Pelteobagrus fulvidraco were cloned and characterized. Bioinformatics analysis revealed that PI3KCa, PI3KC2b and PI3KC3 had different structures in their core promoter regions. The promoter regions of PI3KCa and PI3KC2b had CpG islands but no CAAT and TATA box. In contrast, the promoter of PI3KC3 had the canonical TATA and CAAT box but no CpG island. The binding sites of several transcription factors, such as HNF1, STAT and NF-κB, were predicted on PI3KCa promoter. The binding sites of transcription factors, such as FOXO1, PPAR-RXR, STAT, IK1, HNF6 and HNF3, were predicted on PI3KC2b promoter and the binding sites of FOXO1 and STAT transcription factors were predicted on PI3KC3 promoter. Deletion analysis indicated that these transcriptional factors were the potential regulators to mediate the activities of their promoters. Subsequent mutation analysis and electrophoretic mobility-shift assay (EMSA) demonstrated that HNF1 and IK1 directly bound with PI3KCa and PI3KC2b promoters and negatively regulated the activities of PI3KCa and PI3KC2b promoters, respectively. Conversely, FOXO1 directly bound with the PI3KC2b and PI3KC3 promoters and positively regulated their promoter activities. In addition, AS1842856  incubation significantly reduced the relative luciferase activities of several plasmids of PI3KC2b and PI3KC3 but did not significantly influence the relative luciferase activities of the PI3KCa plasmids. Moreover, by using primary hepatocytes from yellow catfish, AS incubation significantly down-regulated the mRNA levels of PI3KCa, PI3KC2b and PI3KC3 and reduced triacylglyceride (TG) accumulation and insulin-induced TG accumulation, as well as the activities and the mRNA levels of several genes involved in lipid metabolism. Thus, the present study offers new insights into the mechanisms for transcriptional regulation of PI3Ks and for PI3Ks-mediated regulation of lipid metabolism by insulin in fish.In the present study, the length of 360, 1848 and 367 bp sequences of promoters from three subtypes of PI3K family  of yellow catfish  PI3KC2g) from yellow catfish Pelteobagrus fulvidraco [PI3KCa transcription [PI3KCa and PI3KC2b [Phosphatidylinositol-3 kinase (PI3K) is an intracellular transducer with lipid substrate specificity and implicates a wide range of signaling pathways involved in cell survival, proliferation and nutrient metabolism . In mammlvidraco . It involvidraco . At prescription ,7,8, whi PI3KC2b ,10,11. S PI3KC2b ,13. HoweThe members of PI3K family have a pivotal role in the metabolic action of insulin  and theyLipid metabolism is regulated by lipogenesis, lipolysis and lipid transport and many crucial enzymes and transcription factors are involved in these processes. Enzymes related to lipid metabolism include the lipogenic enzymes (such as glucose 6-phosphate dehydrogenase (G6PD), 6-phosphogluconate dehydrogenase (6PGD), isocitrate dehydrogenase (ICDH), malic enzyme (ME) and FA synthase (FAS)) and the lipolytic enzymes (such as adipose triacylglyceride lipase (ATGL) and carnitine palmitoyltransferase 1 (CPT 1), acetylCoA carboxylase b (ACCb)) . SeveralPelteobagrus fulvidraco, an omnivorous freshwater fish, is widely distributed in the inland freshwater waters in several Asian countries including China. The fish is regarded as a good candidate for freshwater culture in China for its delicious meat and high market value. Recently, under intensive aquaculture for this fish species, excess lipid deposition in yellow catfish become more and more widespread, which greatly reduce its taste and economic value. Thus, it is very important to investigate the characteristics of lipid metabolism and explore the pathway for reducing lipid deposition in yellow catfish. PI3Ks implicate a wide range of signaling pathways involved in lipid metabolism. Thus, as a part of our ongoing research into the functional analysis of PI3K members and the mechanism of PI3Ks-mediated regulation of lipid metabolism by insulin in yellow catfish, we identified and characterized the upstream 5′-flanking region of three subtypes of PI3K members  from yellow catfish. Several transcription factors binding sites of their promoters from PI3KCa, PI3KC2b and PI3KC3 were demonstrated by using deletion, mutation and electrophoretic mobility-shift assay (EMSA). We confirmed that PI3Ks were regulated by FOXO1 at the transcriptional level and strong inhibition of FOXO1 binding sites led to transcriptional inactivation of PI3Ks, which in turn impaired insulin-induced lipogenesis in hepatocytes from yellow catfish. Meantime, targeting FOXO1 with AS not only reduced lipogenesis but also lipolysis and lipid transport in hepatocytes from yellow catfish.Yellow catfish PI3KCa, PI3KC2b and PI3KC3 were identified according to our previous studies [PI3KCa, PI3KC2b and PI3KC3 was designated as +1. An intron with 1048 bp sequences was cloned at the position of +370 bp of PI3KCa and no introns were found within +264 bp of PI3KC2b and +343 bp of PI3KC3 (PI3KCa (GenBank accession no.: MG574318), PI3KC2b (GenBank accession no.: MG574319) and PI3KC3 (GenBank accession no.: MG574320) were cloned by using hiTAIL-PCR, respectively  of f PI3KC3 . The 360ectively . A clustpromoter A and thepromoter B and thepromoter C. A TATAredicted C. Severad PI3KC3 . Furtherthprimer .PI3KCa significantly increased the relative luciferase activity. Subsequent deletion to −86 bp significantly decreased the relative luciferase activity  HNF1 binding site did not change the relative luciferase activity, while the mutation of −238/−236 (aMut-HNF1-2) HNF1 binding site significantly increased the relative luciferase activity. However, there is no significant difference after the co-mutation of ((−304/−300) + (−238/−236)) (aMut-HNF1-12) HNF1 binding sites compared with the wild type pGl3-360/+71 of PI3KCa vectors. , −1202/−1198 (2bMut-FOXO1-3) and −947/−943 (2bMut-FOXO1-4) FOXO1 binding sites but showed no significant changes after the mutation of −1231/−1227 (2bMut-FOXO1-2) FOXO1 binding sites. The co-mutation of ((−1708/−1705) + (−947/−943)) (2bMut-FOXO1-14) and ((−1202/−1198) + (−947/−943)) (2bMut-FOXO1-34) FOXO1 binding sites significantly reduced the relative luciferase activity, while the co-mutation of ((−1708/−1705) + (−1231/−1227)) (2bMut-FOXO1-12), ((−1231/−1227) + (−1201/−1198)) (2bMut-FOXO1-23) and ((−1708/−1705) + (−1202/−1198)) (2bMut-FOXO1-13) FOXO1 binding sites showed no significant difference in the relative luciferase activity. In addition, compared with the wild type pGl3-1848/+79 of PI3KC2b vectors, the relative luciferase activity was significantly reduced after the co-mutation of ((−1708/−1705) + (−1231/−1227) + (−1201/−1198)) (2bMut-FOXO1-134) FOXO1 binding sites  significantly increased the luciferase activity when compared with their corresponding wild type  vectors B. Comparng sites C.PI3KC3 promoter, compared with the wild type pGl3-367/+59 of PI3KC3 vectors, the relative luciferase activities were significantly reduced after the mutation of −224/−221 (3Mut-FOXO1-2) FOXO1 binding site and showed no significant difference after the mutation of −347/−344 (3Mut-FOXO1-1) and −183/−179 (3Mut-FOXO1-3) FOXO1 binding sites. Compared with the wild type pGl3-367/+59 of PI3KC3 vectors, the co-mutation of ((−347/−344) + (−224/−221)) (3Mut-FOXO1-12) and ((−347/−344) + (−224/−221) + (−183/−179)) (3Mut-FOXO1-123) FOXO1 binding sites significantly decreased the relative luciferase activities, whereas there are no significant changes after the co-mutation of ((−347/−344) + (−183/−179)) (3Mut-FOXO1-13) and ((−224/−221) + (−183/−179)) (3Mut-FOXO1-23) FOXO1 binding sites  competed for binding when HNF1 binding sequence was used as probe, while the 100-fold unlabeled Mut-HNF1 binding sequence markedly reduced this competition, indicating that this sequence was bound by HNF1 factor (PI3KC2b promoter (PI3KC2b promoter) as probe, suggesting that this sequence was not bound by any factors  at phosphorylation level influenced insulin-induced changes of lipid metabolism in yellow catfish [Although the characterization and tissue expression profile of seven PI3K members from yellow catfish were determined in our previous study [ catfish . HoweverPI3KCa, PI3KC2b and PI3KC3 had different structures. PI3KCa and PI3KC2b had CpG islands but no CAAT and TATA box, while PI3KC3 had the canonical TATA and CAAT box but no CpG island. Moreover, the present study indicated that the TATA box and CAAT box were located at +18 bp and −25 bp of PI3KC3 promoter, respectively, which were distinguished from other report that TATA box was always located at the 5′ upstream region [PI3KCa, PI3KC2b and PI3KC3 promoters were approximately 2, 7 and 45-fold of pGl3-basic empty vector activity, respectively. Thus, we speculated that GC-rich and TATA-less regions probable negatively regulated promoter activity. Bird [PI3KCa, PI3KC2b and PI3KC3 from yellow catfish, which were the basic elements in driving gene transcription. In eukaryotes, the term “core promoter” is often used to focus on the DNA region in the immediate vicinity of the TSS, which is assumed to dock the pre-initiation complex . In the ty. Bird  suggestety. Bird , also exPI3KCa promoter, −1761/−1743 bp of PI3KC2b promoter and −290/−270 bp of PI3KC3 promoter. By using deletion analysis, we also found that the regions from −224/+71 bp of PI3KCa promoter, −1845 to −1522 bp of PI3KC2b promoter and −291 to −85 bp of PI3KC3 promoter positively regulated their corresponding promoter activity, indicating that STAT was a potential positive regulator which mediated PI3K promoter activity from yellow catfish. STAT is a latent cytoplasmic transcription factor that participates in gene regulation and plays important roles in multiple biological processes [PI3KCa promoter, enlightening us that there may be an interaction between STAT and NF-κB in PI3KCa-mediated transcriptional activation. In human, Yang et al. [PI3KCa was transcriptionally regulated through NF-κB pathway. PPARs are key transcriptional factors which mediate the regulation of many enzymes in lipid metabolism [PI3KC2b promoter) is a potential positive regulator in regulating PI3KC2b promoter activity from yellow catfish. In mammals, studies also suggested that PPAR-RXR positively regulated the expression of PI3K regulatory subunit [Identification of transcription factor binding site (TFBS) plays an important role in deciphering the mechanisms of gene regulation . The prerocesses ,13. Simirocesses  also reprocesses . The pretabolism ,32. In t subunit ,34,35.PI3KCa promoter negatively regulated PI3KCa promoter activity and the region from 623 to −388 bp and −144 to −94 bp of PI3KC2b promoter also negatively regulated PI3KC2b promoter activity. Interestingly, two HNF1 binding sites were predicted within the region from −360 to −224 bp of PI3KCa promoter and a HNF6 binding site was predicted within the region from −623 to −388 bp of PI3KC2b promoter and a HNF3 binding site was predicted within −144 to −94 bp of PI3KC2b promoter, indicating that HNFs were the potential negative regulators for PI3K promoter activity in yellow catfish. Moreover, the site-directed mutagenesis and EMSA analysis confirmed that the HNF1 factors directly bound with the sequence from the −249 to −227 bp of PI3KCa promoter and negatively regulated promoter activity. HNF1 is a liver-enriched transcription factor and it plays a key role in regulating liver-specific gene expression [HNF1 and PI3KCa. Similarly, Li [HNF1 was notably decreased in