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Signaling pathway-driven target gene transcription is critical for fate determination of embryonic stem cells (ESCs), but enhancer-dependent transcriptional regulation in these processes remains poorly understood. Here, we report enhancer architecture-dependent multilayered transcriptional regulation at the Halr1–Hoxa1 locus that orchestrates retinoic acid (RA) signaling-induced early lineage differentiation of ESCs. We show that both homeobox A1 (Hoxa1) and Hoxa adjacent long non-coding RNA 1 (Halr1) are identified as direct downstream targets of RA signaling and regulated by RARA/RXRA via RA response elements (RAREs). Chromosome conformation capture-based screens indicate that RA signaling promotes enhancer interactions essential for Hoxa1 and Halr1 expression and mesendoderm differentiation of ESCs. Furthermore, the results also show that HOXA1 promotes expression of Halr1 through binding to enhancer; conversely, loss of Halr1 enhances interaction between Hoxa1 chromatin and four distal enhancers but weakens interaction with chromatin inside the HoxA cluster, leading to RA signaling-induced Hoxa1 overactivation and enhanced endoderm differentiation. These findings reveal complex transcriptional regulation involving synergistic regulation by enhancers, transcription factors and lncRNA. This work provides new insight into intrinsic molecular mechanisms underlying ESC fate determination during RA signaling-induced early differentiation., Graphical Abstract Graphical AbstractEnhancer architecture-dependent multilayered transcriptional regulation at the Halr1–Hoxa1 locus that orchestrates RA signaling-induced early lineage differentiation of ESCs.

The long noncoding RNA HOTAIRM1 reportedly plays important roles in acute myeloid leukemia, gastric cancer and colorectal cancer. Here, we analyzed potential function of HOTAIRM1 in glioma and asked whether it participates in long-range chromatin interactions. We monitored expression of HOTAIRM1 in glioma tissues and correlated levels with patient survival using the TCGA dataset. HOTAIRM1 was highly expressed in glioma tissue, with high levels associated with shortened patient survival time. We then suppressed HOTAIRM1 activity in the human glioblastoma U251 line using CRISPR-cas9 to knock in a truncating polyA fragment. Reporter analysis of these and control cells confirmed that the HOTAIRM1 locus serves as an active enhancer. We then performed Capture-C analysis to identify target genes of that locus and applied RNA antisense purification to assess chromatin interactions between the HOTAIRM1 locus and HOXA cluster genes. HOTAIRM1 knockdown in glioma cells decreased proliferation and reduced expression of HOXA cluster genes. HOTAIRM1 regulates long-range interactions between the HOTAIRM1 locus and HOXA genes. Our work suggests a new mechanism by which HOTAIRM1 regulates glioma progression by regulating high-order chromatin structure and could suggest novel therapeutic targets to treat an intractable cancer.

Temozolomide (TMZ) is a frequently used chemotherapy for glioma; however, chemoresistance is a major problem limiting its effectiveness. Thus, knowledge of mechanisms underlying this outcome could improve patient prognosis. Here, we report that deletion of a regulatory element in the HOTAIR locus increases glioma cell sensitivity to TMZ and alters transcription of multiple genes. Analysis of a combination of RNA-seq, Capture Hi-C, and patient survival data suggests that CALCOCO1 and ZC3H10 are target genes repressed by the HOTAIR regulatory element and that both function in regulating glioma cell sensitivity to TMZ. Rescue experiments and 3C data confirmed this hypothesis. We propose a new regulatory mechanism governing glioma cell TMZ sensitivity.

