receptor subfamily 5 group A member 2 (NR5A2) subgroup, mainly expressed in the liver. Steroidogenic factor-1 ) is part of the NR5A1 subgroup and is mainly expressed in tissues related to steroid synthesis (cyp19a1) in medaka ovarian follicles (cyp19a1 promoter on a conserved binding site to activate cyp19a1 mRNA transcription in chicken ovaries (cyp11a and the steroidogenic acute regulatory protein (STAR), two Leydig cell function markers, was impaired, indicating a defect in androgen biosynthesis (cyp19 gene transcription.Fushi-tarazu factor-1 (FTZ-F1) is a member of the orphan nuclear receptor family discovered in ynthesis . Therefoynthesis . SF-1 poollicles . SF-1 diesis cyp1a1 in medynthesis . CollectFOXL2 has been recognized as a key gene in ovarian differentiation and egg formation in vertebrates  , inflammation (such as nuclear factor of activated T cells (NFAT), prostaglandin-endoperoxide synthase (PTGS2), and code dystrophy 2 (COD2)), and apoptosis/detoxification (such as manganese superoxide dismutase (MnSOD))  of Ad4BP/SF-1 to enhance Ad4BP/SF-1-mediated cyp19a1 transcription  box (FOX) L2 (FOXL2), another important transcription factor discovered in dermatolysis palpebrarum, is a member of the FOX transcription factor family. In recent decades, mammals) . Many FO(MnSOD)) , 13. FOXgulation . In Nilecription . Recent n family . Similarelopment . TherefoFOXL2 cDNA sequences from a giant wrasse and tested both factors in cyp19a1 transcriptional regulation by utilizing two cell lines (HEK293T and COS-7). Examining cyp19a1 promoter deletions and mutations confirmed the SF-1 and FOXL2 binding sites in the cyp19a1 promoter.As an imminent danger species, the fishing industry has sharply decreased the giant wrasse population . HoweverA 2-year-old wild and healthy giant wrasse was purchased from the Hainan Qionghai Fishing Port Terminal and Guangzhou Huangsha Fishery Market. The fish was anesthetized in ice, and samples from the brain and gonads were taken and stored in liquid nitrogen for future use. All animal handling procedures were conducted under the Institutional Animal Care and Use Committee of Hainan University.FOXL2 F1, and FOXL2 R1 primers to amplify the target fragments. For this study, SF-1 open reading frame (ORF) F1, SF-1 ORF R1, FOXL2 ORF F1, and FOXL2 ORF R1 were used to amplify HindIII, XhoI, and XhoI and EcoRI restriction sites. According to the restriction endonuclease site existing in pCDNA3.1, a suitable enzyme that was the same as the fragment multi-band restriction site was selected to double-digest the pCDNA3.1 vector. The SF-1 and FOXL2 ORF sequences were ligated into the pCDNA3.1 plasmid, and then the recombinant plasmid was transferred into the DH5α competent cell. Finally, the recombinant plasmid containing the target fragment was screened and identified, and the promoter sequence was obtained using the GenomeWalker method. The cloning method was performed according to the Universal GenomeWalker™ 2.0 User Manual. RNA was extracted using TRIzol reagent, according to the manufacturer’s instructions. The Prime Script™ RT Reagent Kit with a gDNA Eraser  was used to synthesize cDNA templates. The reverse transcription products were stored at −20°C for later use. cDNA fragments were cloned into PGEM-Teasy plasmids using the SF-1 F1, SF-1 R1, cyp19a1a and cyp19a1b were isolated by genome walking. The online software ALGGEN-PROMO  was used to predict the cyp19a1a and cyp19a1b transcription binding sites of SF-1 and FOXL2. The giant wrasse’s NR5A1 and FOXL2 gene sequences were spliced using DNAMAN software. The ORF was identified using the ORF Finder (https://www.ncbi.nlm.nih.gov/orffinder/). The National Center for Biotechnology Information (NCBI) database (https://www.ncbi.nlm.nih.gov/) was used to identify the coding regions and amino acid sequences. The online software Signal P4.1 was used to predict the signal peptides of NR5A1 and FOXL2 genes in the giant wrasse. In addition, an adjacency (neighbor joining (NJ)) phylogenetic tree was constructed based on amino acid sequence alignment using MEGA 5.0. GeneDoc software was used for multiple sequence alignments.Pomoter regions of the cyp19a1a and cyp19a1b promoters were analyzed using online software, and the SF-1 and FOXL2 binding positions in their sequences were predicted. The promoters were deleted from the analysis. Primers were designed with restriction sites AAGCTT and GGTACC of restriction endonuclease (HindIII and KpnI). Progressive deletion fragments were constructed using a pGL-4.10 fluorescent expression vector. Based on the effect of transcription factors on the progressive deletion of the promoter, the scope of key transcription binding sites was narrowed, and specific primers were designed to clone the full-length sequence of site-directed mutations by fusion PCR. The ORFs of NR5A1 and FOXL2 were ligated using gene-specific primers, restriction endonucleases, and the pCDNA3.1 vector to construct a site-mutant expression vector.In this study, the l-glutamine, 100 U/ml of penicillin, and 0.1 mg/ml of streptomycin in 5% CO2 and cultured at 37°C. The confluent cells were seeded in a 24-well plate. The transfection rate was maintained at ~ 90% at the time of transfection. The specific experimental components were as follows: 1) 250 ng of complete 5′ upstream or deletion constructs  of cyp19a1a and cyp19a1b promoters cloned into the pGL-4.10 (firefly luciferase) vector and 2) 200 ng of pcDNA3.1 expression plasmid  containing ORF cDNAs encoding SF-1 and FOXL2. A 5 ng/well of Renilla luciferase (CMV) vector  was used as an internal control. Luciferase assays were performed 48 h after transfection using the Dual-Luciferase Reporter Assay System (Promega). Renilla luciferase activity was used to normalize firefly luciferase activity. Luminescence was measured using a GloMax®96 microplate luminometer (Promega). The plasmids used in the transfection experiments were prepared using an Endo Free Plasmid Isolation Kit.HEK 293T and COS7 cell lines were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 2 mM of p < 0.05. * represents a significant difference between the data. SPSS and DPS software was used to perform the statistical analyses and constructed graphs using GraphPad Prism version 5.The experimental cell data were set up in four parallels per group. A one-way ANOVA was performed, and the results are expressed as the mean ± SD. Statistical significance was set at FOXL2 cDNA sequences were cloned and sequenced. The gene encoding SF-1 had a 1,467-bp ORF encoding 488 amino acid residues . At the same time, after SF-1 was transfected into COS-7 cells for 48 h, we observed that the transcriptional activities of cyp19a1a and cyp19a1b promoters were significantly increased. However, FOXL2 did not increase the transcriptional activity of the cyp19a1 promoters. Conversely, SF-1 and FOXL2 co-transfection significantly upregulated cyp19a1 promoter activity (p < 0.05). The results of the two cell lines showed that co-transfection of the two transcription factors significantly increased cyp19a1 promoter activity.In the HEK293T cell line, we found that SF-1 significantly upregulated the transcriptional activity of cyp19a1a promoter . We conducted site-directed mutations from −1,265 to −1,256 bp and −119 to −112 bp to determine whether −120 to −112 bp was the key binding site of SF-1 on cyp19a1a. We found that mutating −120 to −112 bp significantly reduced cyp19a1a promoter activity (p < 0.05).With the use of the full length of the cyp19a1b promoter (p < 0.05), the mutations at these sites did not significantly reduce cyp19a1b promoter activity (p < 0.05). Therefore, we determined that these sites