Target therapy aiming at critical molecules within the metastatic signal pathways is essential for prevention of hepatocellular carcinoma (HCC) progression. Hic-5 (hydrogen peroxide inducible clone-5) which belongs to the paxillin superfamily, can be stimulated by a lot of metastatic factors, such as transforming growth factor (TGF-β), hepatocyte growth factor (HGF), and reactive oxygen species (ROS). Previous studies implicated Hic-5 cross-talks with the ROS-c-jun N-terminal kinase (JNK) signal cascade in a positive feedback manner. In this report, we addressed this issue in a comprehensive manner. By RNA interference and ectopic Hic-5 expression, we demonstrated Hic-5 was essential for activation of NADPH oxidase and ROS generation leading to activation of downstream JNK and c-jun transcription factor. This was initiated by interaction of Hic-5 with the regulator and adaptor of NADPH oxidase, Rac1 and Traf4, respectively, which may further phosphorylate the nonreceptor tyrosine kinase Pyk2 at Tyr881. On the other hand, promoter activity assay coupled with deletion mapping and site directed mutagenesis strategies demonstrated the distal c-jun and AP4 putative binding regions  were required for transcriptional activation of Hic-5. Thus Hic-5 was both downstream and upstream of NADPH oxidase-ROS-JNK-c-jun cascade. This signal circuit was essential for regulating the expression of epithelial mesenchymal transition (EMT) factors, such as Snail, Zeb1, E-cadherin, and matrix metalloproteinase 9, involved in HCC cell migration and metastasis. Due to the limited expression of Hic-5 in normal tissue, it can be a promising therapeutic target for preventing HCC metastasis. Tumor metastasis occurs via complicated processes, including epithelial mesenchymal transition (EMT), migration and invasion of primary tumor, followed by intravasation, extravasation, and colonization at the metastatic loci. The tumor microenvironment in HCC contains a lot of metastatic factors, such as hepatocyte growth factor (HGF)2 and transforming growth factorβ (TGFβ)3, capable of triggering HCC progression via a lot of molecular pathways. Target therapy aiming at critical signal molecules within these pathways is essential for prevention of HCC metastasis.Hepatocellular carcinoma (HCC) is one of the most common causes of death from cancer worldwide. The poor prognosis of HCC is due to high recurrence rate mainly caused by intrahepatic metastasis (about 80%) or extrahepatic metastasis (about 20%)4, HGF5, and reactive oxygen species (ROS)6. Previous report demonstrated TGF-β induced Hic-5 expression to promote extracellular matrix degradation and invasion of MCF10A breast cancer cells7. Recent study indicated Hic-5 can regulate EMT in ovarian cancer cells in a TGFβ1-independent manner8. Our study revealed that Hic-5 could be induced by HGF responsible for HCC progression and may serve as a potential HCC prognosis marker5. In addition, Hic-5 may cooperate with paxillin to regulate metastasis of breast cancer9. In the tumor microenvironment, Hic-5 highly expresses in cancer associated fibroblast required for deposition and remodeling of the stromal extracellular matrix (ECM) to promote non-cell autonomous breast tumor progression10.Recently, Hic-5 (hydrogen peroxide inducible clone-5) which belongs to the paxillin superfamily is emerging as a potential target along the metastatic signaling pathway. Hic-5 expression can be stimulated by TGF-β11, as its name suggest, it also has a great impact on ROS generation. Previously, Hic-5 was found to participate ROS generation during endothelial cell migration11. In this context, Hic-5 serves as an adaptor for assembling focal adhesion complex required for NADP oxidase-dependent ROS production11. In the vascular smooth muscle cells, Hic-5 mediated TGFβ-induced activation of NADPH oxidase required for ROS generation and cell adhesion12. Recently, we also found Hic-5 regulated the ROS-c-jun N-terminal kinase (JNK) signaling pathway for HCC progression5. Notably, Hic-5 seems to locate both upstream and downstream of ROS-JNK cascade. However, whether and how Hic-5 cross-talks with the ROS-JNK cascade in a positive feedback manner was not fully elucidated. In this report, we address this issue in a comprehensive manner, demonstrating that Hic-5 interacted with the regulators of NADPH oxidase, including Rac-1, Traf4, and Pyk2, for activating NADPH oxidase-ROS-JNK-c-jun cascade, which in turn was required for Hic-5 transcriptional activation. This positive feedback circuit was essential for elevating mesenchymal transcriptional factors, such as Snail, Zeb1 and the matrix degradation enzyme, matrix metalloproteinases 9 (MMP-9) and decreasing the epithelial marker E-cadherin, involved in HCC cell migration and metastasis.The molecular mechanisms for Hic-5 to trigger EMT and tumor progression appeared to be closely associated with ROS signaling. Not only that Hic-5 gene expression can be induced by reactive oxygen species (ROS)5. To validate whether this is a general pathway for triggering HCC progression, additional patient-derived cells with higher metastatic potential such as HCC413 were employed. Initially, we investigated whether Hic-5 was required for migration and invasion of HCC413. As demonstrated in Fig. 5. Moreover, cell invasion (using matrigel-coated transwell assay) of HCC413 transfected with Hic-5 siRNA decreased by 85% compared with the control siRNA transfected cell  was suppressed in HCC413 transfected with Hic-5 siRNA by 66% compared with the cells with control siRNA . The decrease of phosphorylated JNK in Hic-5 depleted HCC413 was consistent with that observed in the previous report using HCC3295. Moreover, phosphorylation of c-jun, which is the transcription factor downstream of JNK, was also suppressed by 90%  in Hic-5-depleted HCC413 , known to be involved in tumor progression, were also examined. As shown in Fig. p = 0.01, N = 3), respectively, in HCC413 transfected with Hic-5 siRNA for 48 h (compared with those in HCC413 transfected with control siRNA)    Fig.  and prot 5) Fig.  in HCC415, was employed. This cell line is suitable for ectopic expression of Hic-5 followed by observing its impact on ROS-JNK-c-jun pathway. Our previous report has shown overexpression of Hic-5 increased the level of ROS generation in HCC340 using DCF-DA5. Further, we examined whether both H2O2 and superoxide anions (O2−) can be elevated in HCC340 overexpressing Hic-5. Thus Dihydroethidium (DHE), a probe specific for detecting O2− was also included. As demonstrated in Fig. p = 0.04, N = 3), respectively, in HCC340 overexpressing Hic-5 for 48 h compared with those in the p-CMV group   Fig. . In addi. 5 Fig. . Therefo. 5 Fig. , and pri. 5 Fig. . Moreove. 5 Fig. .Fig. 3Ra11. Also, proline rich tyrosine kinase2 (Pyk2), a nonreceptor tyrosine kinase on the focal adhesion is capable of activating NADPH oxidase-dependent ROS generation and on the contrary, activated by NADPH oxidase-dependent ROS signaling22. As shown by IP/western analysis, Hic-5 can be associated with Rac-1, Traf4, and Pyk2, but not with Snail and MMP9 (as negative controls) in HCC413  known to be associated with Hic-5 for ROS generation on the focal adhesionted Fig. . Togetheted Fig. . On the ted Fig. . The effted Fig. . However5, we further examined whether more inhibitors of signal molecules within the NADPH oxidase-ROS-JNK cascade can also suppress Hic-5 expression in HCC413. For this purpose, in addition to DTT (0.3 mM) and SP (10 μM), inhibitor of NADPH oxidase diphenyleneiodonium chloride , an antioxidant Trolox , an inhibitor of MEK (upstream of ERK) PD98059 (10 μM) were employed. Both mRNA  and a JNK inhibitor such as SP600125 (SP) in HCC329RNA Fig.  and protRNA Fig.  of Hic-5RNA Fig.  were supRNA Fig.  and HGF-RNA Fig.  were greRNA Fig. . Also, qRNA Fig. . In addiRNA Fig. .Fig. 4Inp = 0.005, N = 5) but also greatly decreased the amount of Snail, Zeb1, and MMP9 mRNA in HCC413 , containing 1126 bp upstream of translational initiation site, was employed. As shown in luciferase assays of proHic-5(1126), Hic-5 promoter was constitutively active in HCC413 . In addition, an AP4 region TGCCCAGAGCTGCCTCC located at −958 to −941 bp, nearby the c-jun region. Since AP-4 was known to be critical for EMT and progression of a lot of tumors24, the involvement of AP4 binding region was also examined. For this purpose, deletion constructs proHic-5(800), proHic-5(600), proHic-5(400), proHic-5(250) which excluding 300, 500, 700, and 850, respectively, bp from 5′ end of full-length promoter proHic-5(1126) were employed. As demonstrated in Fig. We further investigated the critical transcription factor binding regions on Hic-5 promoter responsible for activation of Hic-5 gene. According to Genomatix software, there is one putative binding motif of c-jun 5. Herein, a lot of missing links in the Hic-5-ROS-JNK signal circuit were clarified and the EMT related genes regulated by Hic-5 for HCC progression were identified. On the upstream, we found Hic-5 interact with the regulators of NADPH oxidase, including Rac-1, Traf4, and pyK2, to activate its enzyme activity required for ROS generation. In the downstream, we confirm that ROS-JNK pathway indeed is responsible for upregulation of Hic-5 gene expression via the transcriptional factor c-jun coupled with AP4. The Hic-5 thus induced in turn reactivate ROS and JNK, establishing a positive feedback signal circuit that regulates the expressions of EMT related genes, including E-cadhedrin, Snail, MMP9, and Zeb-1, required for HCC migration and progression5  development29, methylmercury-induced ER stress39, and Escherichia coli-induced prostatic inflammation40. In spite of these, the detailed transcriptional mechanisms for Hic-5 gene expression induced via different signaling pathway have not been delineated thus far. Previous study indicated Hic-5 promoter contains an evolutionarily