Parasitic wasps produce several factors including venom, polydnaviruses (PDVs) and specialized wasp cells named teratocytes that benefit the survival of offspring by altering the physiology of hosts. However, the underlying molecular mechanisms for the alterations remain unclear. Here we find that the teratocytes of Cotesia vestalis, an endoparasitoid of the diamondback moth Plutella xylostella, and its associated bracovirus (CvBV) can produce miRNAs and deliver the products into the host via different ways. Certain miRNAs in the parasitized host are mainly produced by teratocytes, while the expression level of miRNAs encoded by CvBV can be 100-fold greater in parasitized hosts than non-parasitized ones. We further show that one teratocyte-produced miRNA (Cve-miR-281-3p) and one CvBV-produced miRNA (Cve-miR-novel22-5p-1) arrest host growth by modulating expression of the host ecdysone receptor (EcR). Altogether, our results show the first evidence of cross-species regulation by miRNAs in animal parasitism and their possible function in the alteration of host physiology during parasitism., The moth Plutella xylostella during its larval stage is the host of the endoparasitic wasp Cotesia vestalis. Here the authors show that the parasitoids deliver microRNAs to their hosts through their symbiotic virus and specialized cells leading to induced developmental delay.

Background Parasitoid wasps are well-known natural enemies of major agricultural pests and arthropod borne diseases. The parasitoid wasp Macrocentrus cingulum (Hymenoptera: Braconidae) has been widely used to control the notorious insect pests Ostrinia furnacalis (Asian Corn Borer) and O. nubilalis (European corn borer). One striking phenomenon exhibited by M. cingulum is polyembryony, the formation of multiple genetically identical offspring from a single zygote. Moreover, M. cingulum employs a passive parasitic strategy by preventing the host’s immune system from recognizing the embryo as a foreign body. Thus, the embryos evade the host’s immune system and are not encapsulated by host hemocytes. Unfortunately, the mechanism of both polyembryony and immune evasion remains largely unknown. Results We report the genome of the parasitoid wasp M. cingulum. Comparative genomics analysis of M. cingulum and other 11 insects were conducted, finding some gene families with apparent expansion or contraction which might be linked to the parasitic behaviors or polyembryony of M. cingulum. Moreover, we present the evidence that the microRNA miR-14b regulates the polyembryonic development of M. cingulum by targeting the c-Myc Promoter-binding Protein 1 (MBP-1), histone-lysine N-methyltransferase 2E (KMT2E) and segmentation protein Runt. In addition, Hemomucin, an O-glycosylated transmembrane protein, protects the endoparasitoid wasp larvae from being encapsulated by host hemocytes. Motif and domain analysis showed that only the hemomucin in two endoparasitoids, M. cingulum and Venturia canescens, possessing the ability of passive immune evasion has intact mucin domain and similar O-glycosylation patterns, indicating that the hemomucin is a key factor modulating the immune evasion. Conclusions The microRNA miR-14b participates in the regulation of polyembryonic development, and the O-glycosylation of the mucin domain in the hemomucin confers the passive immune evasion in this wasp. These key findings provide new insights into the polyembryony and immune evasion. Electronic supplementary material The online version of this article (10.1186/s12864-018-4783-x) contains supplementary material, which is available to authorized users.

Background Insecticide resistance is a substantial problem in controlling agricultural and medical pests. Detecting target site mutations is crucial to manage insecticide resistance. Though PCR-based methods have been widely used in this field, they are time-consuming and inefficient, and typically have a high false positive rate. Acetylcholinesterases (Ace) is the neural target of the widely used organophosphate (OP) and carbamate insecticides. However, there is not any software available to detect insecticide resistance associated mutations in RNA-Seq data at present. Results A computational pipeline ACE was developed to detect resistance mutations of ace in insect RNA-Seq data. Known ace resistance mutations were collected and used as a reference. We constructed a Web server for ACE, and the standalone software in both Linux and Windows versions is available for download. ACE was used to analyse 971 RNA-Seq data from 136 studies in 7 insect pests. The mutation frequency of each RNA-Seq dataset was calculated. The results indicated that the resistance frequency was 30%–44% in an eastern Ugandan Anopheles population, thus suggesting this resistance-conferring mutation has reached high frequency in these mosquitoes in Uganda. Analyses of RNA-Seq data from the diamondback moth Plutella xylostella indicated that the G227A mutation was positively related with resistance levels to organophosphate or carbamate insecticides. The wasp Nasonia vitripennis had a low frequency of resistant reads (