were not the core regulatory sites of SF-1 in the cyp19a1b promoter.When we deleted the transcription binding sites that predicted SF-1 in the cyp19a1a promoter.In summary, we concluded that −120 to −112 bp (5′-CAAGGGCAC-3′) was the core regulatory element for SF-1 to regulate the transcriptional activity of the p < 0.05). Based on the predicted results from −2,379 to −871 bp, we performed site-directed mutations at the following three transcription sites: −1,116 to −1,098, −1,035 to −1,027, and −890 to −872 bp. After mutating −890 to −872 bp, cyp19a1b promoter activity decreased significantly (p < 0.05).As cyp19a1b promoter.Therefore, we determined that −890 to −872 bp (5′-AGAGGAGAACAAGGGGAG-3′) was the core regulatory element for FOXL2 to regulate the transcriptional activity of the cyp19a1 is the switch enabling androgen to estrogen conversion, affecting gonadal differentiation and sex ratios by regulating the biosynthesis of gonadal steroid hormones  cooperated with FOXL2 to upregulate cyp19a1a promoter activity (AMH) regulation via functional FOXL2 occurs through protein–protein interactions between FOXL2 and SF-1 in human granulosa cells  and  mammals –26. Furtcription . Similarhe brain . In tilahe brain . Similaractivity . Howeversa cells . In addi tilapia . Researcractions . These rcyp19a1  in the cyp19a1b promoter of tilapia significantly reduced the activity of the promoter .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.


Arabidopsis and fiber crop cotton. In particular, we discuss the latest understanding and insights into the underlying molecular mechanisms of trichomes formation at multiple dimensions, including at the chromatin, transcriptional, post-transcriptional, and post-translational levels. We summarize that the integration of multi-dimensional trichome-associated genes will enable us to systematically understand the molecular regulation network that landscapes the development of the plant trichomes. These advances will enable us to address the unresolved questions regarding the molecular crosstalk that coordinate concurrent and ordered the changes in cotton fiber initiation and progression, together with their possible implications for genetic improvement of cotton fiber.Plant trichomes are specialized epidermal cells that are widely distributed on plant aerial tissues. The initiation and progression of trichomes are controlled in a coordinated sequence of multiple molecular events. During the past decade, major breakthroughs in the molecular understanding of trichome development were achieved through the characterization of various trichomes defective mutants and trichome-associated genes, which revealed a highly complex molecular regulatory network underlying plant trichome development. This review focuses on the recent millstone in plant trichomes research obtained using genetic and molecular studies, as well as ‘omics’ analyses in model plant  Plant trichomes are the unicellular or multicellular appendages originating from epidermal cells and are widely distributed on the surface of different organs of plants, i.e., stems, leaves, petioles, flowers, and seed coats . TrichomArabidopsis thaliana, a model plant, has trichomes that are typically unicellular, non-glandular, and have two to three branches -WD40 repeat (WDR) protein  MIXTA-like transcription factor, GhMML4_D12, is known to regulate the fiber development by binding with GhWDR but not with a bHLH protein  are the main drivers of evolution and domestication because they have the potential to fine-tune traits and improve crops . Previou2[RK]x3Lx6Lx3R is a conserved motif in MYB proteins that is critical for interaction with GLABRA3  are functionally redundant bHLH TFs  regulates early fiber development  biosynthesis and signaling, as well as appropriate concentration of BR promotes cotton fiber elongation (GhFP1 promotes trichome development in Arabidopsis (PACLOBUTRAZOL RESISTANCE 1 (PRE1) is specifically expressed in fiber cells, and core cis-element variation in GhPRE1 contributes to fiber formation  has four WD40 repeats and regulates trichome differentiation; loss of TTG1 function results in a glabrous phenotype  based on the exon-intron structures, similarity of nucleotide sequence, and the presence of additional sequences.  protein and activate the MBW complex to promote GL2 expression, positively regulating trichome development (A highly conserved DNA-binding homeodomain (HD) and leucine zipper (ZIP) motif characterize the HD-ZIP proteins, which constitute one of the largest plant-specific TF families . The ZIPquences. . HD-ZIP quences. . HD-ZIP quences. . GL2 wasrichomes . In gl2 aberrant . GL2 hasth HDG11 . hdg11 mhenotype . The HD-ntiation . GA is kelopment . DELLA pelopment .GL2 homologs MERISTEM LAYER 1 (GbML1), GhHD1, GaHOX1, and GhHOX3 are highly expressed in trichomes  of MYB protein family is the structural basis of interaction between MYB and R/B-like BHLH proteins . In cottelopment . Thus, cArabidopsis, other TFs have also been identified that control trichome development. C2H2 zinc finger proteins, including GLABROUS INFLORESCENCE STEMS (GIS), GIS2, GIS3, ZINC FINGER PROTEIN5 (ZFP5), ZFP6, and ZFP8 are key factors that regulate trichome initiation through GA and CK signaling  complex . In  complex . GCN5 re complex  and reguitiation . Moreove1 mutant . Histone1 mutant . JMJ29 ibidopsis . JMJ29 ditiation .GhHDA5 knockdowns show significantly suppressed fiber initiation and yield. GhH2A12 is a histone H2A gene that controls fiber initiation and early elongation by regulating cell cycle-related genes  controls mRNA fate and expression and is the most abundant and widespread internal mRNA modification. m6A can be dynamically read, written, and erased  regulation and mRNA modification have emerged as important components of post-transcriptional gene expression. ncRNAs include long noncoding RNAs (lncRNAs), small interfering RNAs (siRNAs), and microRNAs (miRNAs). lncRNAs are defined as ncRNAs with transcripts longer than 200 nt; some lncRNAs are known to manipulate fiber initiation . miRNAs d erased . RNA-binArabidopsis (SQUAMOSA PROMOTER BINDING PROTEIN LIKE9 (SPL9), which represses trichome formation on the inflorescence, to regulate trichome development in Arabidopsis  1, LOM2, and LOM3 are targeted by miR171; miR171 over-expression decreases trichome density on Arabidopsis stem and floral organs  1–11 , and IRREGULAR TRICHOME BRANCH 1 (ITB1), which are m6A-modified genes that regulate trichome development  1–11 . ECT2 isbidopsis ; its delbidopsis . ECT2 caelopment . In ect2ranching . These rGreat progress has been made in understanding the regulatory mechanism of the MBW complex in trichome development, but it remains important to clarify the factors that regulate the transcriptional activator complex at the post-translational dimension. Post-translational modifications include (but not limited to), acetylation, glycosylation, ubiquitination, phosphorylation, glycation, SUMOylation, methylation, nitration, and oxidation. The ubiquitin/26S proteasome system (UPS) is an important regulator and mediates the proteasomal degradation of TFs . The UPSArabidopsis, KAKTUS (KAK) encodes a HECT-type E3 ubiquitin ligase that negatively regulates endoreduplication cycles in trichome branching by accelerating GL3 and EGL3 degradation  also controls trichome branching by negatively regulating endoreduplication , the National Special Program for GMO Development of China (No. 2016ZX08009003-003-004 and 2016ZX08005-004), and the Agricultural Science and Technology Innovation Program of Chinese Academy of Agricultural Sciences.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.