ovarian cancer tissues while PI3KCa expression was high in ovarian cancer tissues [PI3KC2b promoter was predicted. IK1 encodes a DNA-binding zinc finger protein and regulates expression of genes involved in important biological pathways [PI3KC2b was one of the target genes of IK1 in leukemia [PI3KC2b promoter activity. Moreover, EMSA assay confirmed that the sequence at −615 to −593 bp of PI3KC2b promoter was a functional IK1 binding locus.In the present study, the deletion analysis indicated that the region from −360 to −224 bp of arly, Li  suggeste tissues ,38. Meanpathways . In mammpathways . Interespathways . Recentlleukemia . In thisPI3KC2b vectors and the pGl3-367/+59 and pGl3-291/+59 of PI3KC3 vectors were significantly reduced after AS incubation. The site-directed mutagenesis assay indicated that the relative luciferase activities of PI3KC2b promoter significantly decreased after site-mutation on the −1708/−1705, −1202/−1198 and −947/−943 FOXO1 binding sites and the similar result was found for PI3KC3 promoter after site-mutation on the −224/−221 FOXO1 binding sites. Furthermore, EMSA assay demonstrated that the −1202/−1198 and −947/−943 FOXO1 binding sites were the functional binding locus on PI3KC2b promoter and also the −224/−221 FOXO1 binding sites was a functional binding locus on PI3KC3 promoter. Similarly, Hui et al. [PI3KCa transcription. However, the present study found no FOXO1 binding site on the PI3KCa promoter, which was probably due to the short length of PI3KCa promoter obtained here.FOXO1 plays a central role in the regulation of the metabolism, stress response and apoptosis  and it aPI3KC2b and PI3KC3 promoter activity, next we explored whether FOXO1 regulated the PI3Ks transcription in yellow catfish. Thus, AS was used to incubation the primary hepatocytes from yellow catfish. The results indicated that AS treatment significantly reduced the transcript levels of PI3KCa, PI3KC2b and PI3KC3 in primary hepatocytes and PI3KC3 was more sensitive to AS than PI3KCa and PI3KC2b. Thus, the present study, for the first time, demonstrated that PI3KCa, PI3KC2b and PI3KC3 were regulated by FOXO1 at the transcriptional levels.After finding that FOXO1 positively regulated FAS, G6PD, 6PGD, ACCa and PPARγ), lipolysis  and lipid transport  [G6PD, 6PGD and PPARγ, suggesting that insulin regulated G6PD, 6PGD and PPARγ gene expressions probably via FOXO1/PI3K pathway in yellow catfish. Similarly, Zou et al. [PPARγ expression in adipocytes. Interestingly, our results showed that the changes of FAS, G6PD and 6PGD activities did not parallel with their mRNA expressions following the AS treatment. Similarly, in our previous study, we also found that insulin changes FAS and G6PD activity but not changes their mRNA level after 24 h incubation on hepatocytes from yellow catfish [CPT1A, PPARα, ACCb, ATGL, CD36, FABP1 and LPL, indicating that AS not only reduced lipogenesis but also the action of lipolysis and lipid transport in yellow catfish. Similarly, several studies pointed out that the inhibition of FOXO1 also reduced the expression of LPL and CD36 [ATGL promoter and increased ATGL expression in adipocytes [Our previous study indicated that insulin regulated lipid metabolism via PI3K pathway . The mecd FABP1) . In thisu et al.  and Liu u et al.  also fou catfish . Chen et catfish  also sugand CD36 ,49. Moreipocytes ,51.PI3KCa, PI3KC2b and PI3KC3 promoters from yellow catfish were cloned and characterized. The different structure of core promoters of PI3KCa, PI3KC2b and PI3KC3 was proposed. Several TFBS of PI3KCa, PI3KC2b and PI3KC3 promoters were demonstrated. In addition, we confirmed that PI3Ks were transcriptionally regulated through FOXO1 pathway. Furthermore, PI3Ks mediated the insulin-induced changes of lipid metabolism by targeting FOXO1 and inhibition FOXO1 with AS not only reduced lipogenesis but also lipolysis and lipid transport in hepatocytes from yellow catfish.In summary, the length of 360, 1848 and 367 bp sequences of Yellow catfish were obtained from Hubei Bairong Fisheries Farm . HepG2 cell lines were obtained from the Cell Resource Center in the Fishery College of Huazhong Agricultural University. Dulbecco’s Modified Eagles Medium (DMEM), 0.25% trypsin-EDTA and fetal bovine serum (FBS) were obtained from Gibco/Invitrogen, USA. AS1842856, dimethyl sulphoxide (DMSO), penicillin, streptomycin, trypan blue and other reagents were purchased from Sigma-Aldrich . We ensured that the experiments were performed in accordance with the experimental protocols of Huazhong Agricultural University (HZAU) and approved by the ethics committee of HZAU PI3KCa, PI3KC2b and PI3KC3 from yellow catfish. Exp. 2 was conducted to determine their potential roles in insulin influencing lipid metabolism.Two experiments were carried out. Exp. 1 was conducted to clone the promoter sequences of PI3KCa (GenBank accession No. KU976455), PI3KC2b (GenBank accession No. KU976460) and PI3KC3 (GenBank accession No. KU976461) from yellow catfish were obtained according to our previous study [PI3KCa, PI3KC2b and PI3KC3. The sequences of PI3KCa, PI3KC2b and PI3KC3 promoters were cloned using the hiTAIL-PCR method [TM II One Step Cloning Kit . The plasmids were named as pGl3-360/+71 of PI3KCa vector, pGl3-1848/+79 of PI3KC2b vector and pGl3-367/+59 of PI3KC3 vector, respectively, according to the distance from their transcription start sites. Plasmids pGl3-224/+71, pGl3-86/+71 of PI3KCa vector, which contained unidirectional deletions of the promoter, were generated with the Erase-a-Base system (Promega) using templates of pGl3-360/+71 of PI3KCa vector. Similarly, plasmids pGl3-1522/+79, pGl3-1263/+79, pGl3-929/+79, pGl3-623/+79, pGl3-388/+79 and pGl3-144/+79 of PI3KC2b vector, as well as pGl3-291/+59 and pGl3-85/+59 of PI3KC3 vectors, were generated with the Erase-a-Base system (Promega) using pGl3-1848/+79 of PI3KC2b vector and pGl3-367/+59 of PI3KC3 vector as templates, respectively. The PCR reactions were performed using the TaKaRa PrimeSTAR® HS DNA Polymerase kit  under the following PCR conditions: 94 °C for 5 min, then 30 cycles of 94 °C for 15 s, 55 °C for 30 s and 72 °C for 1 min, followed by a final extension at 72 °C for 5 min. All plasmids were sequenced for verification in a commercial company . The primer sequences used for plasmid construction are shown in The 5′ cDNA sequences of R method . The spev/v) heat-inactivated FBS  in a humidified atmosphere with 5% CO2 at 37 °C. Before transfections, HepG2 cells were seeded in 24-well cell culture plate at a density of 1.2 × 105 and cultured 24 h to reach 70–80% convergence before the transient transfection. All Plasmids were transiently transfected into HepG2 cells using lipofectamine™ 2000 (Invitrogen) following the manufacture’s protocol. The 500-ng reporter plasmids were used in Opti-MEM (Invitrogen) and they were co-transfected with 25 ng pRL-TK, a Renilla luciferase reporter vector as internal control. After 4 h, the transfection medium was replaced by 10% FBS-DMEM, 10% FBS-DMEM + 0.1 μM AS and 10% FBS-DMEM + 1 μM AS. The concentration of AS was selected according to our preliminary experiments by MTT assay and relative luciferase activity of pGl3-1848/+79 of PI3KC2b vector assay and the results were showed in HepG2 cells were cultured in DMEM medium supplemented with 10% , the JASPAR database (http://jaspar.genereg.net/) and the TFSEARCH database (http://www.cbrc.jp/research/db/TFSEARCH.html). Sequence alignments were assessed with the Clustal-W multiple alignment algorithm. The CpG islands were analyzed by online tool MethPrimer (http://www.urogene.org/methprimer/index1.html) with parameters window 100, shift 1, observed CpG/expected CpG ≥ 0.60 and GC % ≥ 50.For sequence analysis of the promoters of To identify relevant regulatory elements within the promoter regions of PI3KCa, PI3KC2b and PI3KC3 in yellow catfish, site-directed mutagenesis was performed using Quick Change II Site-Directed Mutagenesis Kit  according to the manufacturer’s instructions. For HNF1 mutation analysis, pGl3-360/+71of PI3KCa vectors was used as template. For FOXO1 mutation analysis, pGl3-1848/+79 of PI3KC2b vectors and pGl3-367/+59 of PI3KC3 vectors were used as templates. For IK1 mutation analysis, pGl3-1848/+79, pGl3-1522/+79, pGl3-1263/+79, pGl3-929/+79 and pGl3-623/+79 of PI3KC2b vectors were used as templates. The mutations were conducted at the positions of −304/−300 and −238/−236 for PI3KCa promoter, −1708/−1705, −1231/−1227, −1202/−1198, −947/−943 and −609/−605 for PI3KC2b promoter and −347/−344, −224/−221 and −183/−179 for PI3KC3 promoter. The mutagenesis primers were listed in 2, 0.5 mM DTT, 4% Ficoll, 110 mM KCl, 0.2 μg Poly(dI-dC)) prior to the addition of biotin-labeled double-stranded oligo nucleotide, as listed in EMSA was performed to confirm the functional binding sites of HNF1, IK1 and FOXO1 according to the protocols described in our recent publications . Proteinl-glutamine, 5% (v/v) FBS, penicillin (100 IU/mL) and streptomycin (100 g/mL) in a humidified atmosphere with 5% CO2 at 28 °C. Hepatocytes were counted using a hemocytometer based on the trypan blue exclusion method and only more than 95% cell viability were used for the present experiment. The hepatocytes were plated onto 25 cm2 flasks at the density of 106 cells/mL. The hepatocytes then were incubated with 10 mM PBS (control), 0.1 µM AS, 1 µM AS, 0.1 µM insulin and 1 µM AS + 0.1 µM insulin. The concentration of AS was selected according to our preliminary experiments and the results were showed in Hepatocytes were isolated from yellow catfish according to our previous studies  and were−1 TG mg−1 cellular protein. FAS activity was determined according to the method of Chakrabarty and Leveille [−1 soluble protein. The soluble protein concentration of homogenates was determined by the method of Bradford [Hepatocytes were homogenized in 10 mM PBS. TG was determined by the glycerol 3-phosphate oxidasep-aminophenol (GPO-PAP) method, using a commercial kit  and expressed as μmol LLeveille . G6PD anLeveille . ME actiLeveille . ICDH acLeveille . One uniBradford  using boPI3Ks  and genes involved in lipid metabolism  were examined by quantitative real-time PCR (Q-PCR). Q-PCR assays were performed in a quantitative thermal cycler  with a 20 μL reaction volume containing 10 μL SYBR Premix Ex Taq™ II , 1 μL of diluted cDNA (10-fold), 10 mM each of forward and reverse primers 0.4 μL and 8.2 μL H2O. Primers are given in β-actin, 18s-rRNA, GAPDH, RPL7, HPRT, UBCE and TUBA) were selected in order to test their transcription stability. Two most stable control genes  were selected by using geNorm software [The mRNA levels of software . The relsoftware  when norsoftware ,63.t-test is used to determine the differences between the two groups. For the insulin and AS incubation experiment, the homogeneity of variances among the treatments was tested using Bartlett’s test, they were then subjected to one-way ANOVA followed by Tukey’s multiple range test. Difference was considered significant at p < 0.05.Statistical analysis was performed with SPSS 19.0 software. Results are presented as means ± SEM. For assay of promoters’ activities, student’s