conserved CArG element which is indispensable for myocardin-induced transactivation of Hic-5. Also, they found that serum responsive factor (SRF) binds to this CArG element and is essential for TGF-β-mediated induction of Hic-541. In this study, we found c-jun, the downstream effector of JNK cascade are required for the induction of Hic-5 transcription. Promoter assay also indicated the requirement of the putative c-jun and AP4 binding motif essential for Hic-5 promoter activation. In the future, whether c-jun and AP4 directly bind on the indicated region can be investigated by ChIP assay and EMSA. In addition, we found both c-jun and c-fos  were required for the promoter activity of Hic-5 . Homogenous HCC cell populations were obtained and the sustained proliferation ability (over 20 passages) and metastatic potentials were tested in vitro and in vivo after more than 40 passages. The characteristics of the HCC tumor cell lines were validated by detecting HCC tumor makers such as Glypican 3 (GCP3)46. HGF was from Peprotech . Dithiotheritol was from Sigma . Antibodies for Hic-5, phosphorylated JNK/ERK, c-jun/phosphorylated c-jun, and GAPDH were obtained from Santa Cruz Biotechnology, Inc. . Hic-5 c-DNA expression plasmid (TGFB 1I1) was from OriGene Technologies, Inc. .HCC413 and HCC340, the patient-derived cell lines, were established on January 2014 from parts of HCC tissues obtained from surgery according to the methodology described in our previous reportCells were seeded on a 24-well transwell migration insert  in a complete medium for 24 h. After appropriate treatments, cells that had migrated to the underside of the insert membrane were stained with 0.3% crystal violet. The cells on the topside of the insert membrane were rubbed with a cotton swab. The migrated cells on the underside were imaged using phase contrast microscopy with ×200 magnification. Quantitation of the migrated cell was performed by measuring the intensity of crystal violet staining, using ImageJ software.Cells were seeded on a 24-well transwell migration insert  coated with Matrigel  in a complete medium for 24 h. After appropriate treatments, cells that had invaded to the underside of the insert membrane were stained with 0.3% crystal violet.44. Quantitative real-time qRT-PCR was performed using EZtime Real-Time PCR Premix  with gene-specific primers and probes on a sequence-detection system .Conventional RT-PCR was performed according to that described in our previous report5. The band intensities on the blots were quantified using ImageJ software.A western blots were performed according to our previous studies5. Briefly, intracellular H2O2 and superoxide anions O2− production were monitored by oxidation of permeable dye 2,7-dichlorodihydrofluorescein diacetate (DCF-DA) and Dihydroethidium (DHE), respectively, which react with ROS to form the fluorescent product. The cells were incubated with 20 μmol/L DCFDA or DHE for 30 min and washed twice with PBS. After required treatment, the cells were then harvested for flow cytometry as described previously5. Each determination is based on the mean fluorescence intensity (MFI) of 5000 cells.Cytometric analysis for ROS generation was performed as described in our previous report43.Rac-1 assay was performed according to our recent report2− production will be stimulated with NADPH (200 μM), succinate (5 mM) (with ANM), or xanthine (100 μM). The NADPH-dependent O2− production was determined from the ratio of mean chemiluminescent light units to total protein level.NADPH oxidase activity was measured as described previously. Briefly, the cells were sonicated in 3 × 10-s bursts at 30% power. The cell homogenate was added to the wells of a bioluminescence plate followed by measurement of NADPH oxidase activity in a 50 mM phosphate buffer, containing 1 mM EGTA, 150 mM sucrose, and 10 μM lucigenin. OHic-5 expression was transiently knocked down by 25 nM Hic-5 siRNA  for 48 h, according to the manufacture’s protocol. For overexpression of Hic-5 in HCCs, the cells were transfected with Hic-5 cDNA expression plasmid (TGFB 1I1) driven by CMV promoter (p-Hic-5) for 48 h. Double transfection of p-Hic-5 coupled with Traf4 or Pyk2 siRNA was performed using DharmaFECT Duo Transfection Reagent . The elevation or depletion of Hic-5 was validated by Western blot and RT-PCR.The promoter regions in the full-length promoter plasmids ProHic-5(1126) were amplified from genomic sequence of Hic-5 encompassing 1126 bp upstream of translation start site. The PCR products were ligated into pGL3 vector . The promoter plasmids proHic-5(800), proHic-5(600), proHic-5(400), proHic-5(250) were derived from ProHic-5(1126) by double digestion with various restriction enzymes followed by filling in the restriction site overhangs by Klenow enzyme. Subsequently, the digested DNA fragments were ligated into pGL3 vector.To obtain the site directed mutants of Hic-5 promoter, the core sequence matrix on the indicated transcriptional factor binding regions within wild type Hic-5 promoter proHic-5(1126) were changed into random sequence by KOD-plus-mutagenesis kit according to manufacturer’s protocol .44. Briefly, transfections of the cells with full-length promoter and various truncated formats of Hic-5 promoter inserted in the pGL3 vector were performed using lipofetamin 2000. After required treatments, the cells were harvested and assayed for luciferase activity as described in manual protocol provided by the manufacture . The data were normalized with the cell numbers seeded on 24-well plate for luciferase activity assay.Promoter assay was performed according to our previous reportt test was conducted to evaluate the intensity differences between samples on the Western blot and RT/PCR and the differences in promoter activity between the indicated samples. Quantitative data were expressed as mean ± coefficient variation (CV), indicated by the error bars in each figure.One-way analysis of variance using Student’s Supplemental Fig 1Supplemental Fig 2Supplemental Fig 3Supplemental Fig 4Supplemental Fig 5Supplemental Fig 6


Accurate and efficient transcription termination is an important step for cells to generate functional RNA transcripts. In bacteria, two mechanisms are responsible for terminating transcription: intrinsic (Rho-independent) termination and Rho-dependent termination. Growing examples suggest that neither type of transcription termination is static, but instead are highly dynamic and regulated. Regulatory small RNAs (sRNAs) are key players in bacterial stress responses, are frequently expressed under specific growth conditions, and are predominantly terminated through the intrinsic termination mechanism. Once made, sRNAs can base-pair with mRNA targets and regulate mRNA translation and stability. Recent findings suggest that alterations in the efficiency of intrinsic termination for sRNAs under various growth conditions may affect the availability of a given sRNA and the ability of the sRNA to function properly. Moreover, alterations of mRNA structure, translation, and accessibility by sRNAs have the potential to impact the access of Rho factor to mRNAs and thus termination of the mRNA. Indeed, recent studies have revealed that some sRNAs can modulate target gene expression by stimulating or inhibiting Rho-dependent termination, thus expanding the regulatory power of bacterial sRNAs. Here we review the current knowledge on intrinsic termination of sRNAs and sRNA-mediated Rho-dependent termination of protein coding genes in bacteria. E. coli and many related bacteria, sRNAs often act in concert with the RNA chaperone Hfq. Mutations in hfq are associated with attenuated virulence in many pathogens, suggesting critical roles for sRNAs in pathogenesis  function by base-pairing to target mRNAs, resulting in stimulation or inhibition of mRNA stability and translation. These sRNAs are usually around 100 nt long, and use a short seed region (8–15 nt) to form an RNA duplex with mRNA for regulation  family protein, although SgrS requires transcription termination at its own terminator for function  decreasing pausing and termination  site on the nascent transcript, translocate along the naked RNA and dissociate the elongation complex to terminate transcription  for chitosugars, at its ribosome binding site, inhibit translation and trigger chiP mRNA decay  of mRNAs are another reservoir of potential sites for Rho-dependent termination. Transcriptomic analyses of RNA samples from E. coli treated with sublethal concentrations of Rho inhibitor BCM  that activate rpoS mRNA translation can antagonize Rho-dependent premature termination in the rpoS leader  its distal face binding to an A-rich sequence motif upstream of rut sites in λtR1, and (2) physical interaction of Hfq with Rho protein; this interaction can block Rho binding to other proteins, such as NusG, which has been shown to stimulate Rho termination at some terminators  can positively regulate downstream ORFs by blocking Rho entry.The extent and importance of these levels of regulation are just beginning to be realized. Much still remains to be addressed to fully understand the effect of sRNAs on transcription termination. Unlike translational repression and mRNA degradation, effects on termination must occur rapidly, during the transcription process. This suggests that sRNA pairing with mRNAs must be happening co-transcriptionally; how often does that happen? Are there properties of the particular mRNAs, sRNAs, or transcriptional machinery that will favor this? In addition to the trans-acting sRNAs discussed here, the cis-regulatory riboswitches can also control gene expression through modulation of Rho-dependent transcription termination (Hollands et al., All authors contributed equally and directly to the preparation of this review, and all approved it for publication.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.