This study aimed to investigate the regulation role of fads2 subjected to fatty acid in large yellow croaker and rainbow trout. In vivo, large yellow croaker and rainbow trout were fed a fish oil (FO) diet, a soybean oil (SO) diet or a linseed oil (LO) diet for 10 weeks. The results show that LO and SO can significantly increase fads2 expression (p < 0.05). In vitro experiments were conducted in HEK293T cells or primary hepatocytes to determine the transcriptional regulation of fads2. The results show that CCAAT/enhancer-binding protein α (C/EBPα) can up-regulate fads2 expression. GATA binding protein 3 (GATA3) can up-regulate fads2 expression in rainbow trout but showed opposite effect in large yellow croaker. Furthermore, C/EBPα protein levels were significantly increased by LO and SO (p < 0.05), gata3 expression was increased in rainbow trout by LO but decreased in large yellow croaker by LO and SO. In conclusion, we revealed that FO replaced by LO and SO increased fads2 expression through a C/EBPα and GATA3 dependent mechanism in large yellow croaker and rainbow trout. This study might provide critical insights into the regulatory mechanisms of fads2 expression and LC-PUFA biosynthesis.Δ6 fatty acyl desaturase (Δ6Fads2) is regarded as the first rate-limiting desaturase that catalyzes the biosynthesis of long-chain polyunsaturated fatty acids (LC-PUFA) from 18-carbon fatty acid in vertebrates, but the underlying regulatory mechanism of  In mammals, it is known that the activities of fatty acids desaturases could be affected by diets and hormones . A study crocea) , Japanesponicus) , and grooioides) . The exp and ALA ,19. So fty acids ,17,18,20Siganus canaliculatus). GATA binding protein 3 (GATA3) belongs to the GATA family of transcription factors, which have been implicated as being involved in the regulation of cell fate and differentiation in various cell types [fads2 transcription and LC-PUFA biosynthesis has not been studied.C/EBPα (CCAAT/enhancer-binding protein α) is the first member of the C/EBP family of the basic region leucine zipper proteins which is capable of binding eukaryotic gene promoters and regulating gene transcription ,22. Manyll types ,30. To dOncorhynchus mykiss)), which are both important farming fish species in China, were used as experimental model animals. It has been verified that large yellow croaker has a limited capability to synthesize LC-PUFA as compared to rainbow trout. Hence, they represent suitable model species for the study of LC-PUFA biosynthesis. Fads2 plays an important role in LC-PUFA biosynthesis, but the underlying regulatory mechanism of fads2 in large yellow croaker and rainbow trout has not been comprehensively understood. Therefore, this study was designed to explore the transcriptional regulation of fads2 subjected to fatty acid and its differences between large yellow croaker and rainbow trout in order to provide critical insights into the role of molecular regulatory mechanisms in fads2 expression and LC-PUFA biosynthesis.In the present study, a marine teleost (large yellow croaker) and a freshwater teleost, . Subsequently, total RNA was extracted following the manufacturer’s protocol. The integrity of RNA was detected by electrophoresis using 1.2% denatured agarose gel. The cDNA was reverse transcribed from RNA by PrimeScript RT reagent kit  following the protocol supplied by the manufacturer.β-actin, gapdh, and rpl17 were selected to test their suitability for the normalization of gene expression levels in various tissues of large yellow croaker and rainbow trout. No significant differences in β-actin expression were detected among all treatments, suggesting that β-actin could be used as a reference gene. Further, the mRNA expression of each gene was normalized to that of β-actin using the 2−ΔΔct method [The mRNA expression levels were quantified in a quantitative thermal cycler Mastercycler ep realplex  with SYBR Green real-time PCR kit . Absolute quantification of PCR and RT-qPCR was carried out according to the method as previously described ,33; the t method . Westernt method ,36. The 6 cells/ml and were maintained in a DMEM/F12 medium  containing 15% FBS  and incubated at 18 °C. Microscopic examination ensured that hepatocytes progressively reassociated throughout the culture to form two-dimensional aggregates, in agreement with earlier reports [6 cells/mL and were maintained in DMEM/F12 medium  containing 15% FBS  and antibiotics in a 5% CO2 atmosphere at 28 °C. The hepatocytes were incubated with ALA, LA, EPA, and DHA to confirm the effect of fatty acids on the transcription of the fads2 in vitro. The solvent group was set as the control group. The fatty acid  was supplemented to cells on 6-well plates in the form of BSA/fatty acid complexes that were prepared at 100 μM concentration, incubated for 12 h. After incubation, cells were lysed in the wells and harvested for RNA extraction.In order to simulate the in vivo experiments and further verify the results, we carried out in vitro experiments. The methods of primary culture of hepatocytes of rainbow trout were described previously . Hepatoc reports ,39. As f reports . Hepatocfads2 which had been cloned by our laboratory. PCR was conducted using TransStart FastPfu Fly DNA Polymerase  to get the promoter of fads2 of large yellow croaker (996 bp). Then, the promoter was subcloned into pEASY-T1 vector  for sequencing. The recombinant pEASY-T1 plasmid with the right sequence was used as the template for following PCR cloning. To identify the core promoter region of fads2 of large yellow croaker, a deletion mutant assay was conducted. The recombinant pEASY-T1 plasmid was used as a template for PCR cloning with different primers . Then, the 7 obtained DNA fragments were inserted into the pGL3.0-basic firefly luciferase reporter vector which has no promoter. The constructed reporter vectors were named as LD6D1, LD6D2, LD6D3, LD6D4, LD6D5, LD6D6, and LD6D7. The reporter vectors named OD6D1, OD6D2, OD6D3, OD6D4, OD6D5, OD6D6, and OD6D7 in rainbow trout were obtained through the same methods in large yellow croaker. All plasmids were confirmed by DNA sequencing and prepared using an EasyPure HiPure Plasmid MiniPrep kit . Large yellow croaker and rainbow trout genomic DNA was extracted by DNAiso Reagent  according to the manufacturer’s instructions. Primers used to clone the promoters were designed according to the promoter sequence of  primers  to get d2. Cells were inoculated into 24-well cell culture plates at 2 × 105 cells per well and cultured for 24 h to 80–90% confluence before being transfected. To determine the promoter activities, HEK293T cells were co-transfected with deletion mutational recombinant vectors or pGL3-Basic and phRL-CMV plasmid using lipofectamine 2000  according to the manufacturer’s instructions. To confirm the effects of transcription factors  on the fads2 promoter, HEK293T cells were co-transfected with the fads2 promoter, expression plasmids, and phRL-CMV plasmid. The promoter-less pGL3-basic vector was used as a negative control for each transfection assay. All assays were