Isl1 hypomorphic line and mice with Isl1 ablated in neural crest lineages. During early development, ISL1 is required for sympathetic neuronal fate determination, differentiation, and repression of glial differentiation, although it is dispensable for initial noradrenergic differentiation. ISL1 also plays an essential role in sympathetic neuron proliferation by controlling cell cycle gene expression. During later development, ISL1 is required for axon growth and sympathetic neuron diversification by maintaining noradrenergic differentiation, but repressing cholinergic differentiation. RNA-seq analyses of sympathetic ganglia from Isl1 mutant and control embryos, together with ISL1 ChIP-seq analysis on sympathetic ganglia, demonstrated that ISL1 regulates directly or indirectly several distinct signaling pathways that orchestrate sympathetic neurogenesis. A number of genes implicated in neuroblastoma pathogenesis are direct downstream targets of ISL1. Our study revealed a temporal requirement for ISL1 in multiple aspects of sympathetic neuron development, and suggested Isl1 as a candidate gene for neuroblastoma.Malformations of the sympathetic nervous system have been associated with cardiovascular instability, gastrointestinal dysfunction, and neuroblastoma. A better understanding of the factors regulating sympathetic nervous system development is critical to the development of potential therapies. Here, we have uncovered a temporal requirement for the LIM homeodomain transcription factor ISL1 during sympathetic nervous system development by the analysis of two mutant mouse lines: an  The SNS is derived from trunk neural crest (NC) cells. As NC cells commit to the sympathetic lineage, they downregulate the expression of genes characteristic of NC cells such as Sox10, and express several sympathetic neuron-specific genes such as Phox2b and markers of catecholamine biosynthesis . The newly generated sympathetic neurons transiently exit the cell cycle, and then reenter the cell cycle and are proliferating. Thereafter, an increasing number of sympathetic neurons exit the cell cycle, which peaks at embryonic day 14.5 (E14.5), and undergo post-mitotic differentiation5.Neurons of the sympathetic nervous system (SNS) innervate visceral organs, glands, and blood vessels, and play important roles in maintaining homeostasis. Aberrant development of the SNS causes neurocristopathies associated with cardiovascular instability, gastrointestinal dysfunction, as well as neuroblastoma that arises from developing sympathetic neurons9. BMPs initiate sympathetic neurogenesis by inducing sequential expression of a number of transcription factors4, including Phox2a/2b, Mash1, Hand2, Gata2/3, and Insm1. In Phox2b knockout mice, NC progenitors fail to initiate sympathetic differentiation and express Hand2, Gata3, TH, and DBH10. Gata2/3, Insm1, and Hand2 are BMP-induced transcription factors essential for sympathetic neuron proliferation and differentiation downstream of Mash1 and Phox2b. Together, these factors constitute an early transcriptional network essential for sympathetic neuron development4.Bone morphogenetic protein (BMP) and Notch signals are required for induction and coordination of early sympathetic differentiation and progenitor maintenance. Enhanced Notch signaling results in increased Sox10+ progenitors at the expense of differentiated neurons in sympathetic ganglia12. Recent studies have uncovered a cross-repressive and maintenance mechanism for sympathetic neuron diversification, which involves antagonistic actions between pro-cholinergic factors Ntrk3 (TrkC)/Ret/Tlx3 and pro-noradrenergic factors Hmx1/Ntrk1 (TrkA)14. TrkC and Ret repress Hmx1 expression and induce cholinergic differentiation. Conversely, Hmx1 represses Tlx3 and Ret, induces TrkC, and maintains noradrenergic phenotypes.Segregation of initially bimodal sympathetic neurons into distinct noradrenergic and cholinergic neurons occurs relatively later during development, and is controlled by intrinsic as well as target derived signals15. ISL1 plays an essential role in sensory neuron differentiation by activating genes critical for sensory neuron phenotype, and repressing genes expressed earlier during sensory neuron development15. ISL1 is required for early sympathetic neuron development16; however, genes and signaling pathways regulated by ISL1 and direct targets of ISL1 in sympathetic neurons remain unexplored. In this study, we uncovered a temporal requirement for ISL1 in various aspects of SNS development. ISL1 regulates directly or indirectly a number of genes essential for sympathetic neuron proliferation and differentiation, many of which have been implicated in neuroblastoma pathogenesis, suggesting Isl1 as a candidate gene for neuroblastoma.ISL1 is an LIM-homeodomain transcription factor expressed in all peripheral neuronsWnt1-Cre; Isl1f/f;Rosa26-LacZ mutant (CKO) and Wnt1-Cre;Isl1+/+;Rosa26-LacZ control (ctrl) embryos, and found that primary sympathetic column/chain of Isl1 CKO and ctrl embryos appeared comparable up until E11  in either Isl1 CKO or control sympathetic ganglia  and E12.5 (~7% vs. 30%)  Fig. . In addi2.5 Fig. .Isl1 CKO and control embryos at E11.5–12, a stage when minimal cell loss occurred in Isl1 CKO sympathetic ganglia. We identified 358 downregulated and 555 upregulated transcripts in Isl1 CKO mutants  . These included, e.g., Mki67 , Ccnd1 (G1-S phase), Smc2/4, Bub1 (M phase), Arhgap11a, Pbk   Fig. . Selecteng) Fig. . Reduceding Fig. .Fig. 2RNSox10, Foxd3, Snai1, Sox2, and Crabp2) and glial  progenitors   Fig. , arrow. on) Fig. , arrow. sed Fig. .Fig. 3Im9. We found that ablation of Isl1 during early sympathetic development resulted in reduced BMP signaling (Bmpr1b), but upregulated BMP antagonist signaling (Ctgf and Fst), and a significant increase in expression of Notch ligands  and downstream target genes 17 , chromatin modification , and RNA binding , many of which are known to play critical roles in sympathetic neuron development4 , neurotransmission , and altered expression of genes of neurotransmitters or related enzymes 18  at E14.5, and formed a complex nerve plexus around the dorsal aorta at E17.5 Fig. , arrow. 7.5 Fig. , arrow. 7.5 Fig. .Isl1 hypomorphic sympathetic ganglia  in lia Fig. . Howevernts Fig. .Isl1 hypomorphic and wildtype sympathetic ganglia at E14.5 and identified 641 upregulated and 526 downregulated genes (p < 0.05)  and later (7.1%) developmental stages  and noradrenergic genes 19. Alterations in their expression were confirmed by qPCR, in situ and immunostaining  and 186  genes that were directly regulated by ISL1 during early and later sympathetic development, respectively , axon growth, and neurotransmission . Similarly, many direct targets of ISL1 downregulated in Isl1 hypomorphic sympathetic ganglia were involved in neuronal differentiation  and neurotransmission . Intersection of direct targets of ISL1 found in Isl1 CKO and hypomorphic mutants revealed 41 common targets of ISL1, many of which, e.g., Lmo1, Egln3, Tbx20, were positively regulated by ISL1 at both early and late stages  in mouse sympathetic neurons that, by whole-genome alignment, overlaps with the super-enhancer of LMO1 identified in human neuroblastoma20.Notably, a number of targets of ISL1 downregulated in 5. From E14.5 onward, a majority of sympathetic neurons undergo cell cycle withdrawal and terminal differentiation and maturation; however, the mechanisms underlying these temporal events remain largely unknown. At early developmental stages, ablation of Isl1 resulted in reduced sympathetic neuron proliferation and reduced expression of cell cycle genes, including Ccnd1 and Foxm1. Expression of a number of transcription factors critical for sympathetic neuron proliferation was also downregulated in Isl1 CKO mutants27, including Gata3, Insm1, and Prox1. Although mechanisms by which these transcription factors control sympathetic neuron proliferation are unclear, these data suggest that ISL1 may regulate sympathetic neuron proliferation by controlling cell cycle gene expression.Sympathetic neurons continue to proliferate after the acquisition of pan-neuronal and noradrenergic properties3, including Mash1, Phox2b/2a, Hand2, Gata2/3, and Insm1. We found that ISL1 is downstream of Phox2b, but upstream of Gata2/3, Insm1/2, and Hand1. ISL1 acts parallel to Hand2, since Hand2 expression is not changed in Isl1 CKO mutants and vice versa28. ISL1 is dispensable for initial noradrenergic differentiation, since expression of TH and DBH is not altered in Isl1 CKO mutants. But instead, ISL1 directly or indirectly regulates sets of genes essential for sympathetic neuron development, axon growth, and neurotransmission, suggesting that ISL1 is essential for generic sympathetic neuronal differentiation during early sympathetic neuron development.Differentiation of noradrenergic and pan-neuronal phenotypes is controlled by an early gene regulatory networkIsl1 expression in Isl1 hypomorphic mice caused impaired sympathetic innervations, delayed cell cycle withdrawal, and reduced expression of genes involved in axon growth and neurotransmission, demonstrating a continued requirement for ISL1 in sympathetic neuron differentiation and maturation at later development stages. Analyses of ChIP-seq and RNA-seq data sets from both Isl1 CKO and hypomorphic mutants revealed ISL1 regulates common, yet distinct gene programs during early and later sympathetic neuronogenesis. Tlx3, Prox1, and several cell cycle-related genes were among the direct targets of ISL1 that were downregulated in Isl1 CKO but upregulated in Isl1 hypomorphic mutants, suggesting that ISL1 may act both as an activator and a repressor in a context-dependent manner during sympathetic neuron development. Interestingly, a comparison of ISL1 targets in different cell types  revealed few, if any, common targets that could account for the observed phenotypes in each of these mutants, suggesting that the action of ISL1 is cellular context-dependent.Reduced 32  critical for neuronal development. In the heart, ISL1 physically interacts with Kdm6a to promote H3K27me3 demethylation at the enhancers of target genes encoding key cardiac transcription factors29. It is tempting to speculate that ISL1 might regulate the expression of sympathetic developmental genes via epigenetic mechanisms.Molecular mechanisms of ISL1 in sympathetic neurons remain unknown. Interestingly, ablation of Isl1 causes downregulation of a number of epigenetic regulators6. Ablation of Isl1 resulted in increased Notch signaling but decreased BMP signaling. Accordingly, genes associated with NC and glial progenitors were significantly upregulated, suggesting that during initial sympathetic neurogenesis, ISL1 is required for coordination of lineage differentiation of sympathetic neurons and glia by modulating BMP and Notch pathways. Interestingly, previous studies have revealed a role of ISL1 in cell fate specification and determination in sensory neurons of the dorsal root ganglion and spinal motor neurons. Ablation of Isl1 in NC cells leads to reduced expression of genes essential for nociceptive neuron differentiation, but a failure in developmental downregulation of genes characteristic of sensory progenitors15. In spinal motor neurons, reduced Isl1 expression leads to conversion of motor neurons to V2 interneurons, although underlying mechanism remains unknown18.BMP and Notch pathways are essential for coordinating progenitor maintenance and neuronal differentiation during sympathetic nneurogenesisNtrk3/Ret/Tlx3) and pro-noradrenergic (Hmx1/Ntrk1) factors14. In Isl1 hypomorphic sympathetic ganglia, we observed significantly reduced expression of genes essential for noradrenergic differentiation , but increased expression of genes essential for