Hippo signaling controls cellular processes that ultimately impact organogenesis and homeostasis. Consequently, disease states including cancer can emerge when signaling is deregulated. The major pathway transducers Yap and Taz require cofactors to impart transcriptional control over target genes. Research into Yap/Taz-mediated epigenetic modifications has revealed their association with chromatin-remodeling complex proteins as a means of altering chromatin structure, therefore affecting accessibility and activity of target genes. Specifically, Yap/Taz have been found to associate with factors of the GAGA, Ncoa6, Mediator, Switch/sucrose nonfermentable (SWI/SNF), and Nucleosome Remodeling and Deacetylase (NuRD) chromatin-remodeling complexes to alter the accessibility of target genes. This review highlights the different mechanisms by which Yap/Taz collaborate with other factors to modify DNA packing at specific loci to either activate or repress target gene transcription. Recruitment and/or activation of transcription factors and proteins capable of altering target gene transcriptional activity is a hallmark of many signaling pathways. Alterations in transcriptional activity can be achieved via recruitment of proteins capable of remodeling the chromatin structure through the modification of nucleosome positioning and histone proteins at regulatory regions of target genes ,2,3. TraDrosophila . T. TDrosopyki overexpression coupled with knockdown of brm (or other BAP-specific components) was shown to result in wing disc overgrowth, which was exacerbated as compared to yki overexpression alone. This demonstrates that BAP can limit yki-driven tissue overgrowth. Hyperproliferative wing discs were characteristic of larvae overexpressing Yki or depleted of BAP subunits. Additional malignant features included defects in cell polarity and induction of secreted matrix metalloproteinase 1, which promotes basement membrane degradation [decapentaplegic (dpp) and Wingless (Wg) [dpp and Wg was found to augment the tumor-forming phenotype observed in yki overexpression/brm knockdown wing discs [Tissue growth regulation by Brahma–Yki interactions provides the potential for this protein complex to cause cancer and affect tumor growth. Indeed, dysregulation of both SWI/SNF complexes and Hippo pathway activity can result in cancer phenotypes ,51. Thisradation ,52. Furtess (Wg) . Ectopicng discs . This reDrosophila, cooperation is evident in mammalian cells as well. Investigation into human mammary epithelial cell (MEC) lineage switching between basal and luminal cell fates has implicated TAZ as important in controlling this process. Specifically, TAZ depletion in basal cells, where it is localized to the nucleus, results in lineage switching towards a luminal cell fate. Conversely, ectopic nuclear TAZ expression in luminal cells switches their identity to a more basal cell type [In addition to SWI/SNF–Hippo signaling interactions in ell type . To deteell type . Drosophila and mammals [Brahma-associated factor 53a (BAF53A) has been found to be amplified in head and neck squamous cell carcinoma (HNSCC) along with p63. BAF53A and p63, a DNA-binding transcription factor, form a complex to activate the expression of target genes that promote proliferation and prevent differentiation in HSNCC [BAF53A-p63 repression of WWC1 , which is a regulator of the Hippo–YAP signaling pathway [WWC1 expression was achieved through a BAF53A-p63-mediated reduction in chromatin accessibility upstream of the WWC1 transcription start site. Reduced levels of WWC1 subsequently resulted in increased YAP activity  gene in Drosophila and was initially thought to act as a transcriptional activator [The chromatin protein GAGA factor (GAF) associates with transcriptional machinery components and regulatory protein complexes to alter chromatin structure. GAF is encoded by the ctivator . GAF hasctivator ; includictivator ,58,59,60ctivator ,62,63. Fctivator . The Medctivator . The Medctivator ,66. Drosophila, GAF has been shown to act as a pertinent cofactor for the Yki/Sd–dE2f1 transcriptional program driving cell proliferation  characterization . DNA-binpression .Similar to GAF, the Mediator complex has also been shown to alter DNA packaging via recruitment of chromatin-remodeling complexes ,66. The Drosophila Yki and mammalian YAP to methylate H3K4 and activate transcription [Posttranslational modifications to histones, including methylation, aid in regulation of transcriptional activity. Histone H3 methylation in particular promotes alterations to chromatin structure that affect transcriptional activity . In thiscription ,26. Drosophila S2R+ cells [diap1. Consistent with a functional role, knockdown of Ncoa6 resulted in decreased expression of diap1 in adult wings. Furthermore, increased eye size induced by Yki overexpression was ameliorated by knockdown of Ncoa6 [Ncoa6, a subunit of the Trithorax-related H3K4 methyltransferase complex, was found to be a positive regulator of the Yki–Sd-driven Hippo-responsive transcriptional reporter in R+ cells . Ncoa6 wR+ cells . Overexpof Ncoa6 . These pof Ncoa6 . Drosophila S2 cells and Ncoa6 and Trr were both shown to interact with Yki. Binding of Trr on genomic DNA overlaps with Yki-bound regions, suggesting co-occupancy at Yki target genes. As reported in a similar study, Ncoa6 recruitment was sufficient to drive Yki transcriptional activity [A similar study investigating Yki recruitment of a histone methyl transferase to induce target gene transcription also found that ChIP-seq peaks for Yki overlapped with peaks for the H3K4me3 histone modification. Furthermore, targeting Yki to a novel chromosomal locus induced H3K4me3 modifications in a WW domain-dependent manner . Yki wasactivity ,26 and wWhile the cases documented above describe how Hippo signaling components collaborate with chromatin-remodeling factors to increase gene expression, alterations of chromatin structure to decrease underlying gene activity are also critical for proper genome regulation. One such method of decreasing chromatin accessibility and transcriptional activity is through recruitment of the repressive nucleosome-remodeling and deacetylase (NuRD) complex . The NuRA regulatory complex comprised of Hippo effectors YAP/TAZ–TEAD, TGF-β effectors SMAD2/3, and the pluripotency regulator OCT4, termed the TEAD–SMAD–OCT4 (TSO) complex, has been implicated in governing the switch of human embryonic stem cells (hESCs) to either maintain their pluripotency status or specify their fate towards a mesendoderm (ME) cell type. Cell fate is regulated in part through differential interactions of the NuRD repressor complex to first inhibit expression of loci involved in ME specification until fate induction is triggered, at which point the TSO complex is remodeled to allow for gene activation . The intYAP overexpression revealed transcripts that both increased and decreased under these conditions. This represents another example where YAP/TAZ, which have canonically been thought of as transcriptional activators, are capable of repressing target gene expression. This transcriptional corepressive role of YAP/TAZ was found to depend on TEAD binding, and YAP/TAZ–TEAD were found to directly bind repressed target loci [YAP/TAZ–TEAD collaboration with the NuRD complex to repress target gene transcription has also been verified in recent studies. A screen for genes directly regulated by get loci . The obsget loci . These rThe Hippo signaling pathway has been documented to control tissue growth and homeostasis through its regulation of downstream effectors Yki/YAP and TAZ ,7. CanonDrosophila studies of Hippo and Brahma complexes highlight some of the complexity. Investigations into Yki recruitment of BAP–SWI/SNF chromatin-remodeling complexes demonstrated Yki–Sd interaction with the BAP–SWI/SNF subunit Brahma and regulation of Brahma protein stability by the Hippo kinase [One area of needed investigation is to better define the mechanisms that underlie the specificity of interactions between Hippo components and chromatin-remodeling complexes. o kinase . Brahma–o kinase . Intereso kinase ,33. MoreOf relevance to this issue, recent work has demonstrated a role of the activator protein 1 (AP-1) in driving YAP/TAZ/TEAD activity at distal enhancers . ActivatInteractions of Yki with the GAGA factor and the Mediator complex provide a coactivator function in facilitating Yki target transcription ,30. DespGene repressive activity by YAP/TAZ has been shown to depend on NuRD complex recruitment. The NuRD complex, which has both nucleosome-remodeling and histone deacetylase activity, can be recruited by YAP/TAZ–TEAD to render chromatin inaccessible ,27. The Additional questions regarding Hippo signaling and chromatin remodeling center on the relative timing of interactions between YAP/TAZ–TEAD (or other partners), chromatin-remodeling complexes, and DNA. Do chromatin-remodeling complexes recruit YAP/TAZ–TEAD/other to DNA or vice versa? Is enzymatic activity or target specificity altered by these interactions? What cell states facilitate the protein interactions? Importantly, can the relationships between Hippo components and chromatin remodeling be targeted for therapeutic purposes, particularly with regard to cancer and tissue repair following injury? Cumulatively, the documented collaborations of Yki/YAP/TAZ with chromatin-remodeling factors reviewed herein provides insight into the diverse mechanisms by which Hippo signaling controls gene regulation. With ongoing investigations probing cell-type and tissue-specific roles of the Hippo pathway in governing transcription, it is likely that additional interactions between Yki/YAP/TAZ and the machinery that manages chromatin structure will be discovered.