performed with 3 independent transfections. The luciferase activity was measured using a TransDetect double-luciferase reporter assay kit .The human embryonic kidney cell line (HEK293T cell line) was cultured in high-glucose DMEM supplemented with 10% FBS , 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C and 5% COSmall interference RNAs (siRNA) were designed and synthesized for gene knockdown according to the C/EBPα and GATA3 sequence by GenePharma Biotech . C/EBPα and GATA3 gene knockdown was performed using Xfect RNA Transfection Reagent  according to the protocol supplied by the manufacturer. To determine the potential functions of C/EBPα and GATA3 sites on the promoter activity of large yellow croaker and rainbow trout, site-directed mutation of recombinant plasmids was carried out by PCR reaction with the mutation sites designed in the primers . The recfads2 promoter and expression plasmids (pCS2+ -C/EBPα-flag or pCS2+ -GATA3-flag) were co-transfected into HEK293T cells at a ratio of 1:2. After 24 h of transfection, the HEK293T cells were cross-linked with 1% formaldehyde at 37 °C for 10 min. Protein lysates collected from these cells were incubated with anti-IgG or anti-flag antibodies, then they were analyzed using ChIP assay as per ChIP Kit protocol . Bound target DNA fractions were analyzed by PCR with the specific primers . The sequences were as follows: forward, 5′-CATGCTTCATGTCGAAACAGACAAATGATG-3′, and reverse, 5′-CATCATTTGTCTGTTTCGACATGAAGCATG-3′ of large yellow croaker; forward, 5′-AGAGTCACACCATTTCACCACGTAGGTGTG-3′, and reverse, 5′-CACACCTACGTGGTGAAATGGTGTGACTCT-3′ of rainbow trout. The sequences of the competitive probes were identical to those of the biotin-labeled probes, but there was no biotin-labeled at each end. The sequences of mutative competitive probes were as follows: forward, 5′-CATGCTTCATGTCACGACAGACAAATGATG-3′, and reverse, 5′-CATCATTTGTCTGTCGTGACATGAAGCATG-3′ of large yellow croaker; forward, 5′-AGAGTCACACGAGCTATCTACGTAGGTGTG-3′, and reverse, 5′-CACACCTACGTAGATAGCTCGTGTGACTCT-3′ of rainbow trout. The Annealing Buffer for DNA Oligos  was adopted for DNA oligonucleotide annealing according to the manufacturer’s instructions. The annealed probes were stored at −20 °C until use. According to the instructions of the LightShift Chemiluminescent EMSA Kit , binding reactions and chemiluminescent detection were conducted. For the competition experiment, a 40-fold molar excess of the unlabeled probe was included in the preincubation mixture before the addition of the labeled probe. The nuclear protein was prepared using NE-PER Nuclear and Cytoplasmic Extraction Reagents . The C/EBPα sequence in the t-test. The data of absolute quantification PCR were analyzed by both one-way ANOVA and independent t-test. p < 0.05 was applied as a significant difference and p < 0.01 as a highly significant difference.All data were presented as means ± SEMs. The data on fatty acid composition and Western blotting were analyzed by one-way ANOVA with the help of SPSS 19.0 followed by Tukey’s multiple comparison test. The data of dual-luciferase trial and site-directed mutation were analyzed by independent p < 0.05), such as C18:2n-6 and C18:3n-3 in the liver, muscle, and intestine of large yellow croaker and rainbow trout. The relative content of LC-PUFA in tissues of large yellow croaker and rainbow trout fed LO and SO was significantly lower than the fish fed FO (p < 0.05) except in the liver of rainbow trout. The data of the diet and liver/muscle in large yellow croaker were already published in our previous study [The diet was rich in ALA, LA, DHA, and EPA in linseed oil (LO), soybean oil (SO), and fish oil (FO) diet, respectively. The fatty acid composition of tissues reflected the fatty acid composition of the diet . FO subsus study .p < 0.05), and gene expression in the liver was also significantly up-regulated by SO (p < 0.05). The results were the same in rainbow trout. Furthermore, the fads2 expression in rainbow trout was higher than in large yellow croaker. To be more specific, in the liver, the gene expression in rainbow trout had a highly significant difference in FO and LO group (p < 0.01) and a significant difference in the SO group (p < 0.05) compared with large yellow croaker (p < 0.01) compared with large yellow croaker  (fads2 expression was significantly increased in the ALA and DHA groups (p < 0.05), and had no significant differences in the LA and EPA groups (p > 0.05) in rainbow trout  . The fadow trout .fads2, seven recombinant reporter vectors of large yellow croaker and rainbow trout contained different promoter sequence length were constructed respectively. The activities of progressive deletion mutants were analyzed by dual-luciferase reporter assays. As the results suggested /OD6D1 (PGL3-O-Δ6), were co-transfected into HEK293T cells and the dual-luciferase assay was conducted, using pGL3-Basic and pCS2+ as controls. The results showed that the activity of rainbow trout fads2 promoter was significantly higher than large yellow croaker (p < 0.05) (fads2 promoter activity in large yellow croaker (p < 0.01) but had no regulatory activity on fads2 promoter of rainbow trout. The transcription factor USF1 and USF2 could significantly up-regulate fads2 promoter activity in rainbow trout (p < 0.05) but had no regulatory activity on fads2 promoter of large yellow croaker. The transcription factor NFYB, PAX5, and PPARγ had no regulatory activity on fads2 promoter of both large yellow croaker and rainbow trout. The transcription factor C/EBPα could highly significantly up-regulate fads2 promoter activity in both large yellow croaker and rainbow trout (p < 0.01). The transcription factor GATA3 could highly significantly down-regulate fads2 promoter activity in large yellow croaker (p < 0.01) but up-regulate the activity in rainbow trout (p < 0.05)  a. The tr < 0.05) b,c. The  < 0.05) d,e.fads2 promoter activity in both large yellow croaker and rainbow trout. Transcription factor GATA3 was the only one that could significantly up-regulate the promoter activity in rainbow trout but down-regulate the promoter activity in large yellow croaker. It seemed to be one of the reasons for the difference in fads2 expression between large yellow croaker and rainbow trout. In order to further investigate the function of C/EBPα and GATA3, we first conducted the absolute quantitative analysis and Western blotting. It was obvious that the c/ebpα expression in the liver (p < 0.05) and intestine (p < 0.05) of rainbow trout was higher than in large yellow croaker (p < 0.05) in large yellow croaker and rainbow trout. There were no significant differences between LO group and SO group in tissues of large yellow croaker and liver of rainbow trout (p > 0.05). However, the C/EBPα protein levels in muscle and intestine of rainbow trout were significantly higher in SO group compared with LO group (p < 0.05) (gata3 expression in intestine and muscle of large yellow croaker was significantly decreased in SO and LO group than FO group (p < 0.05) (p > 0.05) (The transcription factor C/EBPα could highly significantly