cholinergic differentiation , demonstrating that ISL1 is required for repression of cholinergic characteristics and maintenance of noradrenergic characteristics during later developmental stages. Previous studies have shown that, in forebrain and spinal cholinergic motor neurons, ISL1, complexed with LHX3 or LHX8, directly binds to and activates the expression of genes of cholinergic traits33. However, we did not find direct binding of ISL1 to the enhancers/promoters of Chat, Vacht, or other cholinergic genes.Segregation of initially bimodal sympathetic neurons into noradrenergic and cholinergic neurons occurs later during development and involves antagonistic actions between pro-cholinergic 38, many of which are direct targets of ISL1. Notably, LMO1 is a neuroblastoma oncogene associated with more proliferative and aggressive phenotype39. A polymorphism within a super-enhancer in the first intron of LMO1 that preserves an evolutionarily conserved GATA factor binding motif predisposes to neuroblastoma. Knockdown of GATA3 results in decreased LMO1 expression and suppression of neuroblastoma cell growth20. We found that ISL1 binds to an evolutionary conserved region within the first intron of Lmo1 (chr7:116289070-116289656) in mouse sympathetic neurons that overlaps with the super-enhancer of LMO1 identified in human neuroblastoma, suggesting co-regulation of LMO1 expression by ISL1 and GATA3 and a potential role of ISL1 in controlling neuroblastoma cell proliferation.Neuroblastoma is an aggressive pediatric tumor arising from developing sympathetic neurons. ISL1 is overexpressed in most types of neuroblastoma, particularly in poorly differentiated neuroblastomas that indicates a poor prognosisIsl1 as a candidate gene for neuroblastoma.Taken together, our study uncovered a temporal requirement for ISL1 during sympathetic neurogenesis  have been reported previously18. To facilitate the visualization and lineage tracing of sympathetic neurons, Isl1f/f mice were crossed onto a Rosa26-LacZ or -Tomato reporter background40. All the experiments involving mice were carried out in accordance with protocols approved by the Animal Committee of Tongji University School of Medicine (TJmed-010-10).Generation of 30. Fragments amplified from mouse cDNA were used to generate the RNA probes for Insm2, Tlx3, Tfap2b, Hand1, and Lmo1. Riboprobe for mouse Vip, Sst, and Ret were kindly provided by Dr. Leping Cheng (Chinese Academy of Sciences). For BrdU staining, pregnant mice were injected with 300 μl of BrdU . For cell counting, sections of sympathetic ganglia of the appropriate developmental stages were cut at 10 μm, every fourth section was stained, and positive cells were counted. For proliferation, the total number of BrdU+ sympathetic neurons (Phox2b+) were counted and expressed as percentage of total Phox2b+ neurons. For cell death assay, the total number of Casp-3+ neurons (Tomato+) were counted and expressed as percentage of total Tomato+ neurons. At least 5–8 matched sections were analyzed and three samples per genotype per time point were analyzed. Detailed information of antibodies is available in the In situ hybridization, immunostaining, and X-gal staining were performed as describedWnt1-Cre;Isl1f/f (CKO) and Wnt1-Cre;Isl1+/+ (ctrl) with Rosa26-tdTomato reporter background. SCGs of E12 Isl1 CKO and control embryos on Rosa-tdTomato background were cultured in matrigel (BD Biosciences) in media containing NGF (20 ng/ml) and NT3 (20 ng/ml). Detailed protocol of sympathetic ganglion dissection and culture are available in the Sympathetic ganglia were dissected from E11.5–12 embryos of genotype 30. ChIP-seq was performed as previously described41. The complete RNA-seq and ChIP-seq data sets are available from the Gene Expressing Omnibus database (http://www.ncbi.nlm.nih.gov/geo/) under the accession number GSE93308. For experimental and data analysis details, see the Online Data Supplement.RNA-seq analyses with two biological repeats were performed as describedt-test was used for two-group comparisons. Differences were considered statistically significant at a value of p ≤ 0.05*, p ≤ 0.01**. For experimental details, see the online All experiments were performed for at least three independent times and respective data were used for statistical analyses. Data were presented as mean ± s.d., and a two-tailed Supplementary methods, Figure and Table LegendsSupplementary figure 1Supplementary figure 2Supplementary figure 3Supplementary figure 4Supplementary figure 5Supplementary figure 6Supplementary Table 1Supplementary Table 2Supplementary Table 3Supplementary Table 4Supplementary Table 5Supplementary Table 6Supplementary Table 7Supplementary Table 8Supplementary Table 9


There is increasing evidence suggesting the contribution of non-coding RNAs (ncRNAs) to the phenotypic and physiological complexity of organisms. A novel ncRNA species has been identified near the transcription boundaries of protein-coding genes in eukaryotes, bacteria, and archaea. This review provides a detailed description of these transcription boundary-associated RNAs (TBARs), including their classification. Based on their genomic distribution, TBARs are divided into two major groups: promoter-associated RNAs (PARs) and terminus-associated RNAs (TARs). Depending on the sequence length, each group is further classified into long RNA species (>200 nt) and small RNA species (<200 nt). According to these rules of TBAR classification, divergent ncRNAs with confusing nomenclatures, such as promoter upstream transcripts (PROMPTs), upstream antisense RNAs (uaRNAs), stable unannotated transcripts (SUTs), cryptic unstable transcripts (CUTs), upstream non-coding transcripts (UNTs), transcription start site-associated RNAs (TSSaRNAs), transcription initiation RNAs (tiRNAs), and transcription termination site-associated RNAs (TTSaRNAs), were assigned to specific classes. Although the biogenesis pathways of PARs and TARs have not yet been clearly elucidated, previous studies indicate that some of the PARs have originated either through divergent transcription or via RNA polymerase pausing. Intriguing findings regarding the functional implications of the TBARs such as the long-range “gene looping” model, which explains their role in the transcriptional regulation of protein-coding genes, are also discussed. Altogether, this review provides a comprehensive overview of the current research status of TBARs, which will promote further investigations in this research area. In the initial phase of genome sequencing, annotated protein-coding genes were thought to be a major contributor to the development of phenotypic and physiological complexity of organisms. However, this viewpoint was challenged soon afterwards. The application of high-throughput sequencing (HTS) in transcriptomics studies uncovered the intriguing phenomenon in which transcription signals were detected across the entire genomes . NotablySaccharomyces cerevisiae) (Drosophila melanogaster), chicken  (Mus musculus) (Homo sapiens) , and Arabidopsis  in budding yeast (evisiae) , fruit f gallus) , mouse (usculus) , human (sapiens) , Arabidothaliana , bacterithaliana , and arcthaliana . Additiothaliana , mouse  of a protein-coding gene, the final output is not always a full-length coding sequence. This is because various kinds of genetic and epigenetic factors, such as binding sites of RNA Pol II or transcription factors (TFs) and chromatin modifications, interrupt transcription initiation and/or elongation . PrematuIn a pioneering study, Drosophila  identified in human and mouse have been previously named as PASRs . These sof 27 nt . In bactectively . In muriectively . Thus, sectively . Anotherectively . The sizin vitro experiments have shown that the pausing and backtracking of Pol II is used to generate 6–14 nt fragments  and protein factors, some of the PASRs target specific promoter regions of genes for epigenetic modifications that affect gene transcription . HoweverTSSaRNAs have been shown to play an important role in the tissue-specific epigenetic regulation of transcription initiation of target genes . In bactTable 1). In contrast to PARs, reports on TARs, especially terminus-associated long RNAs (TALRs), are limited. In one case of TALRs, progesterone receptor gene. In another study using human K562 cells, In addition to the PARs, transcription signals have also been detected at the 3′ ends of protein-coding genes   ranging from 22 to 200 nt in length are highly conserved in human and mouse , 2010. TRPL26 . AnotherRPL26 . The enrRPL26 ; these TRPL26. Similarly, the transcription of another 3′ sense terminus-associated ncRNA, snR93, relies on the promoter activity of the upstream RPL29 gene. These data suggest that this kind of transcriptional regulation is a conserved mechanism underlying TASR biogenesis  tails have been reported in human cells . Becauseogenesis .To investigate the regulatory roles of TARs overlapping the 3′ termini of protein-coding genes in the human genome, Arabidopsis, a striking association of UNTs with promoters of many protein-coding genes has been observed , which alters the transcription status of the PR gene via the “gene looping” mechanism . In this system, we first classified TBARs, based on their genomic position, as PARs and TARs. Then, based on their sequence length, both PARs and TARs were divided into long (>200 nt) and short (<200 nt) species. Using this annotation system, most of the recently reported TBARs could be assigned to one of the categories. For further classification, we suggest that additional important features of TBARs, such as biogenesis pathways, modes of action, and biological functions, should be taken into consideration.Owing to the heterogeneity of the TBAR population, many aspects of TBARs including sequence characteristics, biogenesis pathways and biological functions are poorly understood. One of the most basic and pressing issues derives from their classification. Some of the TBARs such as PASRs, TSSa-RNAs, and tiRNAs exhibit similar genomic locations and sizes in animals. It is unclear whether these sRNAs belong to distinct classes or to the same class . It is aTo advance our understanding of TBARs, further research is needed in a few areas described below. First, it has been suggested that sRNAs mapped to the transcription boundaries of protein-coding genes are degraded remnants of mRNAs . HoweverSecond, recent evidence shows that a genetic mutation within a protein-coding locus may affect not only the gene of interest but also the associated ncRNAs. Notably, some of these affected ncRNAs, in turn, alter the chromatin state through long-range interactions . Thus, wThird, as mentioned above, the high frequency of poly(A) signals on PROMPTs is related to their rapid degradation, which has been proposed as one of the mechanisms to ensure unidirectional transcription elongation . Since mFourth, in addition to the TBARs associated with protein-coding genes, whether TBARs exist on lncRNAs needs to be revisited. Conserved secondary structures are detected at the ends of certain lncRNAs ; howeverOverall, investigations into all of the above-mentioned challenging but intriguing research areas are needed. Results of these investigations are expected to increase our knowledge of TBARs.DY, XM, HW, and YM: wrote the manuscript. ZZ, DY, and XM: prepared the figure and table. All authors read and approved the final manuscript.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.