In contrast to mammals, adult fish display a remarkable ability to fully regenerate central nervous system (CNS) axons, enabling functional recovery from CNS injury. Both fish and mammals normally undergo a developmental downregulation of axon growth activity as neurons mature. Fish are able to undergo damage-induced “reprogramming” through re-expression of genes necessary for axon growth and guidance, however, the gene regulatory mechanisms remain unknown. Here we present the first comprehensive analysis of gene regulatory reprogramming in zebrafish retinal ganglion cells at specific time points along the axon regeneration continuum from early growth to target re-innervation. Our analyses reveal a regeneration program characterized by sequential activation of stage-specific pathways, regulated by a temporally changing cast of transcription factors that bind to stably accessible DNA regulatory regions. Strikingly, we also find a discrete set of regulatory regions that change in accessibility, consistent with higher-order changes in chromatin organization that mark (1) the beginning of regenerative axon growth in the optic nerve, and (2) the re-establishment of synaptic connections in the brain. Together, these data provide valuable insight into the regulatory logic driving successful vertebrate CNS axon regeneration, revealing key gene regulatory candidates for therapeutic development. However, even under these growth-enhanced conditions, regeneration mostly occurs only in a subset of RGCs2, with the majority of axons rarely growing beyond a few millimeters. Furthermore, the regenerating axons frequently grow in an undirected manner, resulting in the “regenerated” axons terminating growth far from their appropriate brain targets9. As such, there remains a gap in our understanding of the genetic programs that drive RGC axon regeneration culminating in target re-innervation and recovery of visual function.Damage to nerves in the CNS as a result of disease or injury most often results in a permanent loss of function in humans. The loss of function stems from the failure of adult mammalian CNS neurons to support regenerative axon growth. In adult mammalian retinal ganglion cell (RGC) neurons, genetic and pharmacologic manipulations of neuron-intrinsic pathways have shown promise in activating a regenerative state after optic nerve injury11, ultimately leading to functional recovery12. It is well known that proteins regulating RGC axon growth and guidance in the developing visual system are highly conserved across vertebrate species, and are transcriptionally down-regulated once the mature circuitry has been established14. In addition, like mammals, optic nerve injury in adult zebrafish induces the expression of axon growth attenuators such as socs3 in RGCs15, suggesting that expression of such negative regulators of axon growth is a normal part of the regenerative program. One major difference in response to CNS axon injury between mammals and fish is the re-expression of a genetic program that promotes axon growth and guidance17. However, we and others have found that the regulation of axon growth-associated genes differs between development and regeneration20. Thus, the difference between mammals and vertebrate species capable of optic nerve regeneration is their ability to reprogram adult RGCs for axon growth in response to optic nerve injury.In contrast to mammals, zebrafish display a remarkable ability to spontaneously regenerate RGC axons. As opposed to regenerating axons in the growth-enhanced mouse models, regenerating axons in the zebrafish optic nerve successfully navigate across the chiasm and re-innervate target neurons in the optic tectum21. What is less clear are the genome-wide changes in expression within the RGCs over time as they first grow toward their intermediate target of the optic chiasm and then navigate toward their principal brain target, the optic tectum. Here we present the first comprehensive analysis of gene expression changes (RNA-seq) and DNA regulatory element accessibility (ATAC-seq) in zebrafish RGCs at specific time points along the axon regeneration continuum from early growth to target re-innervation. Our analysis reveals that successful CNS axon regeneration is regulated by stage-specific gene regulatory modules, and punctuated by regeneration-associated changes in chromatin accessibility at stages corresponding to axonogenesis and synaptogenesis. Together, these data suggest candidates for gene regulatory targets for promoting successful vertebrate CNS axon regeneration.It has been established that specific regeneration-associated gene expression changes in the adult zebrafish retina begins within the first day after optic nerve crush and persist through the re-innervation of the optic tectum22. To achieve a comprehensive picture of the genetic programming driving successful vertebrate CNS axon regeneration, we used RNA-seq to identify changes in gene expression that accompanied axon growth in regenerating RGCs at critical time points after optic nerve injury. We specifically examined how transcript expression in naïve retinas compared with that in retinas dissected from fish at 2, 4, 7, and 12 days post-injury (dpi). Based on the previously established regeneration chronologies, our chosen time-points  > 4), were detected for six out of the seven temporal clusters . Enriched pathways, based on high stringency criteria .In order to understand the regulatory system that guides the post-injury transcriptional program, we identified putative regulatory DNA elements  in RGCs at different stages of regeneration. Assay for Transposase Accessible Chromatin sequencing (ATAC-seq)26.We detected over 40,000 peaklets representing consensus regions of accessible chromatin in RGCs. The majority of peaks were located in intergenic regions, upstream or downstream from annotated genes Fig. . Of the Surprisingly, overall chromatin accessibility changed very little in response to optic nerve injury. In fact, only 233 consensus regions of accessible chromatin (0.5%) were differentially accessible in injured RGCs compared to control RGCs Fig. . All but27 and used a global differential  to compile a list of transcription factor-encoding transcripts associated with regeneration. We discovered 265 definitive transcription factor encoding genes associated with 339 transcripts that were differentially expressed at one or more post-injury time points . Each of these four families of transcription factors included representatives with peak expression early, middle, and late in the regenerative process Fig. , as well30. For each cluster of temporally expressed transcripts we identified potential regulatory elements in the form of accessible chromatin peaklets that were proximal (peaklet center within ± 1 kb from transcription start site) or distal  to the associated gene. Proximal and distal peaklet sequences were then queried for enrichment of binding motifs of transcription factors with similar temporal expression profiles (Table growth toward the midline cluster), we found that motifs for 17 out of the 31 transcription factors queried were enriched in the surrounding regions of accessible chromatin . By comparison, binding motifs for FOSL1 and JUNB displayed a high frequency of co-occurrence with binding sites for 6–7 other transcription factors . A similar trend was observed among proximal peaklets, although the differences were less marked . These differences suggest the possibility that binding site composition and potential transcription factor interactions may be distinguishing characteristics of regeneration-associated promoters and enhancers.Promoters and enhancers rarely function in response to binding of single transcription factors. This is due both to the propensity of transcription factors within the same family to dimerize, and the existence of multiple transcription factor binding sites within a given promoter or enhancer. To identify potential interactions between regeneration-associated transcription factors, we quantified the frequency with which binding sites for differentially expressed transcription factors co-occurred within putative regulatory elements. For example, although KLF6 and WT1 were the most prevalent binding sites within both proximal and distal peaklets, the co-occurrence rate with binding sites with other transcription factors present in this time window was among the lowest Fig. . Within We further quantified the frequency of specific co-occurring binding sites. The co-occurrence of members of the same family was expected due to similarities in recognition sequence specificity. This was observed most clearly in the frequency of shared enriched peaklets between KLF6 and WT1 binding sites, and between the bZIP factors Fig. . HoweverAlthough chromatin accessibility remains mostly stable in RGCs in response to optic nerve injury, we detected a small number of DNA elements in which chromatin accessibility changed in regenerating neurons compared to controls. Intriguingly, the few differentially accessible chromatin peaklets were not evenly distributed across the time points. Instead, most of the elements that became more accessible in response to injury did so at the earliest time point (2 dpi). This led us to postulate a role for these elements in triggering the regenerative growth program. We hypothesized that transcription factors regulated by such elements would not only regulate biological processes necessary for initiation of axon growth, but would also contribute to the regulation of downstream transcription factors that in turn regulate subsequent phases of regeneration.jun was the only regeneration-associated transcription factor encoding gene that was located within at least 100 kb of a differentially accessible chromatin region. In fact, the jun gene is flanked by three peaklets that are differentially open at 2 dpi with respect to controls  The elements may be responsible for initiating the expression of key regeneration-associated transcription factors, which would subsequently regulate other factors in the hierarchy; and (ii) The elements may serve to shift the higher order chromatin structure to reposition enhancers and promoters for the transitions necessary in adult RGCs to reinitiate programs for axonogenesis (2 dpi), and synaptogenesis (12 dpi).41. Supporting our second hypothesis, we find that roughly half the differentially-accessible regions at both early and late times are enriched in CTCF binding sites. CTCF is a transcription factor that has recently been implicated in mediating chromatin looping and marking the boundaries of topologically associating domains42. A logical next step would be to functionally validate interactions between the predicted jun promoter and enhancers, as well as additional long-range regulatory interactions between stably and differentially accessible elements using chromatin capture and genome editing technologies.Our data contain evidence supporting both hypotheses. Supporting the first hypothesis, we find that the gene encoding transcription factor JUN is flanked by promoter and distal enhancer elements that increase in accessibility during axon regeneration. In fact, JUN involvement in such epigenetic activation of pro-regenerative genes in response to axon injury was suggested in a recent review17. In addition, our temporal approach using RNA-seq has greatly expanded detection of regeneration-associated changes in gene expression over previous studies. We anticipate that functional testing of the networks predicted by this model will substantially expand our understanding of the molecular mechanisms governing successful optic nerve regeneration.The combination of gene expression and genomic accessibility over time provides a powerful model of axon regeneration-associated gene regulatory networks. As with any modeling approach, empirical testing and improved technology are expected to refine and strengthen the predictive power of the model. For example, current methods for associating enhancers with target genes will improve with the functional testing of these types of interactions as described above. Another potential caveat of these studies is that the transcriptomic analysis was carried out on whole retinas, rather than FACS-sorted RGCs that were utilized in examining chromatin accessibility. Because RGCs are the only cells directly injured by optic nerve crush, a comparison of gene expression between injured and uninjured retina should primarily reflect changes in RGCs. Yet, it is possible that some of the transcriptomic changes detected may be occurring in other cells within the injured retina, such as infiltrating microglia or retinal neurons connected to RGCs. However, we find good concordance between our transcriptomic results and microarray studies based on RNA extracted from RGCs isolated by laser capture microdissection43. However, to our knowledge, this is the first gene regulatory network analysis of optic nerve regeneration that couples temporal analysis of gene expression with the identification of putative regulatory interactions based on chromatin accessibility and transcription factor expression. We expect that these findings may be applicable to neurons in other regions of the CNS undergoing regenerative axon growth, such as the spinal cord and brain. In order to facilitate comparisons between our data and those derived from other regenerative models, we have created an interactive web application 44. This app enables users to upload a gene list of interest from any Ensembl-supported species and determine how those genes are expressed and potentially regulated in the context of optic nerve regeneration. Such cross-species and cross-system analyses will facilitate identification of novel pathways specifically associated with successful CNS regeneration.In summary, these data provide a roadmap for the identification of key combinations of transcription factors necessary to reprogram adult RGCs for optic nerve regeneration. Similar approaches have been employed to discover transcription factors necessary for direct reprogramming of somatic cells to produce motor neuronsDetailed methods can be found in supplementary information.Institutional Animal Care and Use Committee (IACUC) and carried out in accordance with the relevant guidelines and regulations thereof. The Tg (Tru.gap43:egfp) mil1 (aka fgap43:egfp) transgenic line and Ekkwill wild type strain were maintained as previously described25. Optic nerve crush (ONC) lesions were performed on adult zebrafish, 7–9 months of age, as previously described21.Zebrafish husbandry and all experimental procedures were approved by the University of Wisconsin – Milwaukee RNA was extracted and purified from retinas dissected from naïve (0 dpi) and regenerating adult fish at 2, 4, 7, and 12 dpi. RNA was extracted using the RNeasy Micro kit (Qiagen) and