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Background LINC00152 (long intergenic non-protein coding RNA 152) was identified as an oncogenic lncRNA in multiple cancers. In the current study, we aimed to explore the transcriptional profile of LINC00152 in oral squamous cell carcinoma (OSCC) and its regulations at the transcriptional level. Methods Bioinformatic analysis was performed by extracting the OSCC subset from The Cancer Genome Atlas (TCGA)-Head and Neck Squamous Cell Carcinoma (HNSC). LINC00152 subcellular localization and its interacting transcriptional factors (TFs) were explored. Dual-luciferase assay and ChIP-qPCR were applied to study transcriptional regulation. In vitro and in-vivo tumor cell growth models were used for functional assays. Results NR_024206.2 was the dominant isoform that accounts for 80% of all transcripts of LINC00152. LINC00152 upregulation was associated with unfavorable survival of patients with OSCC. LINC00152 knockdown significantly impaired OSCC cell growth in vitro and in vivo. RNA FISH assay confirmed nuclear and cytoplasmic distribution of LINC00152. It physically interacted with Upstream Transcription Factor 1 (USF1), a common transcription factor in mammalian cells. USF1 could bind to the promoter region of MRPL52 (Mitochondrial Ribosomal Protein L52) and activate its transcription. LINC00152 could enhance the binding, thereby indirectly elevating MRPL52 expression. USF1 or MRPL52 knockdown slowed the proliferation of OSCC cells and partly canceled LINC00152 mediated growth-promoting effects. Conclusion This study revealed a novel LINC00152-USF1/MRPL52 axis promoting OSCC tumor growth.

Bromodomain and extraterminal domain (BET) proteins are epigenetic readers that regulate gene expression. We investigated whether variants in BET genes are associated with survival outcomes for lung cancer. To do this, the associations between 77 variants in BET family genes and survival outcomes were analyzed in 773 non‐small‐cell lung cancer (NSCLC) patients who underwent surgery (349 and 424 patients in the discovery and validation cohorts, respectively). We found that six variants were significantly associated with overall survival (OS) in the discovery cohort, and one variant (rs2506711C>T) was replicated in the validation cohort. BRD3 rs2506711C>T is located in the repressed area and has a strong linkage disequilibrium with rs2427964C>T in the promoter region. BRD3 rs2427964C>T was significantly associated with worse OS in the discovery cohort, validation cohort, and combined analysis. In a luciferase assay, promoter activity in the BRD3 rs2427964 T allele was significantly higher than that in the BRD3 rs2427964 C allele, which selectively bound with the transcriptional repressor SIN3A. Knockdown of BRD3 with BRD3‐specific siRNA decreased the proliferation and migration of lung cancer cells while also increasing the rate of apoptosis. These results suggest that BRD3 rs2427964C>T increases BRD3 expression through increased promoter activity, which is associated with poor prognosis for lung cancer., In this study, we investigated the association of BET gene variants with survival of patients with non‐small‐cell lung cancer (NSCLC). We observed that the rs2427964C>T SNP in the BRD3 promoter region was associated with poorer survival outcome. BRD3 promoter activity was higher in rs2427964_T than in rs2427964_C, which selectively bound with the transcriptional repressor SIN3A. Additionally, BRD3 silencing decreased the proliferation and migration of NSCLC cells. Our data suggest that elevated BRD3 expression regulated by rs2427964C>T leads to reduced overall survival in patients with NSCLC.

Cotton that produces natural fiber materials for the textile industry is one of the most important crops in the world. Class II KNOX proteins are often considered as transcription factors in regulating plant secondary cell wall (SCW) formation. However, the molecular mechanism of the KNOX transcription factors-regulated SCW synthesis in plants (especially in cotton) remains unclear in details so far. In this study, we show a cotton class II KNOX protein (GhKNL1) as a transcription repressor functioning in fiber development. The GhKNL1-silenced transgenic cotton produced longer fibers with thicker SCWs, whereas GhKNL1 dominant repression transgenic lines displayed the opposite fiber phenotype, compared with controls. Further experiments revealed that GhKNL1 could directly bind to promoters of GhCesA4-2/4-4/8-2 and GhMYB46 for modulating cellulose synthesis during fiber SCW development of cotton. On the other hand, GhKNL1 could also suppress expressions of GhEXPA2D/4A-1/4D-1/13A through binding to their promoters for regulating fiber elongation of cotton. Taken together, these data revealed GhKNL1 functions in fiber elongation and SCW formation by directly repressing expressions of its target genes related to cell elongation and cellulose synthesis. Thus, our data provide an effective clue for potentially improving fiber quality by genetic manipulation of GhKNL1 in cotton breeding. This article is protected by copyright. All rights reserved.

CaWRKY40 in pepper (Capsicum annuum) promotes immune responses to Ralstonia solanacearum infection (RSI) and to high-temperature, high-humidity (HTHH) stress, but how it interacts with upstream signalling components remains poorly understood. Here, using approaches of reverse genetics, biochemical and molecular biology we functionally characterised the relationships among the WRKYGMK-containing WRKY protein CaWRKY27b, the calcium-dependent protein kinase CaCDPK29, and CaWRKY40 during pepper response to RSI or HTHH. Our data indicate that CaWRKY27b is upregulated and translocated from the cytoplasm to the nucleus upon phosphorylation of Ser137 in the nuclear localisation signal by CaCDPK29. Using electrophoretic mobility shift assays and microscale thermophoresis, we observed that, due to the replacement of Q by M in the conserved WRKYGQK, CaWRKY27b in the nucleus failed to bind to W-boxes in the promoters of immunity- and thermotolerance-related marker genes. Instead, CaWRKY27b interacted with CaWRKY40 and promoted its binding and positive regulation of the tested marker genes including CaNPR1, CaDEF1 and CaHSP24. Notably, mutation of the WRKYGMK motif in CaWRKY27b to WRKYGQK restored the W-box binding ability. Our data therefore suggest that CaWRKY27b is phosphorylated by CaCDPK29 and acts as a transcriptional activator of CaWRKY40 during the pepper response to RSI and HTHH.