Retinoic acid (RA) induces rapid differentiation of embryonic stem cells (ESCs), partly by activating expression of the transcription factor Hoxa1, which regulates downstream target genes that promote ESCs differentiation. However, mechanisms of RA-induced Hoxa1 expression and ESCs early differentiation remain largely unknown. Here, we identify a distal enhancer interacting with the Hoxa1 locus through a long-range chromatin loop. Enhancer deletion significantly inhibited expression of RA-induced Hoxa1 and endoderm master control genes such as Gata4 and Gata6. Transcriptome analysis revealed that RA-induced early ESCs differentiation was blocked in Hoxa1 enhancer knockout cells, suggesting a requirement for the enhancer. Restoration of Hoxa1 expression partly rescued expression levels of ∼40% of genes whose expression changed following enhancer deletion, and ∼18% of promoters of those rescued genes were directly bound by Hoxa1. Our data show that a distal enhancer maintains Hoxa1 expression through long-range chromatin loop and that Hoxa1 directly regulates downstream target genes expression and then orchestrates RA-induced early differentiation of ESCs. This discovery reveals mechanisms of a novel enhancer regulating RA-induced Hoxa genes expression and early ESCs differentiation.

Temozolomide (TMZ) is a frequently used chemotherapy for glioma; however, chemoresistance is a major problem limiting its effectiveness. Thus, knowledge of mechanisms underlying this outcome could improve patient prognosis. Here, we report that deletion of a regulatory element in the HOTAIR locus increases glioma cell sensitivity to TMZ and alters transcription of multiple genes. Analysis of a combination of RNA-seq, Capture Hi-C, and patient survival data suggests that CALCOCO1 and ZC3H10 are target genes repressed by the HOTAIR regulatory element and that both function in regulating glioma cell sensitivity to TMZ. Rescue experiments and 3C data confirmed this hypothesis. We propose a new regulatory mechanism governing glioma cell TMZ sensitivity.

Proper expression of Homeobox A cluster genes (HoxA) is essential for embryonic stem cell (ESC) differentiation and individual development. However, mechanisms controlling precise spatiotemporal expression of HoxA during early ESC differentiation remain poorly understood. Herein, we identified a functional CTCF-binding element (CBE+47) closest to the 3'-end of HoxA within the same topologically associated domain (TAD) in ESC. CRISPR-Cas9-mediated deletion of CBE+47 significantly upregulated HoxA expression and enhanced early ESC differentiation induced by retinoic acid (RA) relative to wild-type cells. Mechanistic analysis by chromosome conformation capture assay (Capture-C) revealed that CBE+47 deletion decreased interactions between adjacent enhancers, enabling formation of a relatively loose enhancer–enhancer interaction complex (EEIC), which overall increased interactions between that EEIC and central regions of HoxA chromatin. These findings indicate that CBE+47 organizes chromatin interactions between its adjacent enhancers and HoxA. Furthermore, deletion of those adjacent enhancers synergistically inhibited HoxA activation, suggesting that these enhancers serve as an EEIC required for RA-induced HoxA activation. Collectively, these results provide new insight into RA-induced HoxA expression during early ESC differentiation, also highlight precise regulatory roles of the CTCF-binding element in orchestrating high-order chromatin structure.