concentrated using the RNA Clean & Concentrator kit (Zymo). Three replicates were obtained for each time point. Whole retinas were used for transcriptomic studies because the quality of the RNA was much higher from whole retina. cDNA libraries were generated for each RNA sample using Tru-Seq Stranded Total & mRNA Sample Prep Kits, (Illumina 20020595). Each cDNA library was indexed for multiplexing and subsequently sequenced on four lanes of the Illumina Hiseq2000. Libraries were sequenced at 50 bp, 30–40 million paired-end reads/sample.45.After merging technical replicates of RNA-seq samples across lanes, adapter sequences were trimmed , sequence quality was validated , and transcript abundance was estimated using 500 bootstrap samples 46, and purified using DNA Clean & Concentrator kit .Chromatin was extracted from nuclei isolated from purified populations of RGCs. Retinas were dissected from naïve and regenerating fgap43:egfp fish as described above, and enzymatically dissociated for FACS. Cell suspensions were pooled from multiple retinas at each time point , and sorted for RGCs expressing GFP using a Becton Dickinson FACSAria™ III sorter. Although levels of endogenous Gap43 are low in the naïve adult retina, the GFP transgene tends to be long lived and is detectable by FACS at levels that are above background but below the level of visual detection. This enabled us to sort RGCs from naïve retinas since new RGCs continue to be added throughout the life of the animal and low-levels of GFP persist in RGCs even after these new RGCs have established connections in the tectum. However, we required twice the number of naïve retinas compared with injured retinas to accumulate the 50,000 RGCs required for constructing the ATAC-seq libraries. Three replicates of pooled cells were collected for each time point. ATAC-seq libraries were prepared using the Tn5 transposase system  as previously describedPrior to running the full sequence, sequencing depth was estimated and one sample was below the cut-off criteria and therefore omitted. The remaining fourteen samples were indexed for multiplexing and subsequently sequenced on four lanes of the Illumina Hiseq2000. Libraries were sequenced at 50 bp reads to obtain approximately 25 million paired-end reads/sample.47). Duplicate and multiple mapped reads were removed using samtools (v1.6)48. After concatenating aligned reads across all replicates and time points, MACS249 (v2.1.1.20160309) was used to call peaks from aligned reads. Only peaks with a p-value < 10−10 were retained for subsequent analyses. For each remaining subpeak summit, a 500 bp “peaklet” interval was defined using GenomicRanges (v1.30.3)50. We refer to these peaklets as consensus regions of accessible chromatin. Open chromatin in each replicate of each time point was then quantified using DiffBind  by counting the number of overlapping reads for each retained peaklet.After merging technical replicates of ATAC-seq samples across lanes, adapter sequences were trimmed  and sequence quality was validated . Reads were aligned to GRCz10 as well as the transgene sequence (BWA-MEM (v0.7.9a-r786)51.We used motif analysis to determine potential binding sites of differentially expressed transcription factors within regions of accessible chromatin identified by ATAC-seq. Motif enrichment and discovery was carried out within the MEME Suite of motif-based sequence analysis tools (version 5.0.3)52, either comparing each time point post injury  to the initial time point (0 dpi) using a Wald test statistic or using a likelihood ratio test (LRT) to compare the full model with a time factor versus the null model, controlling the false discovery rate (FDR) at 5% or 1%53. Expression heatmaps  were produced using ComplexHeatmap54 (v1.17.1), where transcript clusters were identified using the K-means algorithm, and hierarchical clustering  was used to cluster rows. The Ingenuity Pathway Analysis tool  was used to analyze enrichment of molecular and functional gene networks within the differentially expressed gene sets (FDR < 0.05) at each time point after injury  compared with the initial time point (0 dpi).Following pseudoalignment and quantification of transcripts, differentially expressed transcripts were identified using Sleuth (v0.29.0)55 was used to identify differentially accessible peaklets in an analogous manner to the RNA-seq analysis described above. Peaklets with FDR-controlled p-values < 0.05 in one of the four comparisons were considered to be differentially accessible. ChIPpeakAnno (v3.12.7)56, the danRer10.refGene UCSC annotation package (v3.4.2), and AnnotationHub (v2.10.1) were used to annotate peaklets with genes. Specifically, non-exonic  peaklets overlapping a transcription start site (TSS) or within 1 kb of a TSS were considered to represent proximal peaks, whereas those greater than 1 kb but less than 100 kb of a TSS were considered distal peaks. All statistical analyses were performed in R (v3.4.3). Integrative Genome Viewer (IGV) was used to visualize RNA-seq and ATAC-seq alignments57.After quantifying peaklet accessibility, DESeq2 (v1.18.1)Supplementary Information


Artemisia annua, is the active ingredient in the treatment of malaria. Jasmonic acid, one of the phytohormones, is an important elicitor of artemisinin biosynthesis by enhancing transcription levels of transcription factors. SPL transcription factors are plant-specific transcription factors of plant growth, development, and secondary metabolism regulation. However, to date, the SPL transcription factors that regulate artemisinin biosynthesis is currently unclear. Here, we show that an SPL transcription factor can positively regulate artemisinin biosynthesis by binding to the promoter of artemisinin biosynthetic pathway genes. We screened AaSPL2 by gene expression profiles analysis in 14 SPL transcription factors. We demonstrated that AaSPL2 can activate the promoter of DBR2 by dual-LUC assy. Moreover, in the AaSPL2 overexpression plants, the artemisinin content was increased by 33–86%, and in the AaSPL2 -RNAi transgenic plants, artemisinin content was decreased by 33–65%. These data suggest that AaSPL2 and DBR2 interact with a “GTAC” cis-element in the DBR2 promoter, mediating the transcriptional activation of DBR2 in response to JA and resulting in the improvement on artemisinin content.Artemisinin, an important compound produced by  Artemisia annua L  has been the frontline treatment for malaria in recent years . Artemis annua L . The art annua L . In rece annua L . Because annua L . Our aimA. annua lies between 0.5–1.2%, while the purification yields are typically in the range of 50–80% of the crude extract   is an important conserved elicitor of plant secondary metabolism. It triggers extensive transcriptional reprogramming and leads to an increase in the expression level of the gene encoding the key enzyme for secondary metabolism biosynthetic pathway . JA can ynthesis . ERF189 se (PMT) . JA is ir of ADS . We haveynthesis . Many tremisinin . The bHLand DBR2 .SQUAMOSA Promoter-Binding Protein-Like (SPL) genes are functionally diverse, including modulation of grain size, grain shape, branching, vegetative phase change, shoot regeneration and flowering time  NaOCl (sodium hypochlorite) solution for 10 min, and rinsed three times with sterile water. Subsequently, the seeds were planted on MS medium (A. annua (about 5–7 cm in height) were cut and used as explants for transformation with the Agrobacterium tumefaciens-mediated leaf disk transformation method  were sown directly on soil (vermiculite: perlite: peat moss = 7: 0.5: 2), 1 week later, the tobacco seedlings were transferred into the new pots and cultured with the same conditions as A. annua. Five weeks later, the leaves of tobacco can be used for A. tumefaciens infiltration assay.Seeds of tobacco  under the driven of CaMV35S promoter to generate CaMV35S:: AaSPL2 (overexpression vector). For RNAi interference assay, a 300 bp AaSPL2 fragment was fused into PENTR vector via the BP reaction  and then introduce into the pHELLSGATE12 vector via the LR reaction  to generate the final RNAi- AaSPL2 vector.AaSPL2 and RNAi constructs AaSPL2-RNAi were introduced into A. tumefaciens strain EHA105 by a conventional freezing-and-melting method . For plant hormone treatment, 4-week-old 00 μM MJ . Then thThe cetyltrimethylammonium bromide (CTAB) method was employed to obtain the genomic DNA of the transgenic plants . The forA. annua plants were used for RNA isolation by plant RNA Kit (Tiangen) according to the manufacturer’s instructions. The isolated total RNA was reverse-transcribed into cDNA using the TaKaRa cDNA synthesis kit. qRT-PCR assays using SYBR Green PCR Master Mix kit (Tiangen) as a detector were performed in Roch LightCycler®96 system according to the manufacturer’s instructions. The comparative cycle threshold (CT) method was estimated relative transcript levels of samples and means of triplicate independent PCRs. The β-actin gene of A. annua was used to estimate relative mRNA levels. The qRT-PCR primers are listed in Young leaves of -ΔΔCT method was used for estimating the relative gene expression level according to the following formula . Equal volume of the GTAC-box forward primer (10 μM) and reverse primer (10 μM)   were mix (10 μM) . Yeast c (10 μM) . The seqDBR2 was fused to firefly luciferase gene on the plasmid pGreenII 0800-LUC. The sequenced plasmid was introduced into A. tumefaciens strain EHA105. Then incubated Agrobacterium cells were harvested by centrifugation and resuspended in MS medium (containing 10 mM MES and 150 mM acetosyringone) to an OD600 nm of 0.6. After 3 h incubation at room temperature, bacteria (reporter strain DBR2pro::LUC mixed with effector strain 35Spro::AaSPL2 1:1) were injected into tobacco leaves. Forty-eight hours’ later, the injected tobacco leaves were collected for dual-LUC assay. The dual-Luciferase Reporter Assay system of Promega was used for dual-Luc assays according to the manufacturer’s instructions.For the dual-LUC assay, the promoter of A. annua were collected at pre-flowering stage and dried at 50°C for 24 h. then all the leaves were ground into powder. A sample of 0.1 g powder was suspended in ethanol (2 mL) for ultrasonication. Sampling was carried out in triplicate. Thirty min later, Samples were centrifuged for 15 min, 4°C, at 12 000 g. The supernatant was filtered through a 0.22 mm pore size filter and then analyzed by the Waters Alliance 2695 HPLC system. Artemisinin standard (98% pure) was purchased from Sigma. The approach for detecting artemisinin using HPLC-ELSD has already been described  in A. annua with the GenBank accession number (2 of 4.89 to 5.3. The details of the gene character of SPLs are shown in the The SQUAMOSA promoter-binding protein (SBP) genes contain a conserved domain, which encodes 76 amino acid residues in length for the interaction with DNA . To idendatabase  using thabase  after treatment with JA (100 μM). To this end, the expression intensity and hierarchical clustering (HCL) of SPL genes were formatted as a heatmap by Mev4.9.0 software.3 Exogenous application of JA to A. annua seedlings resulted in strong activation of both AaSPL2 and AaSPL13  and buds, confirming previous studies that artemisinin was produced in the glandular trichomes   . The Q-P, and L6 .AaSPL2 contains a conserved SBP domain in the 289–471 regions. A phylogenetic tree by using nucleic acid sequences was built to investigate the phylogenetic relationships among the SPL proteins in A. annua, Arabidopsis and rice other species. The results indicated that AaSPL2 belongs to the subfamily of AtSPL2/10/11 transcription factors  promoter. Agroinfiltration method was performed to infiltrate m leaves . Controlm leaves , whereasm leaves .cis-element in the promoter  was employed to detect if the expression of DBR2 can be regulated by the product of the transcription factor AaSPL2  in A. annua. The transgenic plants were detected by PCR. Overexpression of AaSPL2 in A. annua resulted in enhanced AaSPL2 expression upon wild-type  can redirect carbon flux to the artemisinin pathway and increase artemisinin contents for several folds (cis-acting elements in the target genes promoter. Many transcription factors are involved in JA signaling cascades and regulate the biosynthesis of artemisinin. Such as AP2/ERF transcription factors AaERF1 and AaERF2, they bind to the promoter of ADS and CYP71AV1 directly (CYP71AV1, DBR2, and ADS, respectively (AaSPL2 mRNA level was enhanced under the treatment of JA and the content of artemisinin was increased in the AaSPL2 overexpression plants also (Since JA and its cyclic precursors and derivatives play important roles in inducing the genes encoding enzymes involving in the biosynthesis of secondary metabolites in the plant . So JA bal folds . JA enhaal folds . JA imprdirectly . Anotherdirectly , which iectively . AaSPL2 nts also . AtSPL9 nts also . On the nts also . Artemisnts also . In our nts also , suggestSalvia miltiorrhiza (G. hirsutum. SPLs play important roles in fiber initiation, branching, root growth, grain size and yield, suggesting that SPL transcription factors play key roles in G. hirsutum growth and development. The reasons for so many SPLs in the G. hirsutum are the tandem duplication events or by assembly error (Through genome-wide identification, a total of 14 SPLs have been found in iorrhiza . There aiorrhiza . A systely error . In thisly error  and induly error , play imly error . Howeverly error , therefocis-regulatory specific core “GTAC” consensus sequences in the promoters of their target genes. Previously, the core “GTAC” consensus sequence of DFR has been identified as the binding motif of SPL9 (TPS21 promoters and activates its expression to regulate the biosynthesis of β-caryophyllene (A. annua may function similarly as an SPL in Arabidopsis. The phylogenetic tree indicated that AaSPL2 may homologous to the AtSPL3 in Arabidopsis.SPL transcription factors can regulate gene expression through binding to  of SPL9 . Plant t of SPL9 . Terpenephyllene . ArtemisAaSPL2 mainly expressed in the buds (AaSPL2 in A. annua may play the role in the regulation of sesquiterpene artemisinin. AaSPL2-OX and AaSPL2-RNAi results show that SPL2 can modulate artemisinin (AaSPL2 partially increases the transcription levels of artemisinin biosynthetic gene ADS, CYP71AV1, DBR2, and ALDH1 supported the hypothesis that enhancing of AaSPL2 expression level may be a plausible mechanism for increase artemisinin content.The expression patterns indicated that the buds , which iemisinin  and DHAAemisinin . The facZL, KT, WC, and LZ conceived and designed the entire research plans. BP, YL, and ZL performed the most of the experiments. BP and ZL provided the technical assistance. ZL wrote the manuscript. ZL, YW, and LZ helped with the organization and editing.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.