Previously, we showed that during Xanthomonas oryzae phage Xp10 infection, the phage protein P7 inhibits the host RNAP by preventing the productive engagement with the promoter and simultaneously displaces the σ70 factor from the RNAP. In this study, we demonstrate that P7 also inhibits the productive engagement of the bacterial RNAP containing the major variant bacterial σ factor, σ54, with its cognate promoter. The results suggest for the first time that the major variant form of the host RNAP can also be targeted by bacteriophage-encoded transcription regulatory proteins. Since the major and major variant σ factor interacting surfaces in the RNAP substantially overlap, but different regions of σ70 and σ54 are used for binding to the RNAP, our results further underscore the importance of the σ–RNAP interface in bacterial RNAP function and regulation and potentially for intervention by antibacterials.The σ factor is a functionally obligatory subunit of the bacterial transcription machinery, the RNA polymerase. Bacteriophage-encoded small proteins that either modulate or inhibit the bacterial RNAP to allow the temporal regulation of bacteriophage gene expression often target the activity of the major bacterial σ factor, σ Graphical abstract •54.Xp10 phage transcription regulator P7 inhibits transcription by RNAP containing σ•54–RNAP with the promoter DNA.P7 prevents the productive engagement of the σ54–RNAP-promoter complexes.• P7 disrupts preformed σ The isomerization of the RPc to the transcriptionally proficient promoter complex (RPo) is accompanied by large-scale conformational rearrangements in both the DNA and the RNAP, primarily in the β, β′ and σ70 subunits. In the RPo, the DNA duplex is locally melted and the + 1 site on the template strand is positioned at the catalytic centre of the RNAP; the double-stranded DNA, which is downstream of the + 1 site, is cradled in the downstream DNA binding channel that consists of a trough formed by the β′ jaw, β downstream lobe, β′ clamp, and β′ region G non-conserved domain (GNCD)  belong to the major σ70 class and share three regions of conserved sequences . Subregions 2.4 and 4.2 of regions 2 and 4 of E. coli σ70 are responsible for the recognition of the conserved − 10 and − 35 double-stranded promoter sequences, respectively 70 makes extensive contact to the β′ clamp helices, which comprise of a coiled-coil motif and constitutes the major σ docking site in the RNAP. Region 4 makes extensive interactions with β flap domain and the conserved features (notably the β′ zipper and β′ zinc binding domain) in the amino-terminal domain of the β′ subunit  70 and the β and β′ subunit are important for the binding of the holoenzyme to conserved − 35 promoter sequence and during promoter clearance for the appropriate exiting of the nascent RNA from the RNAP Central to the regulation of bacterial gene expression is the bacterial RNA polymerase (RNAP), which is a complex multisubunit enzyme responsible for the transcription of RNA from the DNA template. The catalytic “core” of the RNAP is composed of five subunits α70 from the downstream DNA binding channel to allow RPo formation 70 and structurally remodels it 70 region 4 can no longer bind to the conserved − 35 promoter sequence of host promoters and to the β flap domain of the RNAP. This, in turn, allows another T4 protein, MotA, to interact with the far carboxyl terminal region of σ70 and divert the host RNAP from host promoters to T4 phage middle gene promoters, which do not contain conserved − 35 promoter elements Xanthomonas orzyae infecting Xp10 phage, inhibits the host RNAP by causing the displacement of the σ70 during RPc formation 70 region 4 and the β flap. Thus, upon engagement with the promoter DNA, the σ70 factor becomes displaced from the RNAP, which consequently prevents the formation of the RPc Regulating the activity of the RNAP is a key mechanism in controlling gene expression and is often orchestrated by transcription regulators that interact with the RNAP to modulate its activity. Therefore, the RNAP often serves as a nexus for interaction of transcription regulators to fine-tune gene expression to match cellular requirements. Unsurprisingly, some bacteriophages (phages) have evolved strategies to alter the activity of host RNAP during infection to allow the temporal and coordinated usage of the host and phage RNAP for phage gene expression 54, which is present in many bacterial species, is the major variant bacterial σ factor and is unrelated to the σ70 family in sequence, structure, function, and regulation , which is obligatory for the docking of σ54 to the RNAP, makes extensive contacts to the β′ NTD and the β flap domain  and is therefore resistant to inhibition by P7, we previously constructed a P7-sensitive version of the E. coli RNAP by replacing 6–9 aa of the E. coli β′ subunit with the corresponding residues of the X. oryzae RNAP to study the effect of P7 on σ70-dependent transcription in vitro transcription assay using the well-characterised Sinorhizobium meliloti nifH promoter and the catalytic domain of the E. coli Phage shock protein F (PspF1–275) P7SEσ54 activity. Results revealed that the amount of the UpGpGpG transcript synthesised from S. meliloti nifH promoter by P7SEσ54 was substantially reduced (by ~ 80%) in the presence of just an equimolar amount of P7 to P7SEσ54 . Results in 70-dependent promoters 54 from the holoenzyme, resulting in the formation of a ternary complex core RNAP–nifH–probe–P7 complex (= C4) and the Eσ54–P7 and/or Eσ54 complexes, but never the RPc (core RNAP-σ54-nifH-probe complex) or a complex consisting of core RNAP, σ54, nifH probe, and P7.We next conducted EMSAs with 5454, the precise mechanism underpinning this process is unknown. We propose that the binding of P7 to the β flap/β′ NTD domains could allosterically affect other parts of the RNAP and σ54 associated with promoter recognition and RPc formation. This view is consistent with the previous finding that a mutant form of Eσ54, reconstituted with a mutant variant of the core RNAP containing a deletion of the β flap-tip helix, displayed defects at several steps after holoenzyme formation en route to the transcriptionally proficient RPo 54, which is obligatory for the docking of σ54 to the RNAP and makes extensive contacts to the β′ NTD and the β flap domain (= the P7 binding regions) and thereby indirectly affects promoter DNA binding by the Eσ54. Intriguingly, whereas P7 has no detectable effect on Eσ70 activity after the RPo has formed  of the β′ subunit are substituted with the corresponding residues from the X. oryzae β′ subunit (the major determinant for P7 binding), suggests that P7 can inhibit RPc formation by the major and major variant forms of the X. oryzae RNAP, thus suggesting that the Xp10 transcription programme might require or involves the inactivation of the host transcription machinery containing σ54. The use of σ54 by phages for the execution of their transcriptional programme, although rare, is not unprecedented, since the development of the Pseudomonas aeruginosa phage YuA is strictly dependent on the host σ54 factor The transcriptional programme of the Xp10 phage clearly relies on the coordinated activity of both the host and Xp10 RNAPs. During early stages of infection, Xp10 relies on the Our results further indicate that regardless of the nature of the σ factor–β flap–β′ NTD interface, P7 is able to indiscriminately prevent the productive and efficient engagement of the RNAP with the promoter and thereby underscores the significance of the β flap/β′ NTD domains for bacterial RNAP function and regulation. Since the RNAP is a proven antibacterial target, the σ factor–β flap–β′ NTD interface is potentially an Achilles' heel in the bacterial RNAP for intervention by small molecules to inhibit bacterial transcription.BBSRC and the Wellcome Trust. S.W. and S.M. are recipients of Investigator Awards from the Wellcome Trust.This work was funded by project grants from the


Drosophila FOXO (dFOXO) and the zinc finger transcription factor Kruppel homolog 1 (Kr-h1), one of the key players in juvenile hormone signaling. We found that Kr-h1 mutants show delayed larval development and altered lipid metabolism, in particular induced lipolysis upon starvation. Notably, Kr-h1 physically and genetically interacts with dFOXO in vitro and in vivo to regulate the transcriptional activation of insulin receptor (InR) and adipose lipase brummer (bmm). The transcriptional co-regulation by Kr-h1 and dFOXO may represent a broad mechanism by which Kruppel-like factors integrate with insulin signaling to maintain metabolic homeostasis and coordinate organism growth.Transcriptional coordination is a vital process contributing to metabolic homeostasis. As one of the key nodes in the metabolic network, the forkhead transcription factor FOXO has been shown to interact with diverse transcription co-factors and integrate signals from multiple pathways to control metabolism, oxidative stress response, and cell cycle. Recently, insulin/FOXO signaling has been implicated in the regulation of insect development via the interaction with insect hormones, such as ecdysone and juvenile hormone. In this study, we identified an interaction between  The ability to coordinate growth and development with nutrient availability is critical for the adaptation to fluctuating environment. The main hormonal pathway that regulates insect growth and energy metabolism is insulin/insulin-like growth factor signaling (IIS). Unlike the single insulin, two insulin-like growth factor (IGF) system in mammals, insects have multiple insulin-like peptides4. The activation of insulin/insulin-like growth factor signaling stimulates two major kinase cascades: the PI3K/AKT pathway and MAPK/ERK pathways5. In particular the O subclass of the forkhead transcription factors (FOXO) are substrates of PI3K/AKT. Decreased cellular IIS leads to de-phosphorylation and nuclear translocation of FOXO and the transcriptional activation of FOXO target genes7. Besides IIS, FOXO transcriptional activity is modulated by several other pathways  through post-translational modification (PTM) that modulate FOXO binding to DNA or its co-activators7.Metabolic homeostasis plays important roles in developing animals10. Molting hormone ecdysone regulates developmental timing by inhibiting insulin signaling and promoting the nuclear localization of Drosophila forkhead transcription factor (dFOXO)8. During the non-feeding pupation stages of Bombyx silkworm, 20-hydroxyecdysone (20E) induces lipolysis and promotes transcriptional activation of two adipose lipases via the regulation of FOXO11. On the other hand, the link between JH and insulin signaling was first demonstrated in Drosophila where insulin receptor (InR) mutants were seen to reduced JH biosynthesis12. Recent studies on size control further suggest that JH controls growth rate through Drosophila FOXO10. Interestingly, JH also regulates lipid metabolism via the interactions with FOXO in Tsetse flies9 and diapausing mosquitoes13. Across these studies, it remains unclear how JH interacts with nutrient signaling and whether JH directly acts on FOXO-mediated transcriptional control.FOXO plays a key role in mediating the cross-talk between insulin signaling and other insect hormones (e.g. juvenile hormone (JH) and ecdysteroids) to coordinate insect growth, development and metabolic homeostasis14. The interactions between FOXO and its binding partners contribute to the transcriptional specificity of FOXO and pleotropic functions of insulin/FOXO signaling. For instance, mouse FOXO1 interacts with PGC-1α in liver to modulate insulin-mediated