Background Parasitic insects are well-known biological control agents for arthropod pests worldwide. They are capable of regulating their host’s physiology, development and behaviour. However, many of the molecular mechanisms involved in host-parasitoid interaction remain unknown. Results We sequenced the genomes of two parasitic wasps (Cotesia vestalis, and Diadromus collaris) that parasitize the diamondback moth Plutella xylostella using Illumina and Pacbio sequencing platforms. Genome assembly using SOAPdenovo produced a 178 Mb draft genome for C. vestalis and a 399 Mb draft genome for D. collaris. A total set that contained 11,278 and 15,328 protein-coding genes for C. vestalis and D. collaris, respectively, were predicted using evidence (homology-based and transcriptome-based) and de novo prediction methodology. Phylogenetic analysis showed that the braconid C. vestalis and the ichneumonid D. collaris diverged approximately 124 million years ago. These two wasps exhibit gene gains and losses that in some cases reflect their shared life history as parasitic wasps and in other cases are unique to particular species. Gene families with functions in development, nutrient acquisition from hosts, and metabolism have expanded in each wasp species, while genes required for biosynthesis of some amino acids and steroids have been lost, since these nutrients can be directly obtained from the host. Both wasp species encode a relative higher number of neprilysins (NEPs) thus far reported in arthropod genomes while several genes encoding immune-related proteins and detoxification enzymes were lost in both wasp genomes. Conclusions We present the annotated genome sequence of two parasitic wasps C. vestalis and D. collaris, which parasitize a common host, the diamondback moth, P. xylostella. These data will provide a fundamental source for studying the mechanism of host control and will be used in parasitoid comparative genomics to study the origin and diversification of the parasitic lifestyle.

The effects of phosphate concentration on plant growth and photosynthetic performance were examined in leaves of Zizania latifolia. Plants were grown for four weeks in a solution containing 0, 0.16, 0.64, and 2.56 mM orthophosphate. The results showed that the highest net photosynthetic rate (P N) was achieved at 0.64 mM orthophosphate, which corresponded to the maximum content of organic phosphorus in leaves. Low phosphorus (low-P) content in the culture solution inhibited plant growth, affecting plant height, leaf length, leaf number, tiller number, and fresh mass of leaf, sheath, culm, root, and total plant. In addition, we observed that low-P (0.16 mM) did not hinder the growth of roots but increased the root:shoot ratio, and significantly decreased the chlorophyll content, P N, stomatal conductance, and transpiration rate, but increased the intercellular CO2 concentration. Additionally, low-P significantly decreased the maximum carboxylation rate of Rubisco, the maximum rate of ribulose-1,5-bisphosphate regeneration, the effective quantum yield of PSII photochemistry, photochemical quenching coefficient, and electron transport rate, but increased the nonphotochemical quenching. However, the maximal quantum yield of PSII photochemistry was not significantly affected by low-P. High phosphorus (2.56 mM) caused only a slight decrease in gas-exchange parameters. Therefore, the decrease in growth of P-deficient Z. latifolia plants could be attributed to the lowered photosynthetic rate.

The genomes and transcriptomes of hundreds of insects have been sequenced. However, insect community lacks an integrated, up-to-date collection of insect gene data. Here, we introduce the first release of InsectBase, available online at http://www.insect-genome.com. The database encompasses 138 insect genomes, 116 insect transcriptomes, 61 insect gene sets, 36 gene families of 60 insects, 7544 miRNAs of 69 insects, 96,925 piRNAs of Drosophila melanogaster and Chilo suppressalis, 2439 lncRNA of Nilaparvata lugens, 22,536 pathways of 78 insects, 678,881 untranslated regions (UTR) of 84 insects and 160,905 coding sequences (CDS) of 70 insects. This release contains over 12 million sequences and provides search functionality, a BLAST server, GBrowse, insect pathway construction, a Facebook-like network for the insect community (iFacebook), and phylogenetic analysis of selected genes.