Bmovo gene, Bmovo-1, Bmovo-2, Bmovo-3 and Bmovo-4 from the silkworm ovary and verified that ovarian development was regulated by the BmOVO proteins. Results: To understand the regulatory mechanisms of ovarian development, the regulation of four BmOVO isoforms on the B. mori ovarian tumor (Bmotu) promoter activity was investigated with luciferase reporter assays. The results showed the Bmotu promoter activity was positively regulated by BmOVO-1, BmOVO-2, BmOVO-3 and BmOVO-4 in a dose-dependent manner, of which BmOVO-2 had the highest transcriptional activation. However, the first (A1) and third acidic domains (A3) at the N-terminus of BmOVO-1 are transcriptional repression domains, while the fourth (A4) and fifth acidic domains (A5) are transcriptional activation domains. A recombinant BmOVO zinc-finger domain was found to bind to the GTACCGTTGTA sequence located at the Bmotu promoter. Furthermore, the Bmotu promoter activity was negatively regulated by ‘Tal-like’ peptide, which can trigger BmOVO-1 degradation at the N-terminus.In our previous study, we identified four isoforms of the Bmotu promoter activity and ovarian development in the silkworm.These results will help us to further understand the regulatory mechanisms of BmOVO isoforms on The online version of this article (10.1186/s12864-019-5697-y) contains supplementary material, which is available to authorized users. Drosophila, the ovo locus encodes three isoforms, Svb, OVO-A and OVO-B [2H2 zinc fingers at their carboxyl (C) termini, which bind to specific DNA sequences [varian tumor (otu), a target gene of OVO proteins, is essential for the viability and differentiation of the female germline in Drosophila [otu gene expression while OVO-A represses it [OVO proteins belong to members of the zinc finger protein family and serve as transcription factors that regulate gene expression in various differentiation processes. OVOs not only participate in the development of the neural tube and germ cells, but also play an important role in eye regeneration and the maintenance of the eye in adult planarians –6. In Drnd OVO-B . They shequences , but theequences , 10. OVOequences . Ovarianosophila . OVO-B pesses it , 12. Moresses it .Bombyx mori), a model species of Lepidoptera, has many genes which are homologous to those of Drosophila. In our previous studies, four alternatively spliced isoforms of the B. mori ovo (Bmovo) gene, which have been designated as Bmovo-1, Bmovo-2, Bmovo-3 and Bmovo-4 according to their deduced molecular weights from large to small, were identified in the ovary. Sequence comparisons showed that 203 amino acid residues were conserved at the C-terminus among BmOVO-1, BmOVO-2, BmOVO-3 and BmOVO-4, and four common C2H2 zinc fingers were found at the C-termini of BmOVO-1, BmOVO-2 and BmOVO-3, while only one was found in BmOVO-4, suggesting that BmOVOs may be transcription factors [Bmovo-1 overexpression in silkworm ovaries might promote anabolism for ovarian development [The silkworm  is homologous with Drosophila otu, which is essential for female germline differentiation in Drosophila [Bmotu expression level was up-regulated when Bmovo-1 was overexpressed in silkworm ovaries and down-regulated when Bmovo was silenced [Bmotu is a target gene, but to date it is still unknown whether BmOVOs can directly bind to the Bmotu promoter to regulate its expression. Therefore, to elucidate the molecular mechanism of Bmovo gene splicing in regulating the expression of otu gene can help us to further understand the regulatory mechanisms of BmOVO on Bmotu promoter activity and ovarian development in the silkworm, thus providing the theoretical basis and molecular target for regulating ovarian development and increasing silkworm silk production through genetic manipulation.Sex determination and differentiation of osophila . The Bmosilenced , suggestDrosophila, the small peptide Tal can induce selective hydrolysis of the N-terminal transcriptional repression domain of Ovo/Svb, which leads to a change from transcriptional repressor to a transcriptional activator [tal-like gene (NM_0010998471.1) was also found in B. mori, but whether BmOVOs are hydrolyzed by the small peptide encoded by this tal-like gene is unknown. In this research, the regulation of the four BmOVO isoforms on Bmotu promoter activity was investigated with luciferase reporter assays, the binding site of BmOVO to the Bmotu promoter was identified with an electrophoretic mobility shift assay (EMSA) and the BmOVO-1 degradation triggered by the Tal-like small peptide was studied. We found that the BmOVOs are transcriptional activators that directly bind to the Bmotu promoter, and degradation of BmOVO-1 at its N-terminus is mediated by the Tal-like small peptide. These results will help us to further understand the regulatory mechanisms of BmOVO on Bmotu promoter activity and ovarian development in the silkworm.Moreover, in ctivator , 18. A tBmovo gene  were controlled by the B. mori baculovirus ie-1 promoter, while luc gene expression was driven by the promoter of Bmotu  and the reference plasmid pRL-TK (1 × 1010 copies) into BmN cells (105 cells). The results showed that BmOVO-2, BmOVO-3 and BmOVO-4 positively regulated Bmotu promoter activity, with BmOVO-2 having the highest transcriptional activation activity  were constructed. In these vectors, the four spliced isoforms of the BmOVOs are transcription factors with regulatory functions that depend on the N-terminal functional domains. Our previous study indicated that there are six acidic domains A1–A6) and two basic domains (B1 and B2) at the N-terminus of BmOVO-1, two acidic regions (A4 and A5) and two basic domains (B1 and B2) in BmOVO-2 and only one acidic region (A6) in BmOVO-3 while no known functional domains were detected in BmOVO-4. Furthermore, BmOVO-4 has only one zinc-finger (Z4), while BmOVO-1, − 2 and − 3 have four zinc-fingers (Z1–Z4) at their C-termini  . The results showed that both the A1 and A3 domains had transcriptional inhibition on the Bmotu promoter and the both A2 and B1 domains didn’t have significant transcriptional regulation, while both A4 and A5 domains had transcriptional activation activities  Fig. a were coB. mori is unclear. In this study, the tal-like expression vectors  were utilized. The mixture of the tal-like expression vector (1 × 1011 copies) and pFast-potu5-Luc (1 × 1011 copies) was co-transfected into BmN cells with pRL-TK (1 × 1010 copies), and the dual-luciferase reporter assay results showed that the Bmotu promoter activity was significantly reduced in the transfected cells with tal-like expression plasmids compared with the controls, in which the transfected cells did not have tal-like expression plasmids, suggesting that the Bmotu promoter activity was down-regulated by the B. mori Tal-like peptide  (1 × 1011 copies) and pFast-potu5-Luc-ie1-Bmovo1 (1 × 1011 copies) was higher than that in the transfected BmN cells with the tal-like gene expression vector (1 × 1011 copies) alone, but the Bmotu promoter activity in the co-transfected BmN cells with a mixture of pIZT/V5-His-B (1 × 1011 copies) and pFast-potu5-Luc-ie1-Bmovo1 (1 × 1011 copies) was lower than that in the transfected BmN cells with pIZT/V5-His-B (1 × 1011 copies), suggesting that Bmotu promoter activity was negatively regulated by the Tal-like peptide  but could be rescued by BmOVO-1 to a certain extent  gene was co-transfected into BmN cells with each tal-like gene expression vector, respectively . The cells containing pIZT/V5-His-tal or pIZT/V5-His-B produced red fluorescence, but that co-transfected with pIZT/V5-His-5A1–4 + B or pIZT/V5-His-A1–4 + B did not  are involved in important signaling pathway and associated with silkworm growth and development processes [Bmotu promoter, pFast-potu-Luc-ie1-Bmovo2 (1 × 1011 copies) was respectively co-transfected with the pIZT/V5-His, pIZT/V5-His-STAT, pIZT/V5-His-DPP, pIZT/V5-His-Daw, pIZT/V5-His-Ror2 and pIZT/V5-His-BBX-B8 plasmids (1 × 1011 copies). The dual-luciferase reporter assay showed that Bmotu promoter activity clearly decreased in the cells that were co-transfected with pIZT/V5-His-STAT, pIZT/V5-His-DPP, pIZT/V5-His-Daw, pIZT/V5-His-Ror2 or pIZT/V5-His-BBX-B8 compared with the activity of the cells co-transfected with pIZT/V5-His  were predicted by JASPAR CORE (http://jaspar.genereg.net/cgi-bin/jaspar_db.pl) based on the conserved sequence  of the Drosophila OVO target site. Three candidate binding sites, CE1 , CE2  and CE3  were found in the Bmotu promoter sequence  , and we found that the second base T mutated to C  at probe otu-A, causing its ability to bind with the nucleoprotein to be weakened. Additionally, the eleventh base A mutated to G , leading to enhanced binding capacity, while a clear change was not found when the third base A was mutated to G   also showed that three bases  of the CE1 element might play an important role in BmOVO zinc finger binding to CE1   vary in the gonads of the silkworm at different developmental stages [Bmotu is a target gene of the BmOVO proteins [Bmotu gene expression in the ovary of the silkworm at different developmental stages is regulated by different BmOVO isoforms. In the present study, we found that the transcriptional regulatory activity of the four isoforms of BmOVO on Bmotu was different: BmOVO-2, BmOVO-3 and BmOVO-4 are transcriptional activators. However, the positive regulatory effects of BmOVO-1 were not significant in cells transfected with a low dose of the Bmovo-1 expression vector, while the positive regulatory effect was increased in the cells transfected with a