up-regulate  croaker a. The pr < 0.05) . The gat < 0.05) b, but th > 0.05) c.fads2, we conducted the RNAi assay. The results showed that interference with siRNA-1 in rainbow trout and siRNA-3 in large yellow croaker for 48 h decreased the expression of c/ebpα mostly (c/ebpα expression by these siRNA significantly decreased the expression of fads2 (p < 0.05) in rainbow trout and large yellow croaker  and OD6D1 (fads2 promoter of rainbow trout) (p < 0.05) (p < 0.05) (p < 0.05) (In order to further investigate the function of C/EBPα and GATA3 on the  mutated  and dete < 0.05) a. Mutati < 0.05) b, but a  < 0.05) c.fads2 promoter is a direct target of C/EBPα and GATA3, we conducted the ChIP assay. The results showed a significant binding of C/EBPα and GATA3 to the promoter of fads2. No DNA fragment was amplified from the precipitate by control IgG, indicating the specific interaction between C/EBPα and GATA3 with the fads2 promoter (fads2 (To determine whether the promoter . The EMSr (fads2 .fads2 was subject to nutritional regulation and could be up-regulated in fish fed a vegetable oil diet compared to fish fed a fish oil diet [fads2 expression in the liver and intestine of large yellow croaker and rainbow trout, consistent with other studies [The LC-PUFA relative content in the liver of large yellow croaker fed an LO diet was significantly decreased than the fish fed an FO diet, but there was no difference in the liver of rainbow trout, suggesting that rainbow trout had a greater capability to biosynthesis LC-PUFA compared with large yellow croaker. The biosynthesis of LC-PUFA was dependent on dietary C18:3n-3 and C18:2n-6 via desaturases and elongases ,42,43,44oil diet ,48. The  studies ,45,46,48fads2 expression was higher in rainbow trout than in large yellow croaker, whether with the fish oil diet or the vegetable oil diet. The expression level of fads2 generally indicates the capacity of LC-PUFA synthesis in fish [It was consistent with previous studies that marine fish species show less ability to synthesize LC-PUFA from C18:2n-6 and C18:3n-3 ,49,50,51 in fish ,53. Thes in fish . Another in fish ,55.fads2 promoter in fish had gradually advanced [fads2 promoter and dual-luciferase reporter assay was carried out to identify the core promoter of fads2. Two fragments in large yellow croaker and 1 fragment in rainbow trout were identified as the core promoters. Based on the results, it was different from other studies in which just one core promoter was identified [fads2 that are subjected to dietary fatty acids in large yellow croaker, rainbow trout, and Japanese sea bass [fads2 promoter was significantly higher than large yellow croaker, which may be caused by the different regulation of transcription factors. In this study, several transcription factors predicted by JASPAR and Gene Regulation database were used to test the regulation effects on the fads2 promoter. The results showed that different transcription factors played different functions, in which C/EBPα and GATA3 played an important role. In recent years, the understanding of the advanced ,31,52, bentified , indicatentified ,57,58. Ssea bass . LXR andgata3, and fads2 have a correlation in response to the dietary fatty acid composition. Further site-directed mutation, ChIP assay, EMSA assay, and siRNA assay showed that C/EBPα and GATA3 could bind to the fads2 promoter and regulate the transcription directly, also that C/EBPα and GATA3 could regulate the expression of fads2.Previous studies have shown that C/EBPα could be involved in the process of fatty acid and lipid metabolism by regulating some gene transcription, such as FABP1 , FAS, anfads2 in large yellow croaker and rainbow trout and suggested that C/EBPα and GATA3 are powerful transcription factors in the regulation of fads2 transcription. Furthermore, the different regulation of different transcription factors may be one of the reasons for the difference in fads2 expression in large yellow croaker and rainbow trout. This research took C/EBPα and GATA3 as transcription factors involved in fatty acid biosynthesis in vertebrates and may help us to have a comprehensive understanding of LC-PUFA biosynthesis.In conclusion, this study identified the core promoters of


By applying a mixture of topologically triggered templates consisting of the photoresponsive barrier and the T-A·T triplex barrier, the gated transcription of the MG aptamer or the DFHBI-binding aptamer is demonstrated. In addition, a Sr2+-ion/KP topologically triggered DNA tetrahedra promoter-transcription scaffold, for the replication of the MG RNA aptamer, and T7 RNA polymerase are integrated into DNA-based carboxymethyl cellulose hydrogel microcapsules acting as cell-like assemblies. The switchable, reversible transcription of the MG RNA aptamer in a cell-like containment is introduced.Topological barriers control in nature the transcription machinery, thereby perturbing gene expression. Here we introduce synthetically designed DNA templates that include built-in topological barriers for switchable, triggered-controlled transcription of RNA aptamers. This is exemplified with the design of transcription templates that include reversible and switchable topological barriers consisting of a Sr 2+-ion stabilized G-quadruplex units, T-A·T triplex structures and photoisomerizable azobenzene-nucleic acid blockers.Three different topological barriers to switch transcription machineries were introduced including Sr For example, ion-induced formation of G-quadruplexes and their separation in the presence of crown ethers,7,8 the stabilization of T–T and C–C mismatched duplexes by metal ions, such as Hg2+ or Ag+, and their separation by metal-ion binding ligands,9–11 the reversible pH-stimulated formation of i-motif structures,12 the formation of T-A·T triplex structures and their disassembly by fuel strand-driven displacement processes,13 and the photochemical stabilization/destabilization of DNA duplexes by photoisomerizable intercalators, such as azobenzene units,14,15 provide a rich “tool-box” of reconfigurable DNA topologies. Indeed, these switchable DNA structures were applied to assemble DNA switches16 and DNA machines,17–19 such as tweezers,20,21 walkers22–25 or nanocarriers,26–28 to design “smart” materials acting as gated drug carriers for controlled drug release, such as SiO2 nanoparticles,29–31 metal–organic framework nanoparticles32–34 or microcapsules,35,36 and to prepare stimuli-responsive hydrogels exhibiting switchable stiffness properties for shape-memory,37,38 self-healing,39,40 controlled drug-release41 and mechanical actuating applications.42Topological barriers associated with condensed chromatin regulate the transcription of mRNA through the suppression of the accessibility of transcription factors or RNA polymerase to the transcription machinery.2+-ion stabilized G-quadruplex/kryptofix [2.2.2] (KP), T-A·T triplex displacement strands, and photoresponsive trans/cis azobenzene intercalator units. By mixing two scaffolds of topologically reconfigurable transcription machineries composed of trans-azobenzene-duplex