Figure S1. Flow cytometry estimation of the genome size for the M. cingulum. Figure S2. The distribution of 17-mer frequency in M. cingulum genome sequencing reads. Figure S3. Distribution of GC content, CpG Obs/ExpRatios of M. cingulum(Mcin), N. vitripennis(Nvit) and A. mellifera(Amel). Figure S4. COG function classification of the OGS in M. cingulum. Figure S5. KEGG pathway analysis of the OGS in M. cingulum. Figure S6. GO classification of the OGS in M. cingulum. Figure S7. Venn diagram of the homologous protein-coding genes among three wasps (M. cingulum, C. solmsi, N. vitripennis) and fruit fly (D. melanogaster). Figure S8. Phylogenetic relationship of CSP proteins from A. mellifera, C. floridanum, C. solmsi, M. cingulum, N.vitripennis, S.invicta. Figure S9. Phylogenetic relationship of GR proteins from A. mellifera, C. floridanum, C. solmsi, M. cingulum, N.vitripennis, S.invicta. Figure S10. Phylogenetic relationship of IR proteins from A. mellifera, C. floridanum, C. solmsi, M. cingulum, N.vitripennis, S.invicta. Figure S11. Phylogenetic relationship of OR proteins from C. floridanum, D. melanogaster and M. cingulum. Figure S12. Phylogenetic relationship of OBP proteins from A. mellifera, C. floridanum, C. solmsi, M. cingulum, N.vitripennis, S.invicta. Figure S13. Phylogenetic relationship of SNMP proteins from A.mellifera, C. floridanum, C. solmsi, M. cingulum, N.vitripennis, S.invicta. Figure S14. Phylogenetic relationship of GST proteins from A. mellifera, C. floridanum, C. solmsi, M. cingulum, N.vitripennis, S.invicta. Figure S15. Phylogenetic relationship of P450 proteins from N. vitripennis, D. melanogaster and M. cingulum. Figure S16. Phylogenetic relationship of ABC proteins from M. cingulum and D. melanogaster. Figure S17. Different expression levels of miR-14b in different developmental stages of M. cingulum.Table S1. Genome sequencing data of M. cingulum. Table S2. Estimation of M. cingulum genome size using K-mer analysis. Table S3. Summary of the M. cingulum genome assembly. Table S4. The published insect genomes. Table S5. The genome assembly assessment on different insects. Table S6. Classification of repeat sequences identified in the M. cingulum genome. Table S7. Genome features of the M. cingulum, N. vitripennis and A. mellifera. Table S8. Gene features of M. cingulum, N. vitripennis and A. mellifera. Table S9. The insects with OGSs in InsectBase. Table S10. Hemomucin genes in eight wasps. Table S11. The different gene expression of embryo and pseudogerm transcriptomes in KEGG pathway. Table S12. The differently expressed miRNAs in embryo and mixed embryo transcriptomes. Table S13. Comparison of gene numbers for chemoreception in A.mellifera, C. floridanum, C. solmsi, M. cingulum, N. vitripennis and S. invicta. Table S14. Comparison of gene numbers for Gene families associated with insecticide resistance and detoxification in D. melanogaster, A. mellifera, C. floridanum, C. solmsi, M. cingulum, N. vitripennis and S. invicta. Table S15. Comparison of gene numbers of insect immune in A. mellifera, C. floridanum, C. solmsi, M. cingulum, N. vitripennis and S. invicta. Table S16. The PCR primer for target genes of mci-miR-14b. (PDF 6076 kb)

Long non-coding RNA (lncRNA) is a class of noncoding RNA >200 bp in length that has essential roles in regulating a variety of biological processes. Here, we constructed a computational pipeline to identify lncRNA genes in the diamondback moth (Plutella xylostella), a major insect pest of cruciferous vegetables. In total, 3,324 lncRNAs corresponding to 2,475 loci were identified from 13 RNA-Seq datasets, including samples from parasitized, insecticide-resistant strains and different developmental stages. The identified P. xylostella lncRNAs had shorter transcripts and fewer exons than protein-coding genes. Seven out of nine randomly selected lncRNAs were validated by strand-specific RT-PCR. In total, 54–172 lncRNAs were specifically expressed in the insecticide resistant strains, among which one lncRNA was located adjacent to the sodium channel gene. In addition, 63–135 lncRNAs were specifically expressed in different developmental stages, among which three lncRNAs overlapped or were located adjacent to the metamorphosis-associated genes. These lncRNAs were either strongly or weakly co-expressed with their overlapping or neighboring mRNA genes. In summary, we identified thousands of lncRNAs and presented evidence that lncRNAs might have key roles in conferring insecticide resistance and regulating the metamorphosis development in P. xylostella.