high dose of this vector, suggesting that BmOVO-1 is also a transcriptional activator. However, in vitro system cannot fully represent the situation in vivo, only to regulating the expression of endogenous BmOVO can verify the regulatory roles of BmOVO isoforms, which needs to be studied at the individual level. It may be necessary to establish BmOVO transgenic silkworm to solve this problem in the future. In Drosophila, the ovo locus encodes three isoforms, Shaven baby (Svb), OVO-A and OVO-B [otu gene expression, while OVO-A is a repressor [Bmovo locus might also encode a novel BmOVO based on N-terminal domain analysis of the known BmOVO, which may be a transcriptional repressor. Our previous study predicted that BmOVO-2 may be a transcriptional activator [Bmotu promoter, indicating that BmOVO-2 has a positive regulatory effect, which was consistent with the predicted results. BmOVO-3 significantly increased the activity of the Bmotu promoter. The N-terminus of the BmOVO-3 protein has only one acidic domain (A6), so it can be considered that the A6 acidic domain of BmOVO-3 is a transcriptional activation domain. BmOVO belongs to the C2H2-type zinc finger family, and generally three or more of these zinc fingers are required for binding to target DNA. BmOVO-4 only has one zinc finger structure and no known regulatory domain at its N-terminus, but BmOVO-4 positively regulated Bmotu promoter activity; therefore, we hypothesized that BmOVO-4 may interact with other trans-acting factors to lead to the increased expression of target genes. Usually, transcription factors bind to target DNA sequences in homodimers or heterodimers. It was found that the transcriptional activity of the Bmotu promoter could be improved by the interaction of BmOVO-1 with BmOVO-2, BmOVO-3 and BmOVO-4, the interaction of BmOVO-2 with BmOVO-3 and the interaction of BmOVO-2 with BmOVO-4, suggesting that these four BmOVO isoforms compete for binding sites and cross-regulate the expression of target genes as homodimers and heterodimers. Our previous study showed that BmOVO-1, BmOVO-2 and BmOVO-4 are mainly expressed in the ovaries, while BmOVO-3 is mainly expressed in the testes [Previous studies have shown that the expression profiles of the l stages . Bmotu iproteins  that is nd OVO-B . OVO-B pepressor , 12. Howctivator , becausee testes ; therefoDrosophila OVOs, one glycine-rich region, two acidic regions and an extensive glutamine/histidine-rich region comprise the activation region; a charged basic region and a serine-rich domain comprise the repression region [Drosophila OVO, suggesting differences in function between BmOVO and Drosophila OVO. In this study, we found that the A1 and A3 acidic domains at the N-terminus of BmOVO-1 were transcriptional repression regions, and the A4 and A5 acidic domains were transcriptional activation regions, while the transcriptional regulation of the A2 and B1 domains was not significant. The four BmOVO isoforms have different effector domains at their N-termini, which result in differences in transcriptional regulation activities between the BmOVO isoforms; this result was confirmed by dual-luciferase reporter assays. It has been found that when Drosophila OVO-B was fused with the transcriptional repression domain of Drosophila OVO-A, Drosophila OVO-B was changed from a transcriptional activator into a transcriptional repressor [cis-acting factors.OVO-B and OVO-A are transcription factors with opposing regulatory activities due to different effector domains at their N-termini. In n region . The disepressor . A similepressor , 22, 23,Drosophila, the small peptide Tal induces selective hydrolysis of the N-terminal transcriptional repression domain of Ovo/Svb mediated by the proteasome, which leads to a switch of Ovo/Svb from a transcriptional repressor to a transcriptional activator [Bmotu promoter activity that is repressed by the small peptide Tal-like, suggesting that the transcriptional activation of BmOVO-1 is increased by the hydrolysis of the N-terminal transcriptional repression domain of BmOVO-1, triggered by Tal-like. These results indicate that BmOVO protein levels are not only determined by the transcriptional level of the different isoforms, but also by post-translational degradation.In ctivator , 18. TheDrosophila, B. mori, planarians, nematodes, zebrafish and mouse, and they interact with multiple signaling pathways and regulate growth and development [Dpp and Daw, members of the TGF-β family, inhibited the transcriptional activation of BmOVO-2 on the Bmotu promoter. We speculated that over-expression of Dpp and Daw activated the TGF-β signaling pathway [Bmotu promoter together with BmOVO. Additionally, overexpression of BBX-B8, Ror2 and STAT also reduced Bmotu promoter activity. The expression of genes related to growth and development could be regulated by over-expression of the insulin-like peptide BBX-B8, an extracellular peptide [Bmotu promoter activity was not caused by the direct interaction of BBX-B8 with BmOVO. Previous studies have shown that the classical Wnt signaling pathway is mediated by Ror2 in human lung cancer cells [Bmotu promoter activity regulated by BmOVO may have been down-regulated by activating the Wnt signaling pathway mediated by Ror2 in the silkworm. STAT also regulates gene expression by binding to the promoter of target genes [OVO belongs to a large transcription factor family with conservation of function over evolutionary distance. A previous study found that OVO transcription factors are present in elopment , 3, 6. I pathway , and the peptide . Additioer cells . Bmotu pet genes , which mBmotu promoter predicted by bioinformatics which were the binding site of zinc finger protein. Dual-luciferase reporter assay showed that the activity of Bmotu promoters with diverse deletion of CE elements was different for BmOVO-1, BmOVO-2 and BmOVO-3, suggesting that the relationship of epistatic properties of the CE elements in the Bmotu promoter and the activity of Bmotu promoters is complex. Moreover, we found that BmOVO binds to the CE1 element (GTACCGTTGTA) located on the Bmotu promoter, while the nuclear proteins extracted from BmN cells could bind to the Bmotu promoter at three sites  by EMSA. These results suggest that Bmotu gene expression can be regulated by other transcription factors in addition to BmOVO, but these transcription factors need to be further screened and verified. It was also found that T2, A3 and A11 in the CE1 sequence might play an important role in binding of BmOVO to the Bmotu promoter. Bmotu promoter activity was altered with the mutation of the CE1 sequence and the deletion of the CE1 and CE2 elements. This may be related to changes in the Bmotu promoter structure. A study investigating the crystal structure of the zinc finger protein-DNA complex showed that the zinc finger was inserted into the large groove of the DNA double helix through its α-helix to combine with DNA [Bmotu promoter activity. These results are helpful to further understand the regulatory mechanisms of BmOVO on Bmotu promoter activity and ovarian development in the silkworm.There are 3 CE elements  on with DNA . MutatioBmotu promoter activity was positively regulated by four BmOVO isoforms and the BmOVO isoforms could bind to the Bmotu promoter. These results will help us to further understand the regulatory mechanisms of BmOVO on Bmotu promoter activity and ovarian development in the silkworm, thus providing the theoretical basis and molecular target for regulating ovarian development and increasing silkworm silk production through genetic manipulation. In addition, we detected the interaction between the key genes of silkworm important pathway and Bmovo gene, and hope to provide a new clue for the further study of the regulatory role of BmOVO.In conclusion, the The BmN cell line originating from the silkworm ovary was stored in our lab and cultured at 26 °C in TC-100 medium  containing 10% fetal bovine serum (FBS) .5/well in 24-well culture plates. Expression vectors were transfected into the BmN cells using Lips2000 . After 4 h incubation in TC-100 medium without FBS, the medium was replaced with new TC-100 medium with 10% (v/v) FBS, and the cells were incubated for 60 h.Cells were seeded at 1 × 10STAT) gene [Dpp) gene and pIZT/V5-His-Daw containing dawdle (Daw), both of which are members of the transforming growth factor-β(TGF-β family) [Ror2) gene (AK385238.1), which is a Wnt pathway regulatory factor, and pIZT/V5-His-BBX-B8 containing the insulin-like peptide BBx-B8 [The plasmid pRL-TK  was kindly provided by Dr. Wei of Soochow University. We also utilized the following plasmids, which were constructed for a previous study, including pIZT/V5-His-STAT containing the signal transduction and transcription activation factor (AT) gene , pIZT/V5 family) , pIZT/V5e BBx-B8   with the primers Potu5/Potu2–1  to generate pFast-potu5. The luciferase (luc) gene amplified from pGL3  with the primers Luc1/Luc2  respectively with 0.1, 0.15, and 0.2 μg of plasmid pRL-TK, and the co-transfected BmN cells with pFast-potu5-Luc(1.0 μg) and pRL-TK(0.1 μg) plasmids was used as a control.To generate the luciferase expression vector, the polyhedrin promoter and the p10 promoters were removed from the pFastBac™Dual vector by ovo gene . These gBmovo (GU477588) sequences  amplified from Bmovo-1 [Bmovo-1 gene was cloned into the pFast-potu5-Luc-ie1 to construct the vectors pBmovo-T2, pBmovo-T3, pBmovo-TB1, pBmovo-T4, pBmovo-T5 and pBmovo-TB2, respectively. The A1, A1–A2, A1–A3, A1–B1, A1–A4 and A1–A5 regions were deleted in