bridged blocker topologies and the T-A·T triplex, the programmed and reversible gated operation of transcription machineries synthesizing target RNAs is demonstrated. It should be noted that in a recent report,43 the triggered operation of a topologically hindered transcription machinery was reported. In this system, the DNA template was caged in a DNA tetrahedral nanostructure. The separation of the tetrahedral nanostructure by a fuel strand yielded an active template that facilitated the RNA polymerase/NTPs replication of RNAs. This system is, however, not reversible and could not be cycled across “ON”–“OFF” states. In addition, we demonstrate the Sr2+-ions/KP switchable transcription machinery in hydrogel microcapsule cell-like containments as a model system for operating a triggered transcription process in a cell-like microenvironment.44–46The reconfigurable DNA structures allow, in principle, the engineering of DNA templates that include topological barriers for operating reversible and cyclic transcription machineries. Here, we report on the design of switchable transcription machineries driven by Sr2+-ion stabilized G-quadruplex/kryptofix [2.2.2] (KP)-induced switchable transcription of the malachite green (MG) RNA aptamer. The association of MG with its aptamer switches-ON the fluorescence of MG (λem = 665 nm) and, thus, the transcription process is followed by the temporal fluorescence changes of the transcribed MG/aptamer complex. The transcription module includes a DNA duplex template consisting of scaffold (1) and the T7 promoter (2). The strand (1) includes three domains: (a) the T7 promoter binding sequence, (b) the guanosine (G)-rich sequence that self-assembles under appropriate conditions to the G-quadruplex unit, and the domain (c) that is complementary to the MG aptamer sequence. Subjecting the template (1)/(2) to T7 RNA polymerase and the NTPs mixture, in the presence of MG, results in the triggered transcription machinery that replicates the domains (b) and (c) to form the RNA strand (b')/(c'), where (c') corresponds to the MG aptamer. The generation of the MG aptamer is followed by the time-dependent fluorescence changes of the formation of the MG/aptamer complex.472+ ions to the reaction machinery, time marked (i), reconfigures domain (b) into the Sr2+-ion stabilized G-quadruplex structure. The formation of the G-quadruplex topological barrier blocks the transcription process and inhibits the formation of the aptamer, 2+-ion stabilized G-quadruplex blocker units, and the reactivation of the transcription of the MG aptamer, 2+-ion stabilized G-quadruplex, see ESI, Fig. S1 and S22+-ion stabilized G-quadruplex (rather than the common K+-ion stabilized/crown ether switching system). This is due to the fact that the K+-ion stabilized G-quadruplex/crown ether system was found to quench the fluorescence of the MG/aptamer complex, thus perturbing the readout signal of the respective transcription machinery.λem = 500 nm).48 The transcription machinery is composed of the template (2)/(3) where the scaffold (3) includes the domains (a), (d) and (e). Domain (a) corresponds to the T7 promoter (2) binding sites, domain (d) consists of a thymine (T)-rich sequence, being capable of forming a T-A·T triplex structure in the presence of an appropriate trigger, and domain (e) is complementary to the broccoli aptamer sequence. Subjecting the template (2)/(3) to a T7 RNA polymerase and NTPs mixture, in the presence of the DFHBI ligand, activates the transcription machinery and the synthesis of the broccoli RNA aptamer that binds DFHBI. The fluorescence changes of the resulting DFHBI/aptamer complex transduce the transcription process. 2+-ion triggered G-quadruplex blocker unit could not be applied to design a DFHBI-binding aptamer transcription machinery. The DFHBI-binding aptamer includes a G-quadruplex domain that associates with the Sr2+ ions and this perturbs the fluorescence features of DFHBI, thus disturbing the readout of the potentially G-quadruplex switching of the DFHBI transcription machinery.The second switchable transcription process included the T-A·T triplex as the topological blocker unit. This is exemplified in trans/cis azobenzene photoisomerizable blocker units. The trans azobenzene units modifying the nucleic acid strand intercalate into duplex nucleic acid structures, thereby cooperatively stabilizing the duplex DNA structure, whereas the photoisomerized cis-azobenzene tether forms a steric hindrance between the duplex nucleic acid structure thereby destabilizing the duplex structures. Thus, by the appropriate design of a photoisomerizable auxiliary azobenzene-functionalized strand, the light-induced switchable transcription machinery is realized. cis-azobenzene functionalized strand (6c) acts, in the presence of T7 RNA polymerase and NTPs, as the photo-triggered transcription machinery. (The strand (6c) is composed of two domains, one domain hybridizes with (7), and the other domain contains the photoisomerized cis-azobenzene units). The strand (5) being a part of the transcription machinery includes the domains (a), (f) and (c). Domain (a) provides the T7 promoter with a binding site, the domain (f) is engineered to interact with the photoresponsive blocker unit (6)/(7), and domain (c) is complementary to the MG aptamer sequence. The cis-azobenzene (6c)/(7) auxiliary unit does not interact with domain (f) of the transcription machinery; thus subjecting the transcription machinery to a T7 RNA polymerase and NTPs mixture, in the presence of MG, triggers-ON the transcription of the MG aptamer, (c′), which is followed by the fluorescence changes of the MG/aptamer complex. λ > 410 nm, leading to the photoisomerization of the cis-azobenzene-functionalized module (6c)/(7) to the trans-state, (6t)/(7). This results in the hybridization of the trans-azobenzene topological unit to the domain (f) of template (5), forming a topological barrier that switches-OFF the transcription process, λ = 365 nm, photoisomerizes the trans-azobenzene blocker element into the cis-state (6c)/(7), resulting in the separation of the cis-azobenzene (6c)/(7) unit from the template and the reactivation of the transcription process, 49,50 the rigid duplex structure of (6t)/(7) introduces a bulky topological barrier that perturbs and inhibits the transcription of the scaffold.The third topologically triggered transcription machinery involved light-induced control over the transcription machinery using trans-azobenzene blocker and the T-A·T triplex blocker as gating units. The system consists of a mixture of the two scaffolds of transcription machineries, state “O” composed of the (2)/(5) and (6c)/(7) photoresponsive machinery that transcribes the MG aptamer and the T-A·T triplex-responsive template (2)/(3) that transcribes the DFHBI-binding aptamer. Subjecting the mixture to a T7 RNA polymerase and NTPs mixture, in the presence of MG and DFHBI, triggers the two scaffolds of transcription machineries to yield the MG aptamer and DFHBI-binding aptamer, panels IO and IIO, respectively. The irradiation of state “O”, λ > 410 