We compared the interactive effects of temperature and light intensity on growth, photosynthetic performance, and antioxidant enzyme activity in Zizania latifolia Turcz. plants in this study. Plants were grown under field (average air temperature 9.6–25°C and average light intensity 177–375 W m−2) or greenhouse (20–32°C and 106–225 W m−2) conditions from the spring to the early summer. The results indicated that greenhouse-grown plants (GGP) had significantly higher plant height, leaf length, and leaf width, but lower leaf thickness and total shoot mass per cluster compared with field-grown plants (FGP). Tiller emergence was almost completely suppressed in GGP. Significantly higher chlorophyll (Chl) content and lower Chl a/b ratio were observed in GGP than in FGP. From 4 to 8 weeks after treatment (WAT), net photosynthetic rate (PN) was significantly lower in FGP than in GGP. However, from 9 to 12 WAT, PN was lower in GGP, accompanied by a decrease in stomatal conductance (gs) and electron transport rate (ETR) compared with FGP. Suppressed PN in GGP under high temperature combined with low light was also indicated by photosynthetic photon flux density (PPFD) response curve and its diurnal fluctuation 10 WAT. Meanwhile, ETR in GGP was also lower than in FGP according to the ETR — photosynthetically active radiation (PAR) curve. The results also revealed that GGP had a lower light saturation point (LSP) and a higher light compensation point (LCP). From 4 to 8 WAT, effective quantum yield of PSII photochemistry (ΦPSII), photochemical quenching (qP), and ETR were slightly lower in FGP than in GGP. The activities of ascorbate peroxidase (APX), guaiacol peroxidase (POD), glutathione reductase (GR), superoxide dismutase (SOD), and malondialdehyde (MDA) content were significantly higher from 4 to 8 WAT, but lower from 10 to 12 WAT in FGP. However, catalase (CAT) activity was significantly lower in FGP from 4 to 8 WAT. Our results indicated that the growth and photosynthetic performance of Z. latifolia plants were substantially influenced by temperature, as well as light intensity. This is helpful to understand the physiological basis for a protected cultivation of this crop.

The NCBI accession numbers of insect ace-1 and ace-2 genes

The SRA accession numbers of 971 RNA-Seq data used for detecting mutations

Insects undergo metamorphosis, involving an abrupt change in body structure through cell growth and differentiation. Rice stem stripped borer (SSB), Chilo suppressalis, is one of the most destructive rice pests. However, little is known about the regulation mechanism of metamorphosis development in this notorious insect pest. Here, we studied the expression of 22,197 SSB genes at seven time points during pupa development with a customized microarray, identifying 622 differentially expressed genes (DEG) during pupa development. Gene ontology (GO) analysis of these DEGs indicated that the genes related to substance metabolism were highly expressed in the early pupa, which participate in the physiological processes of larval tissue disintegration at these stages. In comparison, highly expressed genes in the late pupal stages were mainly associated with substance biosynthesis, consistent with adult organ formation at these stages. There were 27 solute carrier (SLC) genes that were highly expressed during pupa development. We knocked down SLC22A3 at the prepupal stage, demonstrating that silencing SLC22A3 induced a deficiency in pupa stiffness and pigmentation. The RNAi-treated individuals had white and soft pupa, suggesting that this gene has an essential role in pupal development.

Primers used for RT-PCR validation of three lncRNA genes and their overlapping protein coding genes. (DOCX 12Â kb)

The number of specifically-expressed and differentially-expressed lncRNAs in various populations (DOCX 14Â kb)