pBmovo-T2, pBmovo-T3, pBmovo-TB1, pBmovo-T4, pBmovo-T5 and pBmovo-TB2, respectively. To investigate the function of the A1 domain, Bmovo-2 with the A1 region amplified from the Bmovo-2 gene with the primers ovo-T1/ ovo-A2  and two basic domains  at the N-terminus of BmOVO-1 . In orde Bmovo-1  with thecis-acting elements located on the Bmotu promoter (BABH01009636), different Bmotu promoters that varied in length (potu1 with control element (CE) CE1, CE2 and CE3, nt, 20,464–21,524. potu5 with CE2 and CE3, nt, 20,538–21,524. potu7 with CE3, nt, 20,665–21,524. potu8 with CE1 and CE3, nt, 20,665–21,524) were amplified from silkworm genomic DNA with the primer pairs potu1/potu2–1, potu5/ potu2–1, potu7/potu2–1 and potu8/potu2–1, respectively  has five small open reading frames (sORFs 1A–4A and B), of which 1A, 2A, 3A, 4A and 4B encode 12, 11, 11, 10 and 32 amino acid residues, respectively , a sequence containing the 5′-non coding sequence and the downstream sequence 1A–4A with B (5A1–4 + B) , a sequence containing A1–A4 with B (A1–4 + B)  and a sequence containing B and its downstream non coding sequence (B)  were cloned into the insect cell expression vector pIZT-V5/His (Invitrogen) to construct the tal-like expression vectors pIZT/V5-His-tal, pIZT/V5-His-5A1–4 + B, pIZT/V5-His-A1–4 + B, and pIZT/V5-His-B, respectively [B. mori.The byx mori , 28. Theectively . In thisDrosophila, the small peptide Tal triggers the hydrolysis of the N-terminal transcriptional repression domain of Ovo/Svb, which leads to a change from a transcriptional repressor to a transcriptional activator [tal-like gene in the silkworm, the dsRed reporter gene fused with the N-terminal domain sequence (28 aa) of BmOVO-1 was amplified with the primer pairs ovo1-dsred-F/ovo1-dsred-R  according to the manufacturer’s instructions. Briefly, BmN cells were lysed for 15 min at room temperature using 1× passive lysis buffer which is made from the dilution of the 5 × passive lysis , and then the lysed cells were collected and centrifuged at 12,000 g for 10 min. The supernatant was used for the determination of luciferase activity. Luciferase activity was measured using GloMax Multi Jr. at 490 nm . T-test was performed for statistical analysis by the software Graphpad Prism5.E. coli. The recombinant protein BmOVO zinc-finger domain with 6-His-tagged was purified with Ni-NTA agarose  according to the manufacturer’s instructions. Then, the refolding of the BmOVO zinc-finger domain was carried out by dialysis in TGN buffer .The sequence encoding the last 178 aa containing the four zinc-finger domains of BmOVO was amplified with the primer pairs Bmovo2–3/Bmovo2–2 . The anti-RFP antibody  was used, and immunoblotting with anti-β-tubulin antibody  was conducted as internal control. The signals were measured using an Enhanced Chemiluminescence (ECL) western blot detection kit (Sangon).Drosophila OVO directly binds to the sequence TTACMGTTACA, where M = A/C, of the target promoter [Bmotu promoter using JASPAR CORE (http://jaspar.genereg.net/cgi-bin/jaspar_db.pl). EMSA was performed following the manufacturer’s instructions with the LightShift Chemiluminescent EMSA kit . Briefly, a binding reaction was performed in a final volume of 20 μl, containing 1 μl Poly dI.dC, 1 μl NP-40, 1 μl KCl, 1 μl glycerol, 2 μl 10× binding buffer, 0.75 pM DNA probe and 0.02 mg/ml recombinant BmOVO zinc-finger protein (or 0.175 mg/ml nucleoproteins), followed by incubation for 20 min at room temperature. Then, the recombinant protein binding probe was run on a 6% polyacrylamide gel and transferred to a Hybond-N+ membrane . The membranes were dried and ultraviolet-crosslinked at 120 mJ/cm2 using a UV-light crosslinking instrument. Then, biotin-labeled DNA was detected with the Chemiluminescent Nucleic Acid Detection Module kit . Finally, the signal was visualized by phosphorimaging .promoter . To inveAdditional file 1:Figure S1. Structural domain analysis of BmOVOs. A represents acidic domain. B represents alkaline domain. Z represents zinc-finger domain. Number on domain means number of amino acid residues in a domain. Figure S2. Transcriptional Regulatory activity of BmOVO could be regulated by Dpp, Daw, Ror2, STAT and BBx-B8.The mixture of pFast-potu5-Luc-ie1-Bmovo2(1 × 1011 copies) and pRL-TK (1 × 1010 copies) plasmids was respectively co-transfected with pIZT/V5-His, pIZT/V5-His-STAT, pIZT/V5-His-DPP, pIZT/V5-His-Daw, pIZT/V5-His-Ror2 and pIZT/V5-His-BBX-B8 plasmids (1 × 1011 copies) into BmN cells (105), and the co-transfected BmN cells with pFast-potu5-ie1-Bmovo2 (1 × 1011 copies) and pRL-TK (1 × 1010 copies) plasmids was used as a control. Luciferase activities in the cells were determined at 60 h post-transfection, 100 μg protein from the lysed cells was used for luciferase assay . Figure S3. The Bmotu promoter could bind to nucleoproteins. (a) analysis of the binding of probe otu-A to different concentrations of nucleoproteins, lane1–5: the final concentration of nucleoproteins is 0, 0.175, 0.35, 0.525, 0.7 mg/mL; (b) otu-B and otu-C probes binding to nucleoproteins; (c) otu-A, otuA-mut1, otuA-mut2 and otuA-mut3 binding to the nucleoproteins. Table S1. Different combinations of luc expression plasmids. Table S2. The plasmids used in this study. Table S3. The primers used in this paper. The sequence with underline indicates the enzyme sites, and with wave underline indicates the binding site CE1. Base with frame indicates the mutation. In primer ovo1-dsred-F, the double underline was 5′-terminal sequence of dsred gene, the sequence with boldface were the coding sequence of N- terminus 28 residues of BmOVO-1. Table S4. Sequence of mutant DNA probe. Sequence of mutant DNA probe. The sequence with underline indicates the predicted binding sites, and base with frame indicates the mutation. (DOCX 445 kb)


Saccharomyces cerevisiae, we have recently shownthat GTP levels can also play a role in determining how genes act to respond to changes in cellular energy supply. This review aims to interpret the importance of GTP as an integrative signal in the context of an increasing body of evidence indicating the spatio-temporal complexity of cellular de novo purine nucleotide biosynthesis.This mini-review considers the idea that guanylate nucleotide energy charge acts as an integrative signal for the regulation of gene expression in eukaryotic cells and discusses possible routes for that signal’s transduction. Gene expression is intimately linked with cell nutrition and diverse signaling systems serve to coordinate the synthesis of proteins required for growth and proliferation with the prevailing cellular nutritional status. Using short pathways for the inducible and futile consumption of ATP or GTP in engineered cells of  Life requires energy, and the proliferation of life even more so. The common energy currency in living cells is ATP, generated from oxidative and substrate-level phosphorylation and consumed to drive the fundamental processes of DNA maintenance, synthesis and replication, the expression of genes to produce RNA and proteins, and the transport and movement of chemicals and macromolecules. Of these, gene expression—chromatin remodeling, transcription initiation, transcription elongation, mRNA splicing, and translation—accounts for the majority of cellular energy demand, with ~ 75% frequently offered as an estimate —defined as the relative concentrations of all three phosphorylated adenosine nucleotides [ATP] + 0.5[ADP]/[ATP] + [ADP] + [AMP]  in S. cerevisiae (Kuehner and Brow In prokaryotic systems GTP levels can be directly sensed via influencing the selection of transcription start sites by RNA polymerase (Krásný et al. Drosophila cells, mediating the repression of histone genes and E2F, a key driver of cell proliferation (Kozhevnikova et al. E. coli IMPDH was also shown to exhibit the same sequence-specific DNA-binding activity as the Drosophila enzyme, suggesting that moonlighting as a transcriptional regulator may be a broadly conserved function of this enzyme (Kozhevnikova et al. The unusual dynamic spatial organization of the enzymes required for purine biosynthesis into cellular macrostructures, filamentous cytoophidia (Aughey and Liu Cyanidioschyzon merolae (Imoto et al. Upstream of IMPDH, many of the enzymes required for de novo IMP biosynthesis have been observed to dynamically assemble and disassemble into a multi-enzyme cytoplasmic macrostructure termed the purinosome (Pedley and Benkovic Testing the hypotheses discussed above concerning the mechanisms by which high-energy guanine nucleotide status modulates gene transcription will require multidisciplinary investigations using the latest techniques in molecular biology and fluorescence microscopy. How induction of the ATP- or GTP-consuming pathways affects formation of IMPDH filaments and purinosomes, and how the abundance of activated GTPase switch proteins is influenced are key questions yet to be answered. The synthesis and use of high-energy adenine and guanine nucleotides are intimatelylinked see Fig.  and obtaThe success of future work will depend on the ability to cleanly dissect the in vivo effects of GTP/GEC from those of ATP/AEC, using tools to manipulate the levels of these closely related nucleotides independently from one another. Recent in vitro studies analyzing the filamentation state and activity of human IMPDH enzymes indicate differential allosteric responses to adenine and guanine nucleotides such that IMPDH cytoophidia formation facilitates the accumulation of high levels of guanine nucleotides when the cell requires them (Fernández-Justel et al.