gluconeogenesis15; mammalian FOXO1 binds to Smad2/3 in response to TGF-beta signaling and regulates cell proliferation16; mammalian FOXO transcription factors (FOXO3A and FOXO4) interacts with beta-Catenin of Wnt signaling to modulate cellular oxidative response17. In Drosophila, dFOXO interacts with bZIP transcription factor REPTOR of mechanistic target of rapamycin (mTOR) signaling to regulate growth and energy homeostasis18. Interestingly, recent studies found that FOXO interacts with Ultraspiracle (Usp), a co-factor of the ecdysone receptor, to regulate ecdysone biosynthesis and developmental timing in Drosophila19. To date, factors of JH signaling have not been identified to directly interact with FOXO.FOXO interacts with a number of transcription factors within the nucleus to activate or inhibit transcription of target genes22. JH strongly induces the transcription of Kr-h1 via its receptor Methoprene-tolerant (Met)23. During insect development, Kr-h1 functions as a transcriptional repressor on neurogenesis of mushroom body and photoreceptor maturation25. Kr-h1 belongs to Kruppel-like factors (KLFs) protein family, a group of conserved C2H2 type zinc finger transcription factors. Unlike mammalian KLFs that contain three zinc finger DNA binding domains, Drosophila Kr-h1 has eight zinc finger motifs26. KLFs are also closely related to transcription factor Sp1 (specificity protein 1). At least seventeen KLFs are identified in mammals27. Both KLFs and Sp1-like factors recognized GC-rich DNA elements or CACCC-box in the promoters of target genes27. While KLFs and Sp1 can function as both transcription activator and repressor, the N-terminus of KLFs contains a consensus motif PXDL(S/T) that is thought to interact with transcriptional co-repressor CtBP 29. Some KLFs also interact with transcriptional co-activators to enhance transcriptional activities. For instance, KLF1 is acetylated through its interaction with co-activators p300 and CREB-binding protein (CBP), which leads to elevated induction of target gene beta-globin30.Kruppel-like homolog 1 (Kr-h1) is a key regulator of insect molting and metamorphosis and a major effector in JH signalingDrosophila. While characterizing a Kr-h1 mutant, we found that lipolysis was elevated in fasting mutant larvae. Genetic and molecular analyses revealed that Kr-h1 physically interacts with dFOXO and represses the transcriptional activation of dFOXO target genes, such as insulin receptor (InR) and adipose triglyceride lipase (bmm or brummer). The present study suggests a mechanism by which Kruppel-like factor Kr-h1 integrates with insulin/dFOXO signaling to control lipid metabolism and coordinate organism growth.In this study, we identified an interaction between dFOXO and the zinc finger transcription factor Kr-h1 in Drosophila Kruppel-like factor Kr-h1 in larval development and metabolic control using a P-element-induced hypomorphic allele Kr-h1[7] 31. The P-element insertion is located within the first intron of the Kr-h1 locus and is reported to interfere with the transcription of Kr-h1 isoforms. Kr-h1[7] homozygous mutants are partially viable during embryonic and larval development31. We backcrossed this Kr-h1[7] allele into a ywR background for seven generations. The heterozygotes of cleaned Kr-h1[7] mutants showed prolonged developmental time to pupariation , the major form of lipid storage in the fat body, using larvae at 90 hours after egg laying (AEL). Among fed animals, triglycerides were reduced 2-fold by Kr-h1 mutation, while glycogen was similar among genotypes  . Fasting reduced stored glycogen to the same extent in both genotypes  is a key lipase involved in TAG mobilization32. While transcripts of bmm were slightly up-regulated by fasting in wild-type larvae    . In addition, we observed a reduced expression of FASN1 (Fatty acid synthase 1) in Kr-h1 mutants  Fig. . This reing Fig. . Perilipnts Fig. . FASN1 ints Fig. , it is lbmm, and lipid droplet-associated protein Lsd-1. In contrast, Kr-h1 acts on lipogenesis under normal fed condition. The present study focuses on the role of Kr-h1 in lipolysis under nutrition deprivation.Collectively, these results suggest that Kr-h1 plays an important role in lipid metabolism. Upon fasting, Kr-h1 regulations lipolysis through the transcriptional regulation of triglyceride lipase 35. Here we see that phosphorylation of IIS-regulated kinase AKT was reduced in Kr-h1[7] homozygotes , which are the major DILPs produced from brain neurosecretory cells, known as insulin producing cells (IPCs)  Fig. .Figure 26. Accordingly, mRNA expression of two key dFOXO target genes, 4ebp (eukaryotic translation initiation factor 4E binding protein) and InR were significantly induced in Kr-h1 mutants    Fig. . It is kKr-h1[7] and dfoxo[21]37. Interestingly, we found that dfoxo[21] heterozygotes rescued the delayed pupariation of Kr-h1[7] heterozygous mutants  Kr-h1 and dFOXO in cultured Drosophila cells. We were able to pull down endogenous dFOXO from nuclear and cytoplasmic extracts using an anti-dFOXO antibody. Interestingly, Kr-h1 was detected in the protein complex from the nuclear extracts, but not from the cytoplasmic extracts  in Kc167 cells. The mRNA expression of ain Fig. . Thus, KInR and bmm36, although our recent ChIP-Seq analysis (unpublished) suggests that dFOXO also strongly bound the promoter region near the 5′-UTR of InR . Three putative KLF binding sites denoted P1~P3 in each promoter were identified including sites in 5′-UTR and intronic regions  reported by Kayukawa et al.23.Kr-h1 and dFOXO physically interact and may thus transcriptionally co-regulate metabolic genes. It has been previously shown that dFOXO binds to the promoter regions near transcriptional start sites of ons Fig.  .Binding of dFOXO and Kr-h1 to these putative sites was determined by ChIP-PCR analysis in fasted animals. At ion Fig. , while Kion Fig. . At bmm ion Fig. . The co-bmm and InR. We performed a ChIP-PCR to test this possibility using anti-dFOXO antibody and Kr-h1[7] mutants. dFOXO binding to the InR P1 region was increased from 2.9-fold relative to negative control (Act5C) in fasted wild-type to 8.95-fold in fasted Kr-h1 mutants    Fig. . In cont62) Fig. . At the nts Fig. . These din vivo, we examined the nuclear co-localization of Kr-h1 and dFOXO in larval fat body using our anti-Kr-h1 antibodies. Previous studies showed that Kr-h1 expressed broadly in many tissues during late larval and prepupal stages, including imaginal discs, trachea, central nervous system, ring gland, salivary gland, muscle, gut, and fat body38, and the expression was primarily detected in nuclei. We find that in early L3 larval development, Kr-h1 is expressed at low level and primarily in cytosol  with tissue-specific Gal4 drivers. Knockdown of Kr-h1 in fat body  and muscle  delayed pupariation, while knockdown in gut, IPCs and Corpus allatum (CA) showed no effects on larval development 40. In this capacity, recent studies suggest that JH and Met are involved in not only development programming, but also in metabolic control43, although how JH affects metabolism is largely unknown.The interaction between Kr-h1 and dFOXO has the potential to integrate development and nutrient signaling. Nutrient signaling through FOXO involves insulin, AMPK, SIRT and JNK in both insect and mammals alikebmm transcription. Consistent with previous studies9, triglyceride levels were reduced in flies where the corpora allata were genetically ablated (CAX)  Fig. . ConversAX) Fig. . Additioels Fig.  and up-rRNA Fig. . Met alsbmm Fig. . JH may nts Fig. . Furthernts Fig. . But whiing Fig. . On the ion Fig. . Overall44. Multiple transcription factors interact at their genomic binding sites to enhance transcriptional specificity and pleiotropic functions of metabolic pathways. As a key node in the metabolic network, forkhead transcription factor FOXO has been shown to interact with diverse transcription co-factors and thereby integrate signals to control metabolism and oxidative stress14. Intriguingly, in recent genomic studies47, enriched FOXO binding at specific genes does not always correlate to elevated transcriptional output, suggesting there exists inhibitory or inertial mechanisms to repress the transcriptional activity of DNA-bound FOXO.Transcriptional coordination is a key process contributing to metabolic homeostasisDrosophila Kruppel-like factor Kr-h1 acts as a repressor of dFOXO to modulate induction of two dFOXO target genes, InR and bmm. Like other FOXO interacting partners, Kr-h1 physically binds to dFOXO and inhibits the expression of dFOXO targets by influencing the binding affinity of dFOXO to DNA. The transcriptional activity of FOXO is typically regulated in two layers. The first and probably most important regulation is through PTM, including phosphorylation, acetylation and ubiquitination6. PTM of FOXO proteins can affect its subcellular localization (by phosphorylation), DNA binding affinity (by acetylation) and protein degradation (by ubiquitination). Interestingly, the effects of acetylation on FOXO factors seem to be quite different from those by acetylation on KLFs. Acetylation of FOXO by co-factor CBP/p300 weakens the FOXO binding to its DNA targets48, while CBP/p300 acetylated KLF1 shows increased transcriptional activation of target gene beta-globin30. The second mechanism for the regulation of FOXO activity is through the interaction between FOXO and other transcription factors or co-factors. FOXO factors have been shown to interact with diverse transcription factors  that often potentiate the expression of FOXO target genes14. Kr-h1 identified in our study presents another example for this type of modulatory regulation, although the interaction between Kr-h1 and dFOXO results in transcription repression, instead of activation.Here we find that 49 and with peroxisome proliferator-activated receptor-γ (PPARγ)50. Alternatively, Kr-h1 may act by inhibiting recruitment of dFOXO-coactivators (e.g. SIRT or CBP/p300) or by sequestering these coactivators away from dFOXO. Kr-h1 may also recruit additional co-repressors (e.g. CtBP or Sin3-HDAC) to the dFOXO transactivation sites to block the transcriptional activation of target genes. The N-terminal Q-rich domain of KLFs is crucial for the recruitment of co-repressors CtBP and Sin3A51. In our co-immunoprecipitation assays, the Q-rich domain of Kr-h1 strongly binds to dFOXO, suggesting Kr-h1 might inhibit dFOXO activity through recruiting co-repressors. One possible candidate is an Sds3-like gene (CG14220), which was previously found to co-immunoprecipitate with Kr-h152. Sds3-like gene family proteins can form co-repressor complex with Sin3A and HDAC to inhibit gene transcription via interactions with sequence-specific transcription factors53.While we do not fully resolve how Kr-h1 blocks dFOXO activity, it seems that Kr-h1 can inhibit dFOXO binding to its DNA targets. This result is similar to previous studies showing reduced FOXO-DNA binding upon interaction with androgen receptor (AR)51. The function of Kr-h1 in lipid metabolism and insulin signaling identified in the present study complements recent studies where KLFs function in cellular metabolic regulation, such as gluconeogenesis55. Likewise, KLF15 deletion in mice produces hypoglycemia and impaired amino acid catabolism upon fasting55. Additionally, KLF5 heterozygous mice are resistant to high-fat diet-induced obesity. SUMOylation modulates the transcriptional activities of KLF5 and its association with peroxisome proliferator-activated receptor-delta (PPAR-delta) to control the expression of carnitine-palmitoyl transferase-1b (Cpt1b), uncoupling proteins 2 and 356. Interestingly, KLF4 