Lysine-specific demethylase 1 (LSD1) is an important histone demethylase that mediates epithelial to mesenchymal transition (EMT). The E239K mutation of LSD1 was identified in a luminal breast cancer patient from the COSMIC Breast Cancer dataset. To investigate the functional effects of the E239K mutation of LSD1, a stable LSD1 knockdown MCF7 cell line was generated. Rescue with WT LSD1, but not E239K mutated LSD1, suppressed the invasion and migration of the LSD1 knockdown cells, indicating that the E239K mutation abolished the suppressive effects of LSD1 on the invasion and migration of MCF7 cells. Further analysis showed that the E239K mutation abolished LSD1-mediated invasion and migration of MCF7 cells through downregulation of estrogen receptor α (ERα). Most importantly, the E239K mutation disrupted the interaction between LSD1 and GATA3, which reduced the enrichment of LSD1 at the promoter region of the ERα gene; the reduced enrichment of LSD1 at the promoter region of the ERα gene caused enhanced histone H3K9 methylation, which subsequently suppressed the transcription of the ERα gene. In summary, the E239K mutation abolishes the suppressive function of LSD1 on migration and invasion of breast cancer cells by disrupting the interaction between LSD1 and GATA3.

Objective Genetic variants spanning the ubiquitin-conjugating enzyme E2 L3 (UBE2L3) gene are associated with increased expression of the UBE2L3-encoded E2 ubiquitin-conjugating enzyme, UbcH7, that facilitates activation of proinflammatory NF-κB signaling, and susceptibility to autoimmune diseases. This study aims to delineate how genetic variants carried on the UBE2L3-YDJC autoimmune risk haplotype function to drive hypermorphic UBE2L3 expression. Methods We used bioinformatic analyses, electrophoretic mobility shift assays, and luciferase reporter assays to identify and functionally characterize allele-specific effects of risk variants positioned in chromatin accessible regions of immune cells. Chromatin conformation capture (3C)-qPCR, ChIP-qPCR, and siRNA knockdown assays were performed on patient-derived EBV-transformed B cells homozygous for the UBE2L3-YDJC non-risk or risk haplotype to determine if the risk haplotype increases UBE2L3 expression by altering the regulatory chromatin architecture in the region. Results Five of the seven prioritized variants demonstrated allele-specific increases in nuclear protein binding affinity and regulatory activity. HiChIP and 3C-qPCR uncovered a long-range interaction between the UBE2L3 promoter (rs140490, rs140491, rs11089620) and downstream YDJC promoter (rs3747093) that was strengthened in the presence of the UBE2L3-YDJC risk haplotype, and correlated with the loss of CTCF binding and gain of YY1 binding at the risk alleles. Depleting YY1 by siRNA disrupted the long-range interaction between the two promoters and reduced UBE2L3 expression. Conclusion The UBE2L3-YDJC autoimmune risk haplotype increases UBE2L3 expression through strengthening a YY1-mediated interaction between the UBE2L3 and YDJC promoters.

Transcription activator-like (TAL) effectors are major virulence factors secreted by the type III secretion systems of Xanthomonas oryzae pv. oryzicola (Xoc) and X. oryzae pv. oryzae (Xoo), causing bacterial leaf streak and bacterial blight, respectively, in rice. However, the knowledge of Xoc TAL effector function in promoting bacterial virulence remains limited. Here, we isolated the highly virulent Xoc strain HGA4 from the outbreak region of Huanggang (Hubei, China), which contains four TAL effectors not found in the Chinese model strain RS105. Among these, Tal2b was selected for introduction into RS105, which resulted in a longer lesion length than that in the control. Tal2b directly binds to the promoter region of the gene and activates the expression of OsF3H03g , which encodes 2-oxoglutarate-dependent dioxygenase in rice. OsF3H03g negatively regulates salicylic acid (SA)-related defense by directly reducing SA, and it plays a positive role in susceptibility to both Xoc and Xoo in rice. OsF3H03g interacts with a uridine diphosphate-glycosyltransferase protein (OsUGT74H4), which positively regulates bacterial leaf streak susceptibility and may inactivate SA via glycosylation modification.

Numerous transcription factors (TFs) in ascomycete fungi play crucial roles in cellular processes; however, how most of them function is poorly understood. Here, we identified and characterized a novel TF, CxrC (POX01387), acting downstream of the key TF CxrA, which is essential for plant-biomass-degrading-enzyme (PBDE) production in Penicillium oxalicum. Deletion of cxrC in P. oxalicum significantly affected the production of PBDEs, as well as mycelial growth and conidiospore production. CxrA directly repressed the expression of cxrC after about 12 hr following switch to Avicel culture. CxrC bound the promoters of major PBDE genes and genes involved in conidiospore development. CxrC was found to bind the TSSGTYR core sequence (S: C and G; Y: T and C; R: G and A) of the important cellulase genes cbh1 and eg1. Both N- and C-terminal peptides of CxrC and the CxrC phosphorylation were found to mediate its homodimerization. The conserved motif LPSVRSLLTP (65-74) in CxrC was found to be required for regulating cellulase production. This study reveals novel mechanisms of TF-mediated regulation of the expression of PBDE genes and genes involved in cellular processes in an ascomycete fungus.