nm, transforms the (6c)/(7) module into the trans-azobenzene module (6t)/(7) that hybridizes with domain (f) of template (5), resulting in the blocking of the photoresponsive machinery, state “P”, panel IP, while the triplex responsive machinery is unaffected, panel IIP. That is, the photochemical transition of state “O” to state “P” gates the transcription machinery by blocking the transcription of the MG aptamer, panel IP, while allowing the transcription of the DFHBI-binding aptamer, P. The caged inactive MG aptamer transcription machinery can be, however, switched between “ON” and “OFF” states by the cyclic photoinduced isomerization of the photoresponsive units between the (6c)/(7) (λ = 365 nm) and the (6t)/(7) (λ > 410 nm) units, respectively, panel IP′. Similarly, state “O” can be triggered by the strand (X) that transforms state “O” into state “Q”, where the domain (d) of strand (3) is reconfigured into the topological T-A·T triplex blocked the transcription machinery. That is, subjecting strand (X) to state “O” results in the transcription of the MG aptamer, by the photoresponsive machinery panel IQ, while the transcription of the DFHBI-binding aptamer is blocked, panel IIQ. The T-A·T triplex topologically blocked machinery can be, however, reversibly triggered between “ON” and “OFF” states. The treatment of the system with the anti-blocker strand, (X′), and the subsequent re-treatment of the switched-ON transcription machinery of the DFHBI-binding aptamer with the strand (X′′) result in the cyclic activation/deactivation of the DFHBI-binding aptamer formation, panel IIQ′.The successful design of topologically switchable transcription machineries using different triggers to yield two different aptamers (the MG and DFHBI-binding aptamer) suggested that gated transcription machinery guiding the selective synthesis of target RNAs could be designed. 51,52 polymersomes,53–55 proteinosomes,56–59 colloidosomes60 and coacervate microdroplets.61 Our laboratory developed in the past few years a versatile method to design nucleic acid-based hydrogel microcapsules, and particularly stimuli-responsive microcapsules.62 These microcapsules were loaded with drugs, nanoparticles or proteins and used as functional carriers for the release of the loads. The preparation of the microcapsules involves the impregnation of CaCO3 microparticles with the loads, the functionalization of the particles with promoter-nucleic acid tethers, and the interaction of the modified microparticles with two polymer chains functionalized with two types of pre-engineered hairpins HA and HB. Subjecting the promoter modified microparticles to the hairpin-functionalized polymer chains triggered the inter-polymer hybridization chain reaction (HCR) resulting in a hydrogel coating crosslinked by the inter-hybridized HA/HB duplexes. The subsequent etching of the CaCO3 core templates with ethylenediaminetetraacetic acid, EDTA, yielded substrate-loaded microcapsules. We find that besides using microcapsules as drug carriers, they can be applied as cell-mimicking containments. By appropriate engineering of the hydrogel coating, molecular entities with a molecular weight ca. ≥5 kDa are retained in the cell-like containment with no leakage to the bulk solution, yet a molecular or ionic substrate of a lower molecular weight, ≤5 kDa, can diffuse in-and-out across the microcapsular boundaries.35 We find that the nucleic acid-based hydrogel microcapsules reveal beneficial properties as compared to other protocell containments. The microcapsules revealed high-loading capacities, rapid exchange of low-molecular-weight agents, allowing effective communication with the bulk surrounding solution, low leakage properties towards constituents exhibiting molecular weight ≤ ca. 5 kD, and relatively high stabilities.63 Accordingly, we designed a cell-like containment in which the topologically triggered switchable transcription of the MG aptamer is demonstrated, 3 microparticles. . The loaded CaCO3 particles were, then, coated with polyallylamine hydrochloride, PAH, modified with the nucleic acid promoter (8), and the particles were subjected to two polymer chains PA and PB consisting of carboxymethyl cellulose, CMC, functionalized with the hairpins HA/HB. The hairpin HA was tethered to polymer PA, and hairpin HB was anchored through hybridization with a short tether (9) to polymer PB. (The specific anchoring of HB with (9) is essential for directionality reasons dictating the inter-hybridization of HA/HB). Under these conditions, the promoter strands (8), functionalizing the particles, trigger the HCR process, where (8) opens hairpin HA, and opened HA, opens hairpin HB, and vica versa, resulting in a CMC hydrogel coating of the polymer chains crosslinked by the duplexes comprising inter-cross opened hairpins HA/HB. The subsequent etching of the CaCO3 cores with EDTA resulted in hydrogel-stabilized microcapsules loaded with the tetrahedra-modified (1)/(2a) template, (1)/T-(2a), and T7 RNA polymerase. 3 microparticles coated with the CMC hydrogel before etching. For comparison, the bright field and fluorescence confocal microscopy images of the hydrogel coated CaCO3 particles loaded with T7 RNA polymerase and (1)/T-(2a) prior to etching are provided in panel II and panel III. The fluorescence and bright-field images of the EDTA etched microcapsules are shown in ca. 2.5 μm diameter are formed. The loadings of the microcapsules with the Cy5-labeled (1)/T-(2a) scaffold was estimated to be ca. 2.4 × 10−10 moles in the overall volume of capsules (or 2.6 × 10−16 moles per capsule) and of the T7 RNA polymerase 1.11 × 10−11 moles in the overall mixture of capsules (or 1.23 × 10−17 moles per capsule). The loadings of the microcapsules with fluorescent (1)/T-(2a) was evaluated by following the fluorescence intensities of the resulting microcapsules and concomitantly evaluating the residual fluorescent (1)/T-(2a) in the washing solutions upon the preparation of the microcapsules. The loadings of T7 RNA polymerase were estimated by following the absorbance feature of the protein. /T-(2a) template or with the T7 RNA polymerase. The impregnated microparticles were then subjected to the EDTA etching process under identical conditions used to prepare the microcapsules, and the resulting (1)/T-(2a) solution was analyzed by electrophoresis and compared to a non-etched (1)/T-(2a) solution, Fig. S83 core and the EDTA etching had no effect on the intact structure of (1)/T-(2a). In addition, the activity of the T7 RNA polymerase subjected to the deposition onto the CaCO3 particles and the subsequent etching by EDTA was compared to the parent T7 RNA polymerase activity by comparing the transcription rates of the MG RNA aptamer by the two enzyme systems, Fig. S9.3 impregnation/EDTA etching.The different scaffolds of topologically switchable transcription machineries are operated in a homogeneous buffer solution. Their operation in a cell-like containment could provide a further complex design of transcription processes mimicking native systems. Different cell-like containments acting as cell-like