has recently been identified as a direct target gene of FOXO-mediated transcription during B cell development57, suggesting a potential interaction between KLF and FOXO transcriptional regulatory network.KLFs have well documented roles in cell proliferation, differentiation and apoptosisDrosophila KLF Kr-h1 transcriptionally controls many metabolic genes, including key dFOXO targets (e.g. InR and triglyceride lipase bmm). While it is not known whether Drosophila Kr-h1 could broadly interact with dFOXO across the genome, such a genome-wide interaction between mammalian FOXO factors and KLFs has been suggested by a recent meta-analysis58. Because Kr-h1 plays an important role in morphogenesis during Drosophila development21, the interplay between Kr-h1 and dFOXO raises the possibility that Kr-h1 coordinates growth and development through insulin/dFOXO-mediated metabolic regulation.Our ChIP-PCR studies suggest that bmm is weakly regulated by Kr-h1, although the TAG is significantly reduced in fed Kr-h1[7] mutants. We speculate that decreased TAG in Kr-h1[7] mutants is probably due to the reduction in lipogenesis and fatty acid synthesis, rather than to increased lipolysis. Our preliminary data reveal that the expression of fatty acid synthase is down-regulated in Kr-h1[7] mutants. The potential connection between Kr-h1 and lipogenesis needs to be further examined.We also noticed that the direct interaction between Kr-h1 and dFOXO occurs strongly upon fasting. In the normal fed state, transcriptional regulation of lipase 60. In Tsetse flies, JH and insulin co-regulate the expression of TAG lipase and inhibit lipolysis9, which is consistent with our observation that TAG metabolism and lipase brummer expression are regulated by JH and its receptor Met. Similarly, JH III treatment reduced FOXO levels in diapause-destined female mosquitoes, Culex pipiens13. Although we did not test the paralog of Met, germ cell-expressed (GCE)63, previous studies have demonstrated GCE is not involved in lipid metabolism during lactation state of Tsetse flies9. Finally, JH signaling was recently found to genetically interact with insulin/dFOXO to control larval growth rate and define final body size in Drosophila10. Thus, the transcriptional co-regulation of lipid metabolism by Drosophila Kr-h1 and dFOXO may contribute to a novel mechanism through which JH interacts with insulin signaling to integrate metabolism and growth during larval development.In insects, Kr-h1 is one of the key effectors of JH signaling, an important hormonal pathway governing insect molting, metamorphosis and reproduction. Recent studies reveal that JH also participates in the regulation of carbohydrate and lipid metabolismDrosophila, these transcription factors are likely to co-regulate many key metabolic genes. As well, these metabolic tissues contribute significantly to other insect physiology and organismal functions, such as stress resistance and aging that are tightly regulated by insulin/dFOXO signaling64 and by JH signaling41. We predict that the interplay between Kr-h1 and dFOXO will also contribute to the regulation of these adult physiological processes. In fact, a recent study reported that loss of kruppel-like factors (KLFs) abolished the lifespan extension in multiple longevity paradigms in C. elegans, such as dietary restriction and insulin receptor mutant daf-265. Thus, identifying the key co-factors, mechanisms, and downstream events of the Kr-h1/dFOXO transcriptional network may advance our understanding of how JH and insulin signaling coordinately regulate metabolism, development, and aging.Since both Kr-h1 and dFOXO express highly in metabolic tissues (fat body and muscle) of Kr-h1[7] or Kr-h1 [k04411]31 , Kr-h1 RNAi lines , Kr-h1 EP line #EP228966, UAS-Kr-h1-LacZ25, foxo[21]67, Met[1]68, Met[27]68, r4-gal4 (Bloomington # 33832), Mhc-gal469, Mex-gal470, dilp2-gal471, Aug21-gal472, S106-GS-gal473. Double mutants were made by crossing Kr-h1[7] or Met[27] to foxo[21] respectively. Corpus allatum (CA) ablation flies (named CAX flies) are generated in our laboratory as previously described41. ywR flies were used as wild-type flies in most of the experiments. For methoprene treatment, adult flies were exposed for 24~48 hours to various concentrations of methoprene applied to the side of culture vials.Flies were maintained at 25 °C, 40% relative humidity and 12-hour light/dark. Adults were reared on agar-based diet with 0.8% cornmeal, 10% sugar, and 2.5% yeast (unless otherwise noted). Fly stocks used in the present study are: Kr-h1 [7] mutants. Recombinant Kr-h1α protein was produced and purified using the HaloTag Protein Expression System . Kr-h1α cDNA clone (LD32311) was obtained from DGRC (Drosophila Genomics Resource Center) and cloned into pFN29K His6HaloTag T7 Flexi vector. The Kr-h1α-Halo fusion protein was expressed in Single Step (KRX) Competent Cells and purified using HaloLink Resin .Kr-h1 polyclonal antibody was generated in rabbits against the short peptide sequence ‘LIEHFKRGDLARHG’  and affinity purified . The specificity of Kr-h1 antibody was verified by western blots Fig.  using KrDrosophila Akt antibody (Ser505) , Akt antibody .All western blots were performed per the following procedures: Fly tissues or cells were homogenized in RIPA buffer  with protease inhibitors . Supernatant was incubated with NuPAGE LDS loading buffer  at 70 °C for 10 min. About 20 μg of denatured protein was separated on 4~12% Bis-Tris precast gels  and transferred to PVDF membranes. Following incubation with primary and secondary antibodies, the blots were visualized with Pierce ECL Western Blotting Substrate . Other antibodies used in the present study are Phospho-RpL32 or rp49) by the method of comparative CT. Primer sequences are listed in Supplementary Table Total RNA was extracted using Trizol reagent  from 10~15 synchronously staged larvae or whole adult flies. DNase-treated total RNA was quantified and about 500 ng of total RNA was reverse transcribed to cDNA using iScript cDNA Synthesis Kit . QPCR was performed with an ABI prism 7300 Sequence Detection System . Three to five biological replicates were used for each experimental treatment. mRNA abundance of each gene was normalized to the expression of ribosomal protein L32 (Synchronized eggs were placed on 35 × 10 mm petri dishes containing standard medium (see above) at 20~30 eggs per dish. The numbers of pupae were recorded 2~3 times every day around 120 hr AEL till all larvae molt into pupae.Larval fat body were dissected in 1x PBS and stained with 0.00005% Nile Red  and 1 µg/ml Hoechst 33342  in 75% glycerol for 10 min at room temperature. Fat body was then mounted in 75% glycerol and imaged using an Olympus BX51WI upright epifluorescence microscope.74. For TAG assay, 25 staged larvae or six adult flies were collected and homogenized in 1xPBS containing 0.1% Tween 20 and TAG was quantified using Thermo Scientific™ Triglycerides Reagent . For glycogen measurement, samples were digested with amyloglucosidase  and glucose contents were quantified using Thermo Scientific™ Glucose Hexokinase Reagents . The relative level of each metabolite was obtained by normalizing the metabolites to total protein.All metabolic analyses were performed as previously describedDrosophila Kc167 cells adapted to serum-free culture medium (Drosophila Schneider Medium). Either full-length (Kr-h1 α-isoform) or partial gene products were cloned into Drosophila Gateway Vectors with N-terminal tags (FLAG and HA) following Drosophila Gateway Vectors protocols (https://emb.carnegiescience.edu/Drosophila-gateway-vector-collection). About 1 µg of constructs were transfected to 2 × 106 Kc167 cells using Effectene reagent . Two days after transfection, cells were harvested and lysed in NP-40 lysis buffer  with proteinase inhibitors . To pull-down target proteins, total protein extracts were incubated with proper antibodies and Dynabeads Protein A . Following pull-down, western blotting was performed to examine protein complex. Antibodies used in pull-down and western blots include rabbit anti-Kr-h1 and anti-dFOXO produced in our laboratory, rabbit anti- HA , and mouse anti-FLAG . Nuclear extracts for immunoprecipitation were conducted with a nuclear extraction kit .All the immunoprecipitation and pull-down experiments were conducted in rd instar larvae (90 hr AEL) . Tissue immunostaining were performed as previously described47, using slowFade mounting solution with DAPI . Samples were imaged with a Zeiss 510 laser scanning confocal microscope or an Olympus BX51WI upright epifluorescence microscope equipped with Hamamatsu Flash 4.0 Plus CMOS Camera. Antibodies used in immunohistochemistry included: rabbit anti-Kr-h1 (1:200) (this study), anti-dFOXO (1:200)75, anti-GFP , anti-rabbit IgG-DyLight 488 (1:300) anti-rabbit IgG-Alexa Fluor 594 (1:300) and anti-Guinea pig IgG-DyLight 488 (1:300) . Immunostaining of Kc167 cells was performed following above methods.To examine the co-localization of Kr-h1 and dFOXO, larval fat body was dissected from fed or fasted 347. About 50 staged larvae were used in each sample. Flies were homogenized and cross-linked in 1xPBS containing 1% formaldehyde. The fly nuclear extractions were sonicated using a Branson 450 sonicator to break down the chromatins. Immunoprecipitation was performed using Dynabeads Protein A and anti-Kr-h1 and anti-dFOXO antibodies. Following the wash with LiCl and TE buffer, the DNA-protein complex was eluted, reverse cross-linked, digested with Proteinase K and RNase. Kr-h1-bound or dFOXO-bound DNA fragments were purified and used as templates in qPCR analysis. Binding enrichment was calculated as the fold change between ChIP DNA vs. input DNA (Chromatin extracts before immunoprecipitation). The binding to the coding region of Actin (Act5C) was used as negative controls.ChIP was conducted as previously described76, as modified78, was used to quantify juvenile hormone (JH). Samples were eluted through aluminum oxide columns successively with hexane, 10% ethyl ether–hexane and 30% ethyl ether–hexane. Samples were subjected to a second series of aluminum oxide elutions (30% ethyl ether-hexane then 50% ethyl-acetate–hexane) after derivatization with methyl-d alcohol  and trifluoroacetic acid . Purified samples were analyzed on an HP 7890A Series GC  equipped with a 30 m × 0.25 mm Zebron ZB-WAX column  and coupled to an HP 5975C inert mass selective detector with helium as the carrier gas. MS analysis occurred in the SIM mode, monitoring at m/z 76 and 225 to ensure specificity for the d3-methoxyhydrin derivative of JH III. Total abundance was quantified against a standard curve of derivatized JH III and using farnesol  as an internal standard. The detection limit is approximately 1 pg.For each sample, 197–200 individual flies (7~10-day-old) were placed in 500 µl hexane in a glass vial with a Teflon cap insert and stored at −80 °C prior to analysis. To extract the hormone, the flies were crushed with a Teflon tissue grinder. The resultant homogenate was centrifuged at 3500 rpm for 5 min, and the supernatant was removed to clean vial. Extraction was conducted three times, combining the resultant supernatant from each sample. The gas chromatography/mass spectrometry (GC–MS) methodGraphPad Prism 6  was used for statistical analysis. To compare the mean value of treatment groups versus that of control, either student t-test or one-way ANOVA was performed using Dunnett’s test for multiple comparison. The effects of mutants on starvation responses was analyzed by two-way ANOVA, including Tukey multiple comparisons test.Supplementary Information


