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Transcription factors encoded by the Forkhead (Fox) gene family have diverse, sometimes conserved, regulatory roles in eumetazoan development, immunity, and physiology. Although this gene family includes members that predate the origin of the animal kingdom, the majority of metazoan Fox genes evolved after the divergence of animals and choanoflagellates. Here, we characterize the composition, structure, and expression of Fox genes in the marine demosponge Amphimedon queenslandica to better understand the origin and evolution of this family. The Fox gene repertoire in A. queenslandica appears to be similar to the ancestral metazoan Fox gene family. All 17 A. queenslandica Fox genes are differentially expressed during development and in adult cell types. Remarkably, eight of these, all of which appear to be metazoan-specific, are induced within just 1 h of larval settlement and commencement of metamorphosis. Gene co-expression analyses suggest that these eight Fox genes regulate developmental and physiological processes similar to their roles in other animals. These findings are consistent with Fox genes playing deeply ancestral roles in animal development and physiology, including in response to changes in the external environment.

The nonexpressor of pathogenesis-related (NPR) gene family is well known to play a crucial role in transactivation of TGA transcription factors for salicylic acid (SA)-responsive genes, including pathogenesis-related protein 1 (PR1), during plants' immune response after pathogen attack in the model dicot Arabidopsis thaliana. However, little is known about NPR gene functions in monocots. We therefore explored the functions of NPRs in SA signaling in the model monocot Brachypodium distachyon. BdNPR1 and BdNPR2/3 share structural similarities with A. thaliana AtNPR1/2 and AtNPR3/4 subfamilies, respectively. The transcript level of BdNPR2 but not BdNPR1/3 appeared to be positively regulated in leaves in response to methyl salicylate. Reporter assays in protoplasts showed that BdNPR2 positively regulated BdTGA1-mediated activation of PR1. This transactivation occurred in an SA-dependent manner through SA binding at Arg468 of BdNPR2. In contrast, BdNPR1 functioned as a suppressor of BdNPR2/BdTGA1-mediated transcription of PR1. Collectively, our findings reveal that the TGA-promoted transcription of SA-inducible PR1 is orchestrated by the activator BdNPR2 and the repressor BdNPR1, which function competitively in B. distachyon.

Canonically, EZH2 serves as the catalytic subunit of PRC2, which mediates H3K27me3 deposition and transcriptional repression. Here, we report that in acute leukaemias, EZH2 has additional noncanonical functions by binding cMyc at non-PRC2 targets and uses a hidden transactivation domain (TAD) for (co)activator recruitment and gene activation. Both canonical (EZH2-PRC2) and noncanonical (EZH2-TAD-cMyc-coactivators) activities of EZH2 promote oncogenesis, which explains the slow and ineffective antitumour effect of inhibitors of the catalytic function of EZH2. To suppress the multifaceted activities of EZH2, we used proteolysis-targeting chimera (PROTAC) to develop a degrader, MS177, which achieved effective, on-target depletion of EZH2 and interacting partners (that is, both canonical EZH2-PRC2 and noncanonical EZH2-cMyc complexes). Compared with inhibitors of the enzymatic function of EZH2, MS177 is fast-acting and more potent in suppressing cancer growth. This study reveals noncanonical oncogenic roles of EZH2, reports a PROTAC for targeting the multifaceted tumorigenic functions of EZH2 and presents an attractive strategy for treating EZH2-dependent cancers.

4-1BB [tumor necrosis factor receptor superfamily (TNFRSF9), CD137) is a critical immune stimulator that sustains T cell activity and antitumor immune response. The strategy to eliminate cancers by agonistically targeting 4-1BB is under clinical investigation. As a protein expressed in an inducible manner, 4-1BB is under tight control on both transcription and translation levels to maintain its homeostasis. So far, the mechanisms underlying the transcriptional activation of 4-1BB have been well-interpreted; however, it remains inexplicit how 4-1BB is regulated on the protein level. In this study, we presented experimental evidence supporting that 4-1BB, especially the heavily N-glycosylated (mature) form, is polyubiquitinated and subjected to the ubiquitin-proteasomal system for degradation. By performing proximity-dependent biotin identification screening coupled with biochemical assays, we identified that F-box/LRR-repeat protein 20 acts as the E3 ligase that promotes the polyubiquitination of 4-1BB at the intracellular domain. Our data provided mechanistic insight into 4-1BB regulation on the protein level by unmasking, for the first time, a posttranslational mechanism governing 4-1BB abundance in cells. The findings of this study could potentially guide the development of 4-1BB-targeted therapy for cancers as well as other immune disorders.

High-dose ascorbate confers tubular mitophagy responsible for septic acute kidney injury (AKI) amelioration, yet its biological roles in immune regulation remain poorly understood.

Calvarial bone healing is challenging, especially for individuals with osteoporosis because stem cells from osteoporotic patients are highly prone to adipogenic differentiation. Based on previous findings that chondrogenic induction of adipose-derived stem cells (ASCs) can augment calvarial bone healing, we hypothesized that activating chondroinductive Sox Trio genes (Sox5, Sox6, Sox9) and repressing adipoinductive genes (C/ebp-α, Ppar-γ) in osteoporotic ASCs can reprogram cell differentiation and improve calvarial bone healing after implantation. However, simultaneous gene activation and repression in ASCs is difficult. To tackle this problem, we built a CRISPR-BiD system for bi-directional gene regulation. Specifically, we built a CRISPR-AceTran system that exploited both histone acetylation and transcription activation for synergistic Sox Trio activation. We also developed a CRISPR interference (CRISPRi) system that exploited DNA methylation for repression of adipoinductive genes. We combined CRISPR-AceTran and CRISPRi to form the CRISPR-BiD system, which harnessed three mechanisms (transcription activation, histone acetylation, and DNA methylation). After delivery into osteoporotic rat ASCs, CRISPR-BiD significantly enhanced chondrogenesis and in vitro cartilage formation. Implantation of the engineered osteoporotic ASCs into critical-sized calvarial bone defects significantly improved bone healing in osteoporotic rats. These results implicated the potential of the CRISPR-BiD system for bi-directional regulation of cell fate and regenerative medicine.

Calcific aortic valve disease (CAVD) is an important health burden due to its increasing prevalence and lack of available approaches. Osteogenic transdifferentiation of aortic valve interstitial cells (AVICs) contributes to valve calcification. SRY-related HMG-box transcription factor 5 (SOX5) is essential for cartilage development. Whether SOX5 is involved in AVIC calcification has not been determined. This study aimed to explore the role of SOX5 in warfarin-induced AVIC calcification. Immunostaining showed decreased SOX5 in human calcific AV and warfarin induced mouse calcific AV tissues compared with human noncalcific AV and control mouse AV tissues. In calcific human AVICs (hAVICs) and porcine AVICS (pAVICs), both knockdown and overexpression of SOX5 inhibited calcium deposition and osteogenic marker gene expression. Protein expression assays and ChIP assays showed that overexpression of SOX5 led to increased recruitment of SOX5 to the SOX9 promoter and resulted in increased mRNA and protein expression of SOX9. Coimmunoprecipitation and immunofluorescence showed that SOX5 binds to SOX9 with its HMG domain in nucleus. Blue Native PAGE showed overexpression of SOX5 led to multimeric complex formation of SOX5 and resulted in decreased binding of SOX5 to SOX9 similar to the results of knockdown of SOX5. Further ChIP and western blotting assays showed that both knockdown and overexpression of SOX5 resulted in SOX9 initiating transcription of anti-calcific gene LRP6 in warfarin-treated pAVICs. Knockdown of LRP6 rescues the anti-calcification effect of SOX5 overexpression. We found that both loss and gain of function of SOX5 lead to the same phenotype: decreased warfarin induced calcification. The stoichiometry of SOX5 is crucial for cooperation with SOX9, SOX9 nuclear localization and subsequent binding of SOX9 to LRP6 promoter. These results suggest that SOX5 is a potential target for the development of anti-calcification therapy.

INTRODUCTION Urokinase plasminogen activator (uPA) is a serine protease and it also demonstrates proinflammatory properties. We thus hypothesized that uPA is involved in immunity of Sertoli cells in the rat testis. MATERIALS AND METHODS The uPA gene(PLAU) promoter was cloned by RT-PCR and the transcriptional activation of core domain was screened by using Dual-Luciferase Reporter Assay System. The Sertoli cells were harvested from 20-day-old Sprague-Dawley male rats and total RNA was isolated. The uPA mRNA levels and MyD88 pathway were tested by qPCR. RESULTS We successfully cloned the 1517-bp rat uPA gene and screened the core domain (-455/+40) in five different fragments of uPA promoter. The uPA expression and uPA promoter activity were upregulated in lipopolysaccharide (LPS)-stimulated Sertoli cells. Furthermore, the uPA expression was regulated through the MyD88-independent pathway by interdicting IRF3 and interferon β. CONCLUSION uPA expression is likely induced by LPS through the core promoter domain of uPA. This finding implied that uPA played a role in the immune function of Sertoli cells in rat testis, which might provide the development of new treatments for male infertility.

High levels of histone acetylation are associated with the regulatory elements of active genes, suggesting a link between acetylation and gene activation. We revisited this model, in the context of EGF-inducible gene expression and found that rather than a simple unifying model, there are two broad classes of genes; one in which high lysine acetylation activity is required for efficient gene activation, and a second group where the opposite occurs and high acetylation activity is inhibitory. We examined the latter class in more detail using EGR2 as a model gene and found that lysine acetylation levels are critical for several activation parameters, including the timing of expression onset, and overall amplitudes of the transcriptional response. In contrast, DUSP1 responds in the canonical manner and its transcriptional activity is promoted by acetylation. Single cell approaches demonstrate heterogenous activation kinetics of a given gene in response to EGF stimulation. Acetylation levels modify these heterogenous patterns and influence both allele activation frequencies and overall expression profile parameters. Our data therefore point to a complex interplay between acetylation equilibria and target gene induction where acetylation level thresholds are an important determinant of transcriptional induction dynamics that are sensed in a gene-specific manner., Graphical Abstract Graphical AbstractModel showing how acetylation levels driven by the KDAC-KAT equilibrium differentially affects gene activation.

The karyopherin α2 subunit gene (KPNA2), an oncogene, is involved in metabolic reprogramming in cancer. This study aimed to explore the function of KPNα2 in the growth and glycolysis in colon cancer (CC) cells. Genes from the Oncomine database that were differentially expressed in multiple CC types were screened. Bioinformatics analysis suggested that KPNA2 was highly expressed in CC, and consequently, high expression of KPNA2 was detected in the CC cell lines. Down-regulation of KPNA2 reduced viability and DNA-replication ability, and increased apoptosis of HCT116 and LoVo cells. It also reduced glucose consumption, extracellular acidification rate, and the ATP production in cells. Centromere protein A (CENPA) was confirmed as an upstream transcription activator of KPNA2. There was significant H3K27ac modification in the promoter region of KPNA2. CENPA primarily recruited histone acetyltransferase general control of amino acid synthesis (GCN)-5 to the promoter region of KPNA2 to induce transcription activation. Overexpression of either CENPA or GCN-5 blocked the role of short hairpin KPNα2 and restored growth and glycolysis in CC cells. To conclude, the findings from this study suggest that CENPA recruits GCN-5 to the promoter region of KPNA2 to induce KPNα2 activation, which strengthens growth and glycolysis in, and augments the development of, CC.

Summary Reprogramming of murine female somatic cells to induced pluripotent stem cells (iPSCs) is accompanied by X chromosome reactivation (XCR), by which the inactive X chromosome (Xi) in female somatic cells becomes reactivated. However, how Xi initiates reactivation during reprogramming remains poorly defined. Here, we used a Sendai virus-based reprogramming system to generate partially reprogrammed iPSCs that appear to be undergoing the initial phase of XCR. Allele-specific RNA-seq of these iPSCs revealed that XCR initiates at a subset of genes clustered near the centromere region. The initial phase of XCR occurs when the cells transit through mesenchymal-epithelial transition (MET) before complete shutoff of Xist expression. Moreover, regulatory regions of these genes display dynamic changes in lysine-demethylase 1a (KDM1A) occupancy. Our results identified clustered genes on the Xi that show reactivation in the initial phase of XCR during reprogramming and suggest a possible role for histone demethylation in this process., Graphical abstract, Highlights • Partially reprogrammed iPSCs enabled analyses of early events in XCR • XCR initiates at a subset of genes clustered near the centromere region • XCR occurs before complete shutoff of Xist expression during reprogramming • KDM1A inhibition appears to directly reactivate transcription from the Xi, Aizawa et al. analyzed early events of X chromosome reactivation (XCR) by generation of partially reprogrammed iPSCs. They found XCR initiation at a subset of genes clustered near the centromere region before complete shutoff of Xist expression. Regulatory regions of these genes display dynamic changes in lysine-demethylase 1a (KDM1A) occupancy, suggesting a possible role for histone demethylation in this process.

During embryogenesis, the genome shifts from transcriptionally quiescent to extensively active in a process known as Zygotic Genome Activation (ZGA). In Drosophila, the pioneer factor Zelda is known to be essential for the progression of development; still, it regulates the activation of only a small subset of genes at ZGA. However, thousands of genes do not require Zelda, suggesting that other mechanisms exist. By conducting GRO-seq, HiC and ChIP-seq in Drosophila embryos, we demonstrate that up to 65% of zygotically activated genes are enriched for the histone variant H2A.Z. H2A.Z enrichment precedes ZGA and RNA Polymerase II loading onto chromatin. In vivo knockdown of maternally contributed Domino, a histone chaperone and ATPase, reduces H2A.Z deposition at transcription start sites, causes global downregulation of housekeeping genes at ZGA, and compromises the establishment of the 3D chromatin structure. We infer that H2A.Z is essential for the de novo establishment of transcriptional programs during ZGA via chromatin reorganization., Nature Communications, 12 (1), ISSN:2041-1723

Rhabdomyosarcoma (RMS) is a pediatric malignancy of skeletal muscle lineage. The aggressive alveolar subtype is characterized by t(2;13) or t(1;13) translocations encoding for PAX3- or PAX7-FOXO1 chimeric transcription factors, respectively, and are referred to as fusion positive RMS (FP-RMS). The fusion gene alters the myogenic program and maintains the proliferative state while blocking terminal differentiation. Here, we investigated the contributions of chromatin regulatory complexes to FP-RMS tumor maintenance. We define the mSWI/SNF functional repertoire in FP-RMS. We find that SMARCA4 (encoding BRG1) is overexpressed in this malignancy compared to skeletal muscle and is essential for cell proliferation. Proteomic studies suggest proximity between PAX3-FOXO1 and BAF complexes, which is further supported by genome-wide binding profiles revealing enhancer colocalization of BAF with core regulatory transcription factors. Further, mSWI/SNF complexes localize to sites of de novo histone acetylation. Phenotypically, interference with mSWI/SNF complex function induces transcriptional activation of the skeletal muscle differentiation program associated with MYCN enhancer invasion at myogenic target genes, which is recapitulated by BRG1 targeting compounds. We conclude that inhibition of BRG1 overcomes the differentiation blockade of FP-RMS cells and may provide a therapeutic strategy for this lethal childhood tumor., Nature Communications, 12 (1), ISSN:2041-1723

Carboxylesterase 1 (CES1) is the predominant carboxylesterase in the human liver, involved in metabolism of both xenobiotics and endogenous substrates. Genetic or epigenetic factors that alter CES1 activity or expression are associated with changes in drug response, lipid, and glucose homeostasis. However, the transcriptional regulation of CES1 in the human liver remains uncertain. By applying both the random forest and Sobol’s Sensitivity Indices (SSI) to analyze existing liver RNA expression microarray data (GSE9588), we identified nuclear receptor subfamily 1 group H member 3 (NR1H3) liver X receptor (LXR)α as a key factor regulating constitutive CES1 expression. This model prediction was validated using small interfering RNA (siRNA) knockdown and CRISPR-mediated transcriptional activation of NR1H3 in Huh7 and HepG2 cells. We found that NR1H3’s activation of CES1 is splice isoform-specific, namely that increased expression of the NR1H3-211 isoform increased CES1 expression whereas NR1H3-201 did not. Also, in human liver samples, expression of NR1H3-211 and CES1 are correlated, whereas NR1H3-201 and CES1 are not. This trend also occurs during differentiation of induced pluripotent stem cells (iPSCs) to hepatocytes, where only expression of the NR1H3-211 isoform parallels expression of CES1. Moreover, we found that treatment with the NR1H3 agonist T0901317 in HepG2 cells had no effect on CES1 expression. Overall, our results demonstrate a key role of NR1H3 in maintaining the constitutive expression of CES1 in the human liver. Furthermore, our results support that the effect of NR1H3 is splice isoform-specific and appears to be ligand independent. SIGNIFICANCE STATEMENT: Despite the central role of carboxylesterase 1 (CES1) in metabolism of numerous medications, little is known about its transcriptional regulation. This study identifies nuclear receptor subfamily 1 group H member 3 as a key regulator of constitutive CES1 expression and therefore is a potential target for future studies to understand interperson variabilities in CES1 activity and drug metabolism.

Aryl hydrocarbon receptors (AHRs), a class of ligand-dependent nuclear receptors that regulate cellular responses by inducing the expression of various target genes in response to external signals, are implicated in maintaining retinal tissue homeostasis. Previous studies have shown that the regulation of AHR-induced gene expression requires transcriptional co-regulators. However, it is not yet clear how chromatin remodelers, histone methyltransferases and coactivators interact during AHR-mediated gene expression in human retinal cells. In this study, we reveal that the histone methyltransferase MLL1 and the coactivator FLII are involved in AHR-mediated gene expression in retinal pigment epithelial cells. 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) significantly increased the expression of CYP1A1, CYP1B1 and AHRR in ARPE-19 cells, whereas FLII or MLL1 depletion significantly reduced the expression of these genes induced by TCDD. Mechanistically, FLII binds to AHR in a ligand-dependent manner in ARPE-19 cells. In particular, the binding of FLII to MLL1 occurs through the GelB domain of FLII. In addition, MLL1 binds to AHR in a ligand-independent manner. FLII is involved in the recruitment of the BRG1 chromatin remodeler and MLL1 histone methyltransferase to the AHR-regulated CYP1A1 gene region in ARPE-19 cells and consequently, plays an important role in RNA polymerase II binding and transcriptional activity by modulating chromatin accessibility. Our results identify the functions and mechanisms of action of FLII and MLL1 in AHR-induced gene expression in human retinal pigment epithelial cells.

Glucose transporter type 2 (GLUT2), encoded by the SLC2A2 gene, is an essential component of glucose-stimulated insulin secretion in pancreatic islet β-cells. Like that of the gene encoding insulin, expression of the SLC2A2 gene expression is closely linked to β-cell functionality in rodents, but the mechanism by which β-cell-specific expression of SLC2A2 is controlled remains unclear. In this report, to identify putative enhancer elements of the mouse Slc2a2 gene, we examined evolutional conservation of the nucleotide sequence of its genomic locus, together with ChIP-seq data of histone modifications and various transcription factors published in previous studies. Using luciferase reporter assays, we found that an evolutionarily conserved region (ECR) located approximately 40 kbp downstream of the transcription start site of Slc2a2 functions as an active enhancer in the MIN6 β-cell line. We also found that three β-cell-enriched transcription factors, MafA, NeuroD1, and HNF1β, synergistically activate transcription through this 3’ downstream distal enhancer (ECR3’) and the proximal promoter region of the gene. Our data also indicate that the simultaneous binding of HNF1β to its target sites within the promoter and ECR3’ of Slc2a2 is indispensable for transcriptional activation, and that binding of MafA and NeuroD1 to their respective target sites within the ECR3’ enhances transcription. Co-immunoprecipitation experiments suggested that MafA, NeuroD1, and HNF1β interact with each other. Overall, these results suggest that promoter-enhancer communication through MafA, NeuroD1, and HNF1β is critical for Slc2a2 gene expression. These findings provide clues to help elucidate the mechanism of regulation of Slc2a2 gene expression in β-cells.

Increased lung vascular permeability and neutrophilic inflammation are hallmarks of acute lung injury. Alveolar macrophages (AMϕ), the predominant sentinel cell type in the airspace, die in massive numbers while fending off pathogens. Recent studies indicate that the AMϕ pool is replenished by airspace-recruited monocytes, but the mechanisms instructing the conversion of recruited monocytes into reparative AMϕ remain elusive. Cyclic AMP (cAMP) is a vascular barrier protective and immunosuppressive second messenger in the lung. Here, we subjected mice expressing GFP under the control of the Lysozyme-M promoter (LysM-GFP mice) to the LPS model of rapidly resolving lung injury to address the impact of mechanisms determining cAMP levels in AMϕ and regulation of mobilization of the reparative AMϕ-pool. RNA-seq analysis of flow-sorted Mϕ identified phosphodiesterase 4b (PDE4b) as the top LPS-responsive cAMP-regulating gene. We observed that PDE4b expression markedly increased at the time of peak injury (4 h) and then decreased to below the basal level during the resolution phase (24 h). Activation of transcription factor NFATc2 was required for the transcription of PDE4b in Mϕ. Inhibition of PDE4 activity at the time of peak injury, using intratracheal rolipram, increased cAMP levels, augmented the reparative AMϕ pool, and resolved lung injury. This response was not seen following conditional depletion of monocytes, thus establishing airspace-recruited PDE4b-sensitive monocytes as the source of reparative AMϕ. Interestingly, adoptive transfer of rolipram-educated AMϕ into injured mice resolved lung edema. We propose suppression of PDE4b as an effective approach to promote reparative AMϕ generation from monocytes for lung repair.

Yes‐associated protein 1 (YAP1) and its paralogue PDZ‐binding motif (TAZ) play pivotal roles in cell proliferation, migration, and invasion, and abnormal activation of these TEAD transcriptional coactivators is found in diverse cancers in humans and mice. Targeting YAP1/TAZ signaling is thus a promising therapeutic avenue but, to date, few selective YAP1/TAZ inhibitors have been effective against cancer cells either in vitro or in vivo. We screened chemical libraries for potent YAP1/TAZ inhibitors using a highly sensitive luciferase reporter system to monitor YAP1/TAZ‐TEAD transcriptional activity in cells. Among 29 049 low‐molecular‐weight compounds screened, we obtained nine hits, and the four of these that were the most effective shared a core structure with the natural product alantolactone (ALT). We also tested 16 other structural derivatives of ALT and found that natural ALT was the most efficient at increasing ROS‐induced LATS kinase activities and thus YAP1/TAZ phosphorylation. Phosphorylated YAP1/TAZ proteins were subject to nuclear exclusion and proteosomic degradation such that the growth of ALT‐treated tumor cells was inhibited both in vitro and in vivo. Our data show for the first time that ALT can be used to target the ROS‐YAP pathway driving tumor cell growth and so could be a potent anticancer drug., YAP1 and its paralogue TAZ play pivotal roles in cell proliferation, migration, and invasion, and abnormal activation of these TEAD transcriptional coactivators is found in diverse cancers in humans and mice. We screened chemical libraries for potent YAP1/TAZ inhibitors using a highly sensitive luciferase reporter system to monitor YAP1/TAZ‐TEAD transcriptional activity in cells. We found that alantolactone primarily targets the ROS‐YAP pathway driving tumor cell growth and so could be a potent anticancer drug.

Hypoxia-induced pulmonary microvascular endothelial cell (PMVEC) monolayers hyperpermeability is vital for vascular leakage, which participates in vascular diseases, such as acute lung injury (ALI) and high-altitude pulmonary edema (HAPE). We previously observed that PMVEC permeability was markedly elevated in hypoxia when cocultured with primary type II alveolar epithelial cells (AECII) in which isthmin1 (ISM1) was highly upregulated. However, whether the upregulation of ISM1 plays a role in hypoxia-induced PMVEC hyperpermeability is unclear. In this study, we assessed the role of AECII-derived ISM1 in hypoxia-induced PMVEC hyperpermeability with an AECII/PMVEC coculture system and uncovered the underlying mechanism whereby hypoxia stimulates ISM1 gene expression. We found that ISM1 gene expression was upregulated in cultured AECII cells exposed to hypoxia (3% O2) and that AECII-derived ISM1 participated in hypoxia-induced hyperpermeability of PMVEC monolayers, as small interference RNA (siRNA)-mediated knockdown of ISM1 in AECII markedly attenuated the increase in PMVEC permeability in coculture system under hypoxia. In addition, we confirmed that ISM1 was regulated by hypoxia-inducible factor-1α (HIF1α) according to the evidence that silencing of HIF1α inhibited the hypoxia-mediated upregulation of ISM1. Mechanismly, overexpression of HIF1α transcriptionally activated ISM1 gene expression by directly binding to the conserved regulatory elements upstream of the ism1 locus. We identified a novel HIF-1-target gene ISM1, which involves in hyperpermeability of pulmonary microvascular endothelial cell monolayers under hypoxia. Our in vitro cell experiments implied that the upregulated ISM1 derived from alveolar epithelium might be a vital modulator in hypoxia-induced endothelial hyperpermeability and thereby implicates with hypoxic pulmonary-related diseases.

BackgroundCellular RNA-binding proteins (RBPs) have multiple roles in post-transcriptional control, and some are shown to bind DNA. However, the global localization and the general chromatin-binding ability of RBPs are not well-characterized and remain undefined in hematopoietic cells.ResultsWe first provide a full view of RBPs’ distribution pattern in the nucleus and screen for chromatin-enriched RBPs (Che-RBPs) in different human cells. Subsequently, by generating ChIP-seq, CLIP-seq, and RNA-seq datasets and conducting combined analysis, the transcriptional regulatory potentials of certain hematopoietic Che-RBPs are predicted. From this analysis, quaking (QKI5) emerges as a potential transcriptional activator during monocytic differentiation. QKI5 is over-represented in gene promoter regions, independent of RNA or transcription factors. Furthermore, DNA-bound QKI5 activates the transcription of several critical monocytic differentiation-associated genes, including CXCL2, IL16, and PTPN6. Finally, we show that the differentiation-promoting activity of QKI5 is largely dependent on CXCL2, irrespective of its RNA-binding capacity.ConclusionsOur study indicates that Che-RBPs are versatile factors that orchestrate gene expression in different cellular contexts, and identifies QKI5, a classic RBP regulating RNA processing, as a novel transcriptional activator during monocytic differentiation.

EPAS1 (endothelial PAS domain protein 1), as the major effect gene for the adaptation to chronic hypoxia, is required for hypoxic pulmonary hypertension (HPH). Downregulated EPAS1 ameliorates the development of HPH. We confirmed that EPAS1-specific inhibitor PT2385 ameliorated HPH features, as demonstrated by right ventricle hypertrophy, right ventricular systolic pressure, and pulmonary vascular remodeling. However, the mechanism of EPAS1 in HPH pathogenesis remains unclear. RNA sequencing in human pulmonary artery endothelial cells with EPAS1 knockdown identified EPAS1-regulated genes, including ICAM1 (intercellular adhesion molecule 1), which created a proinflammatory perivascular microenvironment associated with HPH by elevating leukocyte adhesion to the vascular endothelium. Chromatin immunoprecipitation assays revealed that EPAS1 directly bound to ICAM1 promoter. The long noncoding RNA small nucleolar RNA host gene 5 (SNHG5), significantly increased in acute exacerbation period of chronic obstructive pulmonary disease and hypoxic human pulmonary artery endothelial cells, also contributed to the regulation of ICAM1 expression. Endothelial-specific deletion of Snhg5 also rescued HPH in mice. Overexpression of EPAS1 or SNHG5 enhanced, while the depletion of EPAS1 or SNHG5 attenuated, ICAM1 transactivation. SNHG5 was directly regulated by EPAS1, and interestingly, the upregulated SNHG5 could further enhance the levels of EPAS1, which consequently led to hypoxia-induced ICAM1 transactivation. RNA pull-down assay followed by high-throughput sequencing demonstrated that miR-625-5p could bind to SNHG5. Manipulating miR-625-5p altered the levels of EPAS1 during hypoxia. Our data showed a positive feed-forward exists between EPAS1 and SNHG5 signaling during hypoxia-induced ICAM1 transactivation in endothelial cells. Targeting EPAS1 and SNHG5 may provide promising strategies for the prevention of HPH.

Background The process of early development varies across the species-rich phylum Arthropoda. Owing to the limited research strategies for dissecting lineage-specific processes of development in arthropods, little is known about the variations in early arthropod development at molecular resolution. The Theridiidae spider, Parasteatoda tepidariorum, has its genome sequenced and could potentially contribute to dissecting early embryonic processes. Results We present genome-wide identification of candidate genes that exhibit locally restricted expression in germ disc forming stage embryos of P. tepidariorum, based on comparative transcriptomes of isolated cells from different regions of the embryo. A subsequent pilot screen by parental RNA interference identifies three genes required for body axis formation. One of them is a GATA-like gene that has been fast evolving after duplication and divergence from a canonical GATA family gene. This gene is designated fuchi nashi (fuchi) after its knockdown phenotypes, where the cell movement toward the formation of a germ disc was reversed. fuchi expression occurs in cells outside a forming germ disc and persists in the endoderm. Transcriptome and chromatin accessibility analyses of fuchi pRNAi embryos suggest that early fuchi activity regulates chromatin state and zygotic gene activation to promote endoderm specification and pattern formation. We also show that there are many uncharacterized genes regulated by fuchi. Conclusions Our genome-based research using an arthropod phylogenetically distant from Drosophila identifies a lineage-specific, fast-evolving gene with key developmental roles in one of the earliest, genome-wide regulatory events, and allows for molecular exploration of the developmental variations in early arthropod embryos.

Increased abundance of polo-like kinase 1 (PLK1) is observed in various tumor types, particularly in lung adenocarcinoma (LUAD). Here, we found that PLK1 accelerated the progression of LUAD through a mechanism that was independent of its role in mediating mitotic cell division. Analysis of human tumor databases revealed that increased PLK1 abundance in LUAD correlated with mutations in KRAS and p53, with tumor stage, and with reduced survival in patients. In a mouse model of KRAS G12D -driven, p53-deficient LUAD, PLK1 overexpression increased tumor burden, decreased tumor cell differentiation, and reduced animal survival. PLK1 overexpression in cultured cells and mice indirectly increased the expression of the gene encoding the receptor tyrosine kinase RET by phosphorylating the transcription factor TTF-1. Signaling by RET and mutant KRAS in these tumors converged to activate the mitogen-activated protein kinase (MAPK) pathway. Pharmacological inhibition of the MAPK pathway kinase MEK combined with inhibition of either RET or PLK1 markedly suppressed tumor growth. Our findings show that PLK1 can amplify MAPK signaling and reveal a potential target for stemming progression in lung cancers with high PLK1 abundance.

Phosphorylation of estrogen receptor α (ER) at serine 118 (pS118-ER) is induced by estrogen and is the most abundant posttranslational mark associated with a transcriptionally active receptor. Cistromic analysis of pS118-ER from our group revealed enrichment of the GRHL2 motif near pS118-ER binding sites. In this study, we used cistromic and transcriptomic analyses to interrogate the relationship between GRHL2 and pS118-ER. We found that GRHL2 is bound to chromatin at pS118-ER/GRHL2 co-occupancy sites prior to ligand treatment, and GRHL2 binding is required for maximal pS118-ER recruitment. pS118-ER/GRHL2 co-occupancy sites were enriched at active enhancers marked by H3K27ac and H3K4me1, along with FOXA1 and p300, compared to sites where each factor binds independently. Transcriptomic analysis yielded four subsets of