containments were developed in the past few years. These include liposomes,ca. 70% of its activity and the template (1)/T-(2a) retains its intact structure in the microcapsule, cell-like containment and the permeability features of NTPs, MG, Sr2+ ions and KP through the microcapsule boundaries, we applied the system as a functional topologically switchable transcription machinery of the MG RNA aptamer in a cell-like containment assembly, 2+-ion/KP-switchable tetrahedra (2a)/(1) transcription machinery in a homogeneous buffer solution, see Fig. S102+ ions and KP, reversible reconfigurable formation and separation of the G-quadruplex structure, and domain (c) consists of a complementary sequence to the MG RNA aptamer. The treatment of the microcapsules with NTPs and MG triggers-ON the intracapsular transcription of the MG RNA aptamer. Subjecting the microcapsules to Sr2+ ions is, then, anticipated to reconfigure domain (b) of (1) into the Sr2+-ion stabilized G-quadruplex unit that acts as a topological barrier for the transcription process. Under these conditions, the transcription of the MG aptamer is blocked. Further addition of KP to the system separates the G-quadruplex, resulting in the reactivation of the transcription process in the microcapsule. Thus, switchable intramicrocapsule operation of the topologically triggered transcription proceeds in the system. The switchable intramicrocapsule operation of Sr2+-ions/KP-stimulated “ON”/“OFF” transcription process is followed by the time-dependent fluorescence changes of the MG/aptamer complex in the presence of NTPs and MG, and demonstrated in 2+ ions are added to the system. This reconfigures domain (b), of scaffold (1), into a Sr2+-ion stabilized G-quadruplex barrier that blocks the transcription process. At time-interval marked (ii), KP is added to the system. The permeation of KP into the microcapsule separates the Sr2+-ion stabilized G-quadruplex, resulting in the reactivation of the transcription process. At time-interval marked (iii), Sr2+ ions were re-added to the system, and re-blocked the transcription process in microcapsules. It should be noted that the switchable fluorescence changes observed in the microcapsules show noisy fluorescence intensities, as compared to the fluorescence data recorded for an analogous homogeneous buffer system. This is attributed to the fact that microcapsular hydrogel containments represent a microheterogeneous environment that reveals light-scattering that perturbs the fluorescence intensities. Furthermore, the relatively low number of microcapsules used in the total reaction volume, required the operation of the fluorescence spectrophotometer at enhanced sensitivities that resulted in further background noise. The switchable intracapsular transcription of the MG aptamer was further supported by bright field and confocal fluorescence microscopy experiments, t = 0. Only the bright field image of the microcapsule is visible. Panel II depicts the bright field (a) and confocal fluorescence microscopy (b) images of the microcapsules loaded with the (1)/T-(2a) template and T7 RNA polymerase, after a time interval of 24 hours. The green fluorescence of the transcribed MG aptamer loaded microcapsule is observed, indicating the successful transcription of the MG aptamer. Panel III depicts the experimental results corresponding to microcapsules loaded with the (1)/T-(2a) template and T7 RNA polymerase subjected to NTPs, MG and Sr2+ ions, for a time interval of 24 hours. While the bright field image of the capsule is visible (a), no fluorescence of the MG aptamer loaded capsules is observed (b), consistent with the Sr2+-ion stabilized G-quadruplex topological inhibition of the transcription process. Panel IV depicts the images generated upon treatment of the switched-OFF microcapsule system shown in panel III with KP for a time interval of 24 hours. In addition to the bright field image of the microcapsule (a), the green fluorescence of the MG/aptamer complex formed in the microcapsule as a result of switched-ON transcription of the MG aptamer is observed (b). The result is consistent with the KP stimulated separation of the Sr2+-ion stabilized G-quadruplex topological barrier that allows the activation of the transcription process. It should be noted that all fluorescence changes of the MG/aptamer complex are confined to the microcapsule containments. Centrifuged precipitation of the microcapsules does not lead to any detectable fluorescence of the MG/aptamer complex in the surrounding buffer solution, and the loaded precipitated microcapsules can be resuspended by gentle shaking. Specifically, the introduction of a means to gate the transcription machinery, in bulk buffer solutions, suggests that the incorporation of gated transcription/translation machinery into protocell containments could enable the triggered programmed synthesis of target proteins in analogy to natural processes.Realizing that the T7 RNA polymerase preserves 2+-ion stabilized G-quadruplex units, T-A·T triplex structures and photoisomerizable azobenzene-nucleic acid blockers. Other topologically triggered reconfigurable nucleic acid structures, such as redox-triggered ligand/aptamer complexes, enzyme-triggered blocker units consisting of aptamer–ligand complexes or enzyme/DNAzyme nicked duplex blockers could provide a means to switch the transcription processes. In addition, in order to mimic native regulation transcription processes, we introduce synthetic transcription machineries and gated transcription machineries that could lead to dose-controlled and programmed gene expression. The artificial model systems mimicking native topologically modulated transcription machinery introduce a dynamic means to yield programmed and modulated RNAs for possible therapeutic applications. Furthermore, primary attempts to integrate the topologically triggered transcription machinery into cell-mimicking containment microcapsules were provided. While the machinery advances systems chemistry by establishing control over gene expression using supramolecular complexes, important challenges are ahead of us. In particular, the significance of the present results rests on the incorporation of the switchable transcription machinery into artificial cell containments. The co-addition of dynamic networks activating the transcription machinery in cell-like containments or the coupling of the transcription machinery to co-added translation machinery in cell-like containments could be important future steps to design synthetic cell-like containments mimicking native systems.The study introduced an artificial topological means to switch the transcription of RNA. Three different topological barriers to switch the transcription reaction were introduced including SrP. Z., A. F. and I. W. formulated the concepts and methodology of the study. The manuscript was written through the contributions of all authors. All authors participated in the experimental work and have given approval regarding the final version of the manuscript.There are no conflicts to declare.SC-013-D2SC01599D-s001