The third Japanese meeting entitled “Biological Function and Evolution through Interactions between Hosts and Transposable Elements (TEs)” was held on 5–6 September 2016 at National Institute of Genetics (NIG), Mishima, Japan. Supported by NIG, the goal of the meeting was to bring together researchers who study diverse biological phenomena such as schizophrenia, carcinogenesis, cellular reprograming, skin function, placental formation, plant mutagenesis and epigenetics, and small RNA-mediated heterochromatinization, where TEs are involved in various ways. The meeting included 13 invited speakers. Here we present highlights of these invited talks. Transposable elements (TEs) are very important parts of genomes, as they play various roles in biological functions and evolution. Their important roles were evident at the third Japanese meeting entitled “Biological Function and Evolution through Interactions between Hosts and Transposable Elements (TEs)” which was held on 5–6 September 2016 at National Institute of Genetics (NIG), Mishima, Japan, with a beautiful view of Mt. Fuji. Before these TE meetings, the first of which was held in 2013, there were very few opportunities in Japan for TE researchers to gather to exchange their ideas. For the third meeting in September, we had 13 invited speakers in six sessions chaired by Kei Fukuda , Hidenori Nishihara , Toshiki Habu , Hidetaka Ito , Hidetoshi Saze , and the author, Kenji Ichiyanagi . There were many questions and comments from the more than 50 attendees and from the session chairs with time allowed for extensive and profound discussions.Irf7-knockout (KO) mice do not, suggesting an involvement of the interferron-dependent cellular cascade [de novo L1 insertions preferentially reside around genes important for synaptic function, suggesting an involvement of these L1 insertions in pathogenesis of schizophrenia. L1 amplification in the prefrontal cortex is also observed for animal models when the immune system is activated (poly I:C dsRNA injection in mice and EGF treatment of macaques). Again, environmental factors induce L1 retrotransposition in somatic cells. She also presented data showing that L1 is amplified in neurons derived from induced pluripotent stem cells (iPSCs) from patients with the 22q11.2 deletion syndrome with schizophrenia. Thus, genetic factors also play a role. Identifying the type and location of the neurons that accumulate de novo L1 insertions in the brain of schizophrenia patients, which is now feasible by single cell genomics techniques, will provide further insights on the relationship between L1 retrotransposition and psychiatric disorders.Right after the opening remarks by Ichiyanagi, Yukihito Ishizaka  presented an interesting link between HIV infection, LINE-1 (L1) retrotransposition, and neurocognitive disorders in humans. He and his colleagues found that L1 mobility is increased in 6 of 15 blood samples of HIV-1 positive patients, and that the Vpr protein of HIV-1 is responsible for the induction of L1 retrotransposition [ cascade . Taken t cascade . Miki Bu cascade . InteresEvidence has been accumulating that endogenous retroviruses (ERVs) can serve as an enhancer or promoter of host genes, and their expression patterns are likely programmed by a combination of host transcription factors. Kosuke Hashimoto  presented his recent results about a transcriptome study of c-Myc-induced liver tumors, which reveals that transcription from LTR sequences of several mouse ERV families are increased by up to 1000-fold, a situation similar to those in virus-induced tumors . Shuttinpro region of an ERV, termed SASPase (Skin Aspartic Protease), which is expressed in epidermis. For terrestrial vertebrates, the stratum corneum, the uppermost dead cell layer of epidermis constituting the boundary between the body and environment, is crucial to adapt and live in a dry environment. In the stratum corneum of mammals, which have supple and moisturized skin, the profilaggrin protein is hydrolyzed to yield filaggrin monomers, which play a role in barrier function of the stratum corneum and are a major predisposing factor for atopic dermatitis. Interestingly, SASPase is conserved among mammals, and moreover, is involved in profilaggrin hydrolysis. KO mice of this ERV-derived protease show a dry-skin phenotype due to abnormal proteolysis of profilagrin and consequent reduction of filagrin [Sushi-ichi-related retrotransposon homologues (SIRHs). Among 11 SIRH genes in mammals, he and his colleague have shown that Peg10 , Peg11 (Sirh2/Rtl1) and Sirh7 (Ldoc1) are essential for development and reproduction, and implicated in multiple aspects of placental function [Sirh11 (Zcchc16) KO mice exhibit various behavioral abnormalities, possibly related to the noradrenergic system [Sirh11 is retained in three groups of the eutherians , whereas it is heavily mutated in xenarthrans such as armadillo. Moreover, a large variation in the SIRH11 protein is detected even in the three groups in a lineage-specific manner; for example, a loss of a C-terminal CCHC RNA-binding domain that occurred independently in gibbons and megabats and a loss of the N-terminal half that occurred independently in New World monkeys, Hystricognaths (such as Porcupine), and Cetartiodactyla . Therefore, SIRH11 may have contributed to the diversification of eutherians by lineage-specific structural changes and subsequent species-specific functional changes, which have been fixed via complex natural selection events [Examples of TE exaptation, where TEs give rise to functional sequences, are not restricted to DNA segments. TE-encoded proteins can also be exapted. Takeshi Matsui  talked about a protease derived from the filagrin . Thus, efilagrin . Fumitosfunction . In addic system . Interesn events .nDart1, a non-autonomous element associated with a hAT family of DNA transposon aDart1, has generated various mutants under natural growth conditions due to its tendency to insert into genic regions [snow-white leaf1 (swl1), shows variegated albino phenotype. He and his colleague revealed that it is caused by an insertion of nDart1 in the SWL1 gene, which was subsequently revealed to be essential for thylakoid membrane organization during chloroplast development. The nDart1 insertion inhibits the production of the wild-type protein by interfering with transcription from the normal start site. Of interest is that this albino phenotype is variegated. This is because the inserted copy sometimes gets excised by the aDart1 transposase during somatic growth [GmphyA1 and GmphyA2, which encode a photoreceptor, phytochrome A (phyA). They then characterized these genes in both photoperiod-sensitive and insensitive soybean lines [GmphyA2 gene is disrupted by an insertion of a novel Ty1/copia-like retrotransposon named SORE-1 (Soybean Retroelement 1) into the coding region, and this SORE-1 insertion causes photoperiod insensitivity. Interestingly, the SORE-1-inserted allele of GmphyA2 is present only in soybean lines grown in high latitude regions of Japan [GmphyA2 disruption by the SORE-1 insertion led to adaptation to a particular environment, while the buffering effect of gene duplication has sustained the plants, a novel consequence of nonfunctionalization of duplicate genes by TE insertion [SORE-1 is a potentially useful mutagen that induces heritable mutations, because its transcription occurs during the reproductive phase. In addition, SORE-1 appears to be controlled by an epigenetic mechanism involving small RNAs. Thus, artificial control of its epigenetic state may provide a tool to engineer novel traits in soybean.Of course, TE mobility often confers deleterious effects rather than benefits. Kazuo Tsugane  presented molecular mechanisms by which active TE transposition affects the host phenotype. In rice,  regions . Among tc growth . Akira Kan lines . In the of Japan . Why is nsertion . In viewArabidopsis transposon VANDAL21 is silent in wild-type plants due to heavy DNA methylation [VANC21, one of three VANDAL21-encoded genes, is introduced into the wild-type background as a transgene, it reduces the DNA methylation levels of the endogenous copies of VANDAL21, resulting in their active transcription and mobilization [Despite many TE exaptation events, the host-TE interactions present some sorts of arm race, because of the potential harmfulness of TE mobility. For example, TEs are usually transcriptionally silenced by epigenetic mechanisms while a subfamily can emerge that escapes such regulation. Aoi Hosaka , a graduate student in Tetsuji Kakutani’s lab, talked about a TE family that combats epigenetic silencing mechanisms. The hylation . Howeverlization . These dlization . They alDrosophila ovarian somatic cell line (OSC), presented his recent results that revealed that the Piwi protein physically associates with linker histone H1. Indeed, depletion of Piwi decreases the histone H1 density, as well as the heterochromatin protein 1a (HP1a) density, on target loci [Drosophila ovary likely consists of two pathways; (1) modulation of chromatin accessibility by regulating H1 density and (2) establishment and maintenance of heterochromatin by recruiting HP1a.In addition to DNA and histone modifications, small RNAs constitute an important part of the host defense system. Although post-transcriptional gene silencing (PTGS) is a built-in activity for small RNA-mediated regulation, small RNAs are also involved in transcriptional gene silencing (TGS) in many cases. Kuniaki Saito , who has been studying the mechanism of transcriptional repression mediated by Piwi-interacting RNAs (piRNAs) in the get loci . Loss ofMili  causes an almost complete loss of oocyte piRNAs, which results in an increase in transcripts from some specific retrotransposons, similar to what has been observed in male germ cells [Pld6 , encoding a mitochondrial nuclease/phospholipase, decreases the level of piRNAs to only a half of that in wild-type oocytes, which is in stark contrast to the situation in males where the same mutation severely affects piRNA biogenesis [Like in flies, piRNAs of 26–30-nucleotide (nt) are generated in male and female germ cells  in mice. The piRNA pathway in mouse male germ cells is essential for spermatogenesis and repression of some families of retrotransposons. In oocytes, however, the regulation and function of piRNAs are poorly understood. Yuka Kabayama , a graduate student in Hiroyuki Sasaki’s lab, investigated the consequences of loss of piRNA-pathway components in oocytes. She showed that a KO mutation of rm cells , 22. On ogenesis . Becauseogenesis , these rcis-acting RNAi.In fission yeast, heterochromatin is formed and maintained via a small RNA pathway, and this system continues to provide mechanistic insights of small RNA-mediated heterochromatinization. Yota Murakami  presented a mechanistic link between phosphorylation of RNAPII and efficiency of small RNA-mediated heterochromatinization. Previously, he and his colleague revealed that RNAPII is important for siRNA generation and heterochromatinization of the centromeres . The C-tThe meeting was fruitful: the participants enjoyed it, and they exchanged their recent results and ideas throughout the meeting including coffee breaks and banquet. Three months before this meeting, an international TE meeting  was held in Busan, Korea, which was organized by Dr. Heui-Soo Kim  and supported by the Korean Society for Molecular and Cellular Biology. The second international TE meeting is now being planned (chief organizer: Dr. Fumitoshi Ishino), which will be held in Tokyo in 2017. There will be researchers not only from Japan and Korea, but also from other countries in Asia, which will enhance and extend TE research in Asia.