This bioinformatics study aimed to identify ETV4 transcription factor oncogenes and outline anticancer drugs for these genes. First, we collected known 61 ETV4 cancer targets that were framed as two classes of queries to screen against the multiomics resources in GeneMANIA. This method accessed and added functionally similar 20 genes to each set. These data were interpreted by hub genes, network clustering, gene ontology, and pathway analyses, and the results confirmed that all resultant genes were cancer promoters. The ETS-binding motifs were identified from the promoter regions of these genes. Thus, 23 ETV4 targets were figured and those involved in oncogenesis were filtered as the following 16 putative nodes: MMP8, MMP14, KDR, BRIP1, CXCR1, GRB14, SHC2, SHC4, SH2B1, SH2B2, INPPL1, PTPN3, GNG12, SEMA4D, RHOA, and SPSB2. The transcriptional regulation of these oncogenes was coordinated by an extensive miRNA network that found to deregulate many cancer pathways. Using DgIb database, the high quality 6 oncogene-drug combinations (MMP8-CHEMBL1231240, MMP8-Aminomethylamide, CXCR1-Reparixin, SEMA4D-Pepinemab, RHOA-Clausine E, and SPSB2-CHEMBL175296) were proposed. These findings may advance our understanding of novel neoplastic gene nexus of ETV4 and design treatment strategies for its modulation.

Dysregulation of long noncoding RNA SNHG17 is associated with the occurrence of several tumors; however, its role in esophageal squamous cell carcinoma (ESCC) remains obscure. The present study demonstrated that SNHG17 was upregulated in ESCC tissues and cell lines, induced by TGF‐β1, and associated with poor survival. It is also involved in the epithelial‐to‐mesenchymal transition (EMT) process. The mechanism underlying SNHG17‐regulated c‐Myc was detected by RNA immunoprecipitation, RNA pull‐down, chromatin immunoprecipitation, and luciferase reporter assays. SNHG17 was found to directly regulate c‐Myc transcription by binding to c‐Jun protein and recruiting the complex to specific sequences of the c‐Myc promoter region, thereby increasing its expression. Moreover, SNHG17 hyperactivation induced by TGF‐β1 results in PI3K/AKT pathway activation, promoting cells EMT, forming a positive feedback loop. Furthermore, SNHG17 facilitated ESCC tumor growth in vivo. Overall, this study demonstrated that the SNHG17/c‐Jun/c‐Myc axis aggravates ESCC progression and EMT induction by TGF‐β1 and may serve as a new therapeutic target for ESCC., SNHG17 was upregulated and associated with poor survival in ESCC. TGF‐β1 induced SNHG17 involved in the epithelial‐to‐mesenchymal transition process and activated the PI3K/AKT pathway. Mechanistically, SNHG17 was found to directly regulate c‐Myc transcription by binding to c‐Jun protein and recruiting the complex to specific sequences of the c‐Myc promoter region. SNHG17 aggravated ESCC growth in vivo.

Summary With the rapid development of synthetic biology in recent years, particular attention has been paid to RNA devices, especially riboswitches, because of their significant and diverse regulatory roles in prokaryotic and eukaryotic cells. Due to the limited performance and context‐dependence of riboswitches, only a few of them (such as theophylline, tetracycline and ciprofloxacin riboswitches) have been utilized as regulatory tools in biotechnology. In the present study, we demonstrated that a ribosome‐dependent ribo‐regulator, LRR, discovered in our previous work, exhibits an attractive regulatory performance. Specifically, it offers a 60‐fold change in expression in the presence of retapamulin and a low level of leaky expression of about 1–2% without antibiotics. Moreover, LRR can be combined with different promoters and performs well in Bacillus thuringiensis, B. cereus, B. amyloliquefaciens, and B. subtilis. Additionally, LRR also functions in the Gram‐negative bacterium Escherichia coli. Furthermore, we demonstrate its ability to control melanin metabolism in B. thuringiensis BMB171. Our results show that LRR can be applied to regulate gene expression, construct genetic circuits and tune metabolic pathways, and has great potential for many applications in synthetic biology., In the present study, we demonstrated that a ribosome‐dependent ribo‐regulator, LRR, discovered in our previous work, exhibits an attractive regulatory performance. We show its ability to control melanin metabolism in Bacillus thuringiensis BMB171.

ObjectiveIn a recent genome-wide association study, a significant genetic association between rs34330 of CDKN1B and risk of systemic lupus erythematosus (SLE) in Han Chinese was identified. This study was undertaken to validate the reported association and elucidate the biochemical mechanisms underlying the effect of the variant.MethodsWe performed an allelic association analysis in patients with SLE, followed by a meta-analysis assessing genome-wide association data across 11 independent cohorts (n = 28,872). In silico bioinformatics analysis and experimental validation in SLE-relevant cell lines were applied to determine the functional consequences of rs34330.ResultsWe replicated a genetic association between SLE and rs34330 (meta-analysis P = 5.29 × 10-22 , odds ratio 0.84 [95% confidence interval 0.81-0.87]). Follow-up bioinformatics and expression quantitative trait locus analysis suggested that rs34330 is located in active chromatin and potentially regulates several target genes. Using luciferase and chromatin immunoprecipitation-real-time quantitative polymerase chain reaction, we demonstrated substantial allele-specific promoter and enhancer activity, and allele-specific binding of 3 histone marks (H3K27ac, H3K4me3, and H3K4me1), RNA polymerase II (Pol II), CCCTC-binding factor, and a critical immune transcription factor (interferon regulatory factor 1 [IRF-1]). Chromosome conformation capture revealed long-range chromatin interactions between rs34330 and the promoters of neighboring genes APOLD1 and DDX47, and effects on CDKN1B and the other target genes were directly validated by clustered regularly interspaced short palindromic repeat (CRISPR)-based genome editing. Finally, CRISPR/dead CRISPR-associated protein 9-based epigenetic activation/silencing confirmed these results. Gene-edited cell lines also showed higher levels of proliferation and apoptosis.ConclusionCollectively, these findings suggest a mechanism whereby the rs34330 risk allele (C) influences the presence of histone marks, RNA Pol II, and IRF-1 transcription factor to regulate expression of several target genes linked to proliferation and apoptosis. This process could potentially underlie the association of rs34330 with SLE.

Summary Plant ethylene‐responsive factors (ERFs) play essential roles in cold stress response, but the molecular mechanisms underlying this process remain poorly understood. In this study, we characterized PtrERF9 from trifoliate orange (Poncirus trifoliata (L.) Raf.), a cold‐hardy plant. PtrERF9 was up‐regulated by cold in an ethylene‐dependent manner. Overexpression of PtrERF9 conferred prominently enhanced freezing tolerance, which was drastically impaired when PtrERF9 was knocked down by virus‐induced gene silencing. Global transcriptome profiling indicated that silencing of PtrERF9 resulted in substantial transcriptional reprogramming of stress‐responsive genes involved in different biological processes. PtrERF9 was further verified to directly and specifically bind with the promoters of glutathione S‐transferase U17 (PtrGSTU17) and ACC synthase1 (PtrACS1). Consistently, PtrERF9‐overexpressing plants had higher levels of PtrGSTU17 transcript and GST activity, but accumulated less ROS, whereas the silenced plants showed the opposite changes. Meanwhile, knockdown of PtrERF9 decreased PtrACS1 expression, ACS activity and ACC content. However, overexpression of PtrERF9 in lemon, a cold‐sensitive species, caused negligible alterations of ethylene biosynthesis, which was attributed to perturbed interaction between PtrERF9, along with lemon homologue ClERF9, and the promoter of lemon ACS1 gene (ClACS1) due to mutation of the cis‐acting element. Taken together, these results indicate that PtrERF9 acts downstream of ethylene signalling and functions positively in cold tolerance via modulation of ROS homeostasis by regulating PtrGSTU17. In addition, PtrERF9 regulates ethylene biosynthesis by activating PtrACS1 gene, forming a feedback regulation loop to reinforce the transcriptional regulation of its target genes, which may contribute to the elite cold tolerance of Poncirus trifoliata.

Re-activation of fetal haemoglobin (HbF) has been proved to be an effective strategy for the treatment of β-haemoglobinopathies. In this study, we identified TEA domain transcription factor 4 (TEAD4) as a new potential regulator of HbF by integrating public data sets with quantitative polymerase chain reaction analysis in β-thalassaemia patients. Significant negative correlation was observed between the expression of TEAD4 and HbF levels in β-thalassaemia patients. Functional validations of TEAD4 inhibition in both β-thalassaemia CD34+ cells and HUDEP-2 cells indicated that depletion of TEAD4 led to a significant increase of HbF. Finally, we identified a binding motif of TEAD4 on γ-globin gene promoters; its disruption consistently led to de-repression of HbF. Taken together, these results demonstrate that TEAD4 could act as a transcriptional inhibitor of the γ-globin gene through direct binding on its promoter. Our findings demonstrate a novel role of TEAD4 on the regulation of HbF, which may benefit patients with β-haemoglobinopathies.

Atherosclerosis (AS) is the main aetiology of coronary heart disease, cerebral infarction and peripheral vascular disease in humans. Long‐noncoding RNA (LincRNA)‐p21 has been reported to participate in the development of AS. Therefore, this study was designed to investigate the mechanism of LincRNA‐p21 on suppressing the development of AS. We fed ApoE−/− mice with a high‐fat diet to induce an AS mouse model where the lesion area of AS and the extent of lipid deposition were measured. The binding of LincRNA‐p21 and miR‐221 or miR‐221 and SIRT1 was measured using a dual luciferase reporter gene assay and RIP. Following loss‐ and gain‐ function assays, CCK8, EdU, Transwell assay and scratch test were performed to determine the biological processes of human aortic endothelial cells (HAECs). miR‐221 was highly expressed while SIRT1 was poorly expressed in AS. LincRNA‐p21 acted as a sponge for miR‐221. miR‐221 targeted and negatively regulated the expression of SIRT1. LincRNA‐p21 promoted the deacetylation of Pcsk9 by SIRT1 by competitively binding to miR‐221, whereby promoting HAEC proliferation, migration and tube formation. In conclusion, LincRNA‐p21 acted as a molecular sponge for miR‐221 to promote deacetylation of the promoter region of Pcsk9 by SIRT1, therefore preventing the development of AS.

Anthocyanins are common secondary metabolites in plants that impart red coloration to fruits and flowers. The important WRKY transcription factor family plays multifaceted roles in plant growth and development. In this study, we found a WRKY family gene, Pyrus bretschneideri WRKY75, that may be involved in anthocyanin synthesis in pear. Unlike Arabidopsis thaliana WRKY75, PbWRKY75 may be a positive regulator of anthocyanin synthesis. A transient expression assay indicated that PbWRKY75 promoted pear anthocyanin synthesis. The structural genes (PbANS, PbDFR, and PbUFGT) and positive regulators (PbMYB10 and PbMYB10b) of anthocyanin synthesis were significantly upregulated in the fruitlet skins of PbWRKY75-overexpressing "Zaosu" pears. Subsequently, yeast one-hybrid and dual-luciferase assays indicated that PbWRKY75 promoted PbDFR, PbUFGT, and PbMYB10b expression by activating their promoters. These results revealed that PbWRKY75 may promote the expression of both PbMYB10b and anthocyanin late biosynthetic genes (PbDFR and PbUFGT) by activating their promoters, thereby inducing anthocyanin synthesis in pear. This study enhanced our understanding of the mechanism of pear anthocyanin synthesis, which will be beneficial in the improvement of pear peel color.

In Liberibacter asiaticus, PrbP is a transcriptional regulatory protein involved in survival and persistence during host infection. Tolfenamic acid was previously found to inhibit interactions between PrbP and the promotor region of rplK, resulting in reduced survival of L. asiaticus in the citrus host. In this study, we performed transcriptome analyses to elucidate the PrbP regulon in L. crescens, as it is phylogenetically the closest related species to L. asiaticus that can be grown in laboratory conditions. Chemical inhibition of PrbP with tolfenamic acid revealed that PrbP is involved in the regulation of diverse cellular processes, including stress response, cell motility, cell cycle and biofilm formation. In vitro DNA binding and bacterial two-hybrid assays also suggested that PrbP is a global regulator of multiple transcription factors (RpoH, VisN, PleD, MucR, MocR and CtrA) at both transcriptional and/or post-transcriptional levels. Sub-lethal concentrations of tolfenamic acid significantly reduced the attachment of L. crescens during biofilm formation and decreased long-term persistence in biofilm structures. Overall, our findings show the importance of PrbP in regulating diverse biological processes through direct and indirect interactions with other transcriptional regulators in L. crescens.

SummaryThe CI and Cro repressors of bacteriophage λ create a bistable switch between lysogenic and lytic growth. In λ lysogens, CI repressor expressed from thePRMpromoter blocks expression of the lytic promotersPLandPRto allow stable maintenance of the lysogenic state. When lysogens are induced, CI repressor is inactivated and Cro repressor is expressed from the lyticPRpromoter. Cro repressor blocksPRMtranscription and CI repressor synthesis to ensure that the lytic state proceeds. RexA and RexB proteins, like CI, are expressed from thePRMpromoter in λ lysogens; RexB is also expressed from a second promoter,PLIT, embedded inrexA.Here we show that RexA binds CI repressor and assists the transition from lysogenic to lytic growth, using both intact lysogens and defective prophages with reporter genes under control of the lyticPLandPRpromoters. Once lytic growth begins, if the bistable switch does return to the immune state, RexA expression lessens the probability that it will remain there, thus stabilizing the lytic state and activation of the lyticPLandPRpromoters. RexB modulates the effect of RexA and may also help establish phage DNA replication as lytic growth ensues.

Senescence is a gradual physiological process involving the integration of numerous internal and environmental signals. Abscisic acid (ABA) is a well-known inducer of senescence. However, the regulatory mechanisms underlying ABA-mediated senescence remain largely unknown. Here, we report that the citrus homeodomain leucine zipper I (HD-ZIP I) transcription factor CsHB5 functions as a regulator of ABA-triggered senescence. CsHB5 acts as a nucleus-localized transcriptional activator, the expression of which appeared to be closely associated with citrus senescence. Overexpression of CsHB5 in citrus calli upregulated the expression of ABA- and reactive oxygen species (ROS)-related genes, and significantly increased the content of ABA and hydrogen peroxide (H2 O2 ), whereas silencing CsHB5 in citrus calli downregulated the expression of ABA-related genes. Additionally, heterogenous overexpression of CsHB5 in Solanum lycopersicum (tomato) and Arabidopsis thaliana (Arabidopsis) leads to early leaf yellowing under dark-induced senescence conditions. Meanwhile, the levels of ABA and H2 O2 in transgenic tomatoes increased significantly and the lycopene content decreased. Transcriptome analysis of CsHB5-overexpressing citrus calli and tomato showed that CsHB5 was involved in multiple senescence-associated processes, including chlorophyll degradation, nutrient compound biosynthesis and transport, as well as ABA and ROS signal transduction. The results of yeast one-hybrid assays, electrophoretic mobility shift assays and dual luciferase assays indicated that CsHB5 directly binds to the promoters of ABA biosynthetic genes, including β-carotene hydroxylase 1 (BCH1) and 9-cis-epoxycarotenoid dioxygenase 2 (NCED2), thereby activating their transcription. Our findings revealed that CsHB5 participates in senescence, at least partly, by directly controlling ABA accumulation. Our work provides insight into the regulatory mechanisms underlying ABA-mediated senescence.

Cadmium (Cd) at low concentrations has a potential to promote cell proliferation. However, the molecular mechanisms of Cd-induced proliferation are not well understood. Here, we reported that Cd (0-500 nM) significantly promoted the proliferation of HepG2 cells as demonstrated by elevated cell viability, more EdU-positive cells and increased gene expression of KI-67 and COX-2. Meanwhile, the gene expression of DNA methyltransferases was found to be elevated while that of tumor suppressor genes DAPK1 and RASSF1A were decreased under Cd exposure. Correspondingly, the methylation level of promoters in DAPK1 and RASSF1A were increased. Specifically, the CpG sites at -461 (Chr3:50, 374, 481) of RASSF1A promoter, and that at -260 (Chr9:90, 113, 207), -239 (Chr9:90, 113, 228), and -68 (Chr9:90, 113, 399) of DAPK1 promoter, were significantly hypermethylated. Moreover, 5-azacytidine (an inhibitor of DNA methyltransferase) partly impaired Cd-induced promoter hypermethylation of RASSF1A and DAPK1 genes, increased their expressions and slowed down Cd-induced cell proliferation, suggesting that DNA methylation play an essential part in Cd-boosted proliferation. The study showed that Cd caused promoter hypermethylation of RASSF1A and DAPK1, decreasing their expression and leading to higher level of cell proliferation. Furthermore, Cd at low concentrations could influence DNA methylation, which may serve as the proliferative mechanism of Cd.

Set-nup214 is a recurrent fusion gene that is mainly observed in T-cell acute lymphoblastic leukemia (T-ALL). Dysregulation of homeobox (Hox) genes is frequently observed in patients with leukemia. Consistent with this, HoxA genes are upregulated in the set-nup214 + T-ALL cell line and patients. Although SET-Nup214 has been reported to be recruited to the promoter regions of HoxA genes, the detailed mechanisms of how SET-Nup214 specifically binds to HoxA gene promoters and regulates HoxA gene expression are not known. In this study, we demonstrated that SET-Nup214 interacts with MLL via the SET acidic region of SET-Nup214. SET-Nup214 and MLL cooperatively enhance the promoter activity of the HoxA10 gene. Neither the SET region alone nor the Nup214 region alone sufficiently enhanced the HoxA10 gene promoter. Our results indicated that the SET portion of the SET-Nup214 fusion protein is important for interactions with MLL and transcription enhancement of the HoxA10 gene. Thus, our study will contribute to the understanding of how SET-Nup214 and MLL disturb the expression of HoxA10 gene in leukemia.

During the honeybee larval stage, queens develop larger brains than workers, with morphological differentiation appearing at the fourth larval phase (L4), just after a boost in nutritional difference both prospective females experience. The molecular promoters of this caste-specific brain development are already ongoing in previous larval phases. Transcriptomic analyses revealed a set of differentially expressed genes in the L3 brains of queens and workers, which represents the early molecular response to differential feeding females receive during larval development. Three genes of this set, hex70b, hex70c and hex110, are more highly transcribed in the brain of workers than in queens. The microRNAs miR-34, miR-210 and miR-317 are in higher levels in the queens' brain at the same phase of larval development. Here, we tested the hypothesis that the brain of workers expresses higher levels of hexamerins than that of queens during key phases of larval development and that this differential hexamerin genes expression is further enhanced by the repressing activity of miR-34, miR-210 and miR-317. Our transcriptional analyses showed that hex70b, hex70c and hex110 genes are differentially expressed in the brain of L3 and L4 larval phases of honeybee queens and workers. In silico reconstructed miRNA-mRNA interaction networks were validated using luciferase assays, which showed miR-34 and miR-210 negatively regulate hex70b and hex110 genes by directly and redundantly binding their 3'UTR (untranslated region) sequences. Taken together, our results suggest that miR-34 and miR-210 act together promoting differential brain development in honeybee castes by downregulating the expression of the putative antineurogenic hexamerin genes hex70b and hex110.

Homeostasis of the hematopoietic system is achieved in a hierarchy, with hematopoietic stem cells at the pinnacle. Because only hematopoietic stem cells (HSCs) can self‐renew, the size of the hematopoietic system is strictly controlled. In hematopoietic reconstitution experiments, 1 HSC can reconstitute the entire hematopoietic system, whereas 50 multipotent progenitors cannot. This indicates that only HSCs self‐renew, whereas non‐HSC hematopoietic progenitors are programmed to differentiate or senesce. Oncogenic mutations of the mixed lineage leukemia gene (MLL) overcome this “programmed differentiation” by conferring the self‐renewing ability to non‐HSC hematopoietic progenitors. In leukemia, mutated MLL proteins constitutively activate a broad range of previously transcribed CpG‐rich promoters by an MLL‐mediated transcriptional activation system. This system promotes self‐renewal by replicating an expression profile similar to that of the mother cell in its daughter cells. In this transcriptional activation system, MLL binds to unmethylated CpG‐rich promoters and recruits RNA polymerase II. MLL recruits p300/CBP through its transcriptional activation domain, which acetylates histone H3 at lysines 9, 18, and 27. The AF4 family/ENL family/P‐TEFb complex (AEP) binds to acetylated H3K9/18/27 to activate transcription. Gene rearrangements of MLL with AEP‐ or CBP/p300‐complex components generate constitutively active transcriptional machinery of this transcriptional activation system, which causes aberrant self‐renewal of leukemia stem cells. Inhibitors of the components of this system effectively decrease their leukemogenic potential., MLL gene rearrangements generate constitutively active transcriptional machinery that activates a broad range of CpG‐rich promoters. This machinery confers self‐renewing ability to non‐stem cells to cause leukemia. Therefore, promoting self‐renewal is a novel hallmark of cancer, especially featured in hematopoietic malignancies.

Trichomes function in plant defenses against biotic and abiotic stresses; examination of glabrous lines, which lack trichomes, has revealed key aspects of trichome development and function. Tests of allelism in 51 glabrous rice (Oryza sativa) accessions collected worldwide identified OsSPL10 and OsWOX3B as regulators of trichome development in rice. Here, we report that OsSPL10 acts as a transcriptional regulator controlling trichome development. Haplotype and transient expression analyses revealed that variation in the approximately 700-bp OsSPL10 promoter region is the primary cause of the glabrous phenotype in the indica cultivar WD-17993. Disruption of OsSPL10 by genome editing decreased leaf trichome density and length in the NIL-HL6 background. Plants with genotype OsSPL10WD-17993 /HL6 generated by crossing WD-17993 with NIL-HL6 also had fewer trichomes in the glumes. HAIRY LEAF6 (HL6) encodes another transcription factor that regulates trichome initiation and elongation, and OsSPL10 directly binds to the HL6 promoter to regulate its expression. Moreover, the transcript levels of auxin-related genes, such as OsYUCCA5 and OsPIN-FORMED1b, were altered in OsSPL10 overexpression and RNAi transgenic lines. Feeding tests using locusts (Locusta migratoria) demonstrated that non-glandular trichomes affect feeding by this herbivore. Our findings provide a molecular framework for trichome development and an ecological perspective on trichome functions.

Summary The WUSCHEL‐related homeobox (WOX) transcription factors WOX11 and WOX12 regulate adventitious rooting and responses to stress. The underlying physiological and molecular regulatory mechanisms in salt stress tolerance remain largely unexplored. Here, we characterized the roles of PagWOX11/12a from 84K poplar (Populus alba × P. glandulosa) and the underlying regulatory mechanism in salt stress. PagWOX11/12a was strongly induced by salt stress in roots. Overexpression of PagWOX11/12a in poplar enhanced salt tolerance, as evident by the promotion of growth‐related biomass. In contrast, salt‐treated PagWOX11/12a dominant repression plants displayed reduced biomass growth. Under salt stress conditions, PagWOX11/12a‐overexpressed lines showed higher reactive oxygen species (ROS) scavenging capacity and lower accumulation of hydrogen peroxide (H2O2) than non‐transgenic 84K plants, whereas the suppressors displayed the opposite phenotype. In addition, PagWOX11/12a directly bound to the promoter region of PagCYP736A12 and regulated PagCYP736A12 expression. The activated PagCYP736A12 could enhance ROS scavenging, thus reducing H2O2 levels in roots under salt stress in PagWOX11/12a‐overexpressed poplars. The collective results support the important role of PagWOX11/12a in salt acclimation of poplar trees, indicating that PagWOX11/12a enhances salt tolerance through modulation of ROS scavenging by directly regulating PagCYP736A12 expression in poplar.

Fusarium oxysporum is the causal agent of the devastating Fusarium wilt by invading and colonizing the vascular system in various plants, resulting in substantial economic losses worldwide. Target of rapamycin (TOR) is a central regulator that controls intracellular metabolism, cell growth, and stress responses in eukaryotes, but little is known about TOR signalling in F. oxysporum. In this study, we identified conserved FoTOR signalling pathway components including FoTORC1 and FoTORC2. Pharmacological assays showed that F. oxysporum is hypersensitive to rapamycin in the presence of FoFKBP12 while the deletion mutant strain ΔFofkbp12 is insensitive to rapamycin. Transcriptomic data indicated that FoTOR signalling controls multiple metabolic processes including ribosome biogenesis and cell wall‐degrading enzymes (CWDEs). Genetic analysis revealed that FoTOR1 interacting protein 4 (FoTIP4) acts as a new component of FoTOR signalling to regulate hyphal growth and pathogenicity of F. oxysporum. Importantly, transcript levels of genes associated with ribosome biogenesis and CWDEs were dramatically downregulated in the ΔFotip4 mutant strain. Electrophoretic mobility shift assays showed that FoTIP4 can bind to the promoters of ribosome biogenesis‐ and CWDE‐related genes to positively regulate the expression of these genes. These results suggest that FoTOR signalling plays central roles in regulating hyphal growth and pathogenicity of F. oxysporum and provide new insights into FoTOR1 as a target for controlling and preventing Fusarium wilt in plants., Genetic analysis revealed that FoTOR1 interacting protein 4 (FoTIP4) acts as a new component of FoTOR signalling to regulate hyphal growth and pathogenicity of Fusarium oxysporum.

Cytokinins regulate diverse aspects of plant growth and development, primarily through modulation of gene expression. The cytokinin-responsive transcriptome has been thoroughly described in dicots, especially Arabidopsis, but much less so in monocots. Here, we present a meta-analysis of five different transcriptomic analyses of rice (Oryza sativa) roots treated with cytokinin, including three previously unpublished experiments. We developed a treatment method in which hormone is added to the media of rice seedlings grown in sterile hydroponic culture under a continuous airflow, which resulted in minimal perturbation of the seedlings, thus greatly reducing changes in gene expression in the absence of exogenous hormone. We defined a core set of 205 upregulated and 86 downregulated genes that were differentially expressed in at least three of the transcriptomic datasets. This core set includes genes encoding the type-A response regulators (RRs) and cytokinin oxidases/dehydrogenases, which have been shown to be primary cytokinin response genes. GO analysis revealed that the upregulated genes were enriched for terms related to cytokinin/hormone signaling and metabolism, while the downregulated genes were significantly enriched for genes encoding transporters. Variations of type-B RR binding motifs were significantly enriched in the promoters of the upregulated genes, as were binding sites for other potential partner transcription factors. The promoters of the downregulated genes were generally enriched for distinct cis-acting motifs and did not include the type-B RR binding motif. This analysis provides insight into the molecular mechanisms underlying cytokinin action in a monocot and provides a useful foundation for future studies of this hormone in rice and other cereals.

In honey bees, the process of producing two female castes, including queens and workers, is nutritionally controlled by differential feeding royal jelly to newly emerged larvae. Although they have almost identical genetic blueprints, these castes show striking differences in their morphologies, longevities and reproductive capabilities. DNA methyltransferase 3 (Amdnmt3) gene is involved in the regulatory network for honeybee caste differentiation. Due to the role of two zinc fingers containing transcription factors, SP1 and SP3 in controlling mammalian Dnmts, this study aimed to determine a similar interaction of SPs with Amdnmt3 in the honeybee. We confirmed that the promoter region of Amdnmt3 contained multiple predicted SP1/SP3 binding sites and then investigated the role of AmSP3 in queen-worker differentiation network. We observed that the expression level of Amsp3 was significantly higher in worker larvae than that in queen larvae at 48 h, 84 h and 120 h. Knockdown of Amsp3 expression by RNAi in worker larvae significantly reduced the expression level of Amdnmt3 and caused morphological changes in adult bees towards a queen-like phenotype. However, the expression levels of Amsp3 and Amdnmt3 were repressed by juvenile hormone (JH). Our results suggest that AmSP3 is an important part of the queen-worker differentiation network and supports the role of Amdnmt3 in determining the phenotypic outcome of developing larvae.

AIM We investigated differential DNA methylation in gingival tissues in periodontal health, gingivitis, and periodontitis, and its association with differential mRNA expression. MATERIALS AND METHODS Gingival tissues were harvested from individuals and sites with clinically healthy and intact periodontium, gingivitis, and periodontitis. Samples were processed for differential DNA methylation and mRNA expression using the IlluminaEPIC (850 K) and the IlluminaHiSeq2000 platforms, respectively. Across the three phenotypes, we identified differentially methylated CpG sites and regions, differentially expressed genes (DEGs), and genes with concomitant differential methylation at their promoters and expression were identified. The findings were validated using our earlier databases using HG-U133Plus2.0Affymetrix microarrays and Illumina (450 K) methylation arrays. RESULTS We observed 43,631 differentially methylated positions (DMPs) between periodontitis and health, and 536 DMPs between gingivitis and health (FDR

Agrobacterium tumefaciens is a soil-borne bacterium that is known for its DNA delivery ability and widely exploited for plant transformation. Despite continued interest in improving the utility of the organism, the lack of well-characterized engineering tools limits the realization of its full potential. Here, we present a synthetic biology toolkit that enables precise and effective control of gene expression in A. tumefaciens. We constructed and characterized six inducible expression systems. Then, we optimized the one regulated by cumic acid through amplifier introduction and promoter engineering and evaluated its 15 cognate promoters. To establish fine-tunability, we constructed a series of spacers and a promoter library to systematically modulate both translational and transcriptional rates. We finally demonstrated the application of the tools by co-expressing genes with altered expression levels using a single signal. This study provides precise expression tools for A. tumefaciens, facilitating rational engineering of the bacterium for advanced plant biotechnological applications.

OBJECTIVE To study the telomerase reverse transcriptase (TERT) mutation and the p53 pathway in T1 urinary bladder cancer (UBC). MATERIALS AND METHODS This prospectively performed population-based study included all patients in the Southeast Healthcare Region in Sweden with T1 UBC registered in the period 1992-2001, inclusive. Given that p53 and TERT are important factors for tumour proliferation, although their interrelationships are unknown, we assessed both the TERT and the p53 mutations. Furthermore, we conducted a p53 immunohistochemistry (IHC) analysis using two thresholds for p53 positivity: 10% of tumour cells and 50% of tumour cells (p53 IHC50%). Cox proportional hazards analysis and Kaplan-Meier curves were used to study time to tumour progression. RESULTS Out of 158 patients, we observed the TERT mutation in 74 (47%), the p53 mutation in 48 (30%), and p53 IHC50% positivity in 72 patients (46%). The TERT mutation was more common in p53 mutation-positive patients (P = 0.009), and the latter group also had more patients with p53 IHC50%-positive tumour cells (P = 0.02). In the TERT mutation-negative tumours a p53-positive mutation was associated with a shorter time to progression (P = 0.03) compared to patients with p53-negative mutation. In contrast, in tumours with both TERT mutation positivity and p53 mutation positivity, a longer time to progression was observed in the group with p53 IHC50% positivity compared to the group with p53 IHC50%-negative tumours. CONCLUSIONS In stage T1 UBC, the combination of the TERT mutation and the p53 mutation was associated with tumour progression. A protective effect of the TERT promotor mutation against tumour progression induced by the p53 mutation and subsequent p53 accumulation in tumour cells might be possible, but further investigations are necessary.

NKX2-5, a master cardiac regulatory transcription factor was the first known genetic cause of congenital heart diseases (CHDs). To further investigate its role in CHD pathogenesis, we performed mutational screening of 285 CHD probands and 200 healthy controls. Five coding sequence variants were identified in six CHD cases (2.1%), including three in the N-terminal region (p.A61G, p.R95L, and p.E131K) and one each in homeodomain (HD) (p.A148E) and tyrosine-rich domain (p.P247A). Variant-p.A148E showed tertiary structure changes and differential DNA binding affinity of mutant compared to wild type. Two N-terminal variants-p.A61G and p.E131K along with HD variant p.A148E demonstrated significantly reduced transcriptional activity of Nppa and Actc1 promoters in dual luciferase promoter assay supported by their reduced expression in qRT-PCR. Nonetheless, variant p.R95L affected the synergy of NKX2-5 with serum response factor and TBX5 leading to significantly decreased Actc1 promoter activity depicting a distinctive role of this region. The aberrant expression of other target genes-Irx4, Mef2c, Bmp10, Myh6, Myh7, and Myocd is also observed in response to NKX2-5 variants, possibly due to the defective gene regulatory network. Severely impaired downstream promoter activities and abnormal expression of target genes due to N-terminal variants supports the emerging role of this region during cardiac-developmental pathways.

In embryonic stem (ES) cells, oxidative stress control is crucial for genomic stability, self-renewal and cell differentiation. Heme oxygenase-1 (HO-1) is a key player of the anti-oxidant system, and is also involved in stem cell differentiation and pluripotency acquisition. We found that the HO-1 gene is expressed in ES cells and induced after promoting differentiation. Moreover, downregulation of the pluripotency transcription factor (TF) OCT4 increased HO-1 mRNA levels in ES cells, and analysis of ChIP-seq public data revealed that this TF binds to the HO-1 gene locus in pluripotent cells. Finally, ectopic expression of OCT4 in heterologous systems repressed a reporter carrying the HO-1 gene promoter and the endogenous gene. Hence, this work highlights the connection between pluripotency and redox homeostasis.

Ajuba is a multiple LIM domain‐containing protein and functions as a transcriptional coregulator to modulate many gene expressions in various cellular processes. Here, we describe that the LIM domain of Ajuba interacts with Twist, and the Twist box is a pivotal motif for the interaction. Biologically, Ajuba enhances transcription of target gene N‐cadherin as an obligate coactivator of Twist. The enhancement is achieved by binding to the E‐box element within N‐cadherin promoter as revealed by luciferase reporter and chromatin immunoprecipitation assays. Mechanistic investigation demonstrates that Ajuba recruits CBP and Twist to form a ternary complex at the Twist target promoter region and concomitantly enhances histone acetylation at these sites. These findings identify that Twist is a new interacting protein of Ajuba and Ajuba/Twist/CBP ternary complex may be a potential treatment strategy for Twist‐related tumour metastasis.

Interleukin-32 (IL-32), a multiple pro-inflammatory cytokine, played a vital role in immune-related diseases and infectious diseases. The promoter region of IL-32, which showed functional polymorphism, could regulate IL-32 expression. Although IL-32 rs28372698 polymorphism was related to a lot of inflammatory diseases, the association of IL-32 rs28372698 polymorphism with hepatic flare of chronic HBV infection was not been reported. In the present study, IL-32 rs28372698 polymorphism was genotyped in 104 chronic HBV carriers and 151 active chronic hepatitis B (CHB) patients. The frequency of IL-32 T/T genotype in patients with active CHB was significantly higher than that in chronic HBV carriers. Furthermore, the frequency of the T allele at IL-32 rs28372698 in active CHB patients was also higher than that in chronic HBV carriers. Serum level of IL-32 in active CHB patients was higher than that in chronic HBV carriers. Our results imply that IL-32 T/T polymorphism might be associated with hepatic flare of chronic HBV infection. This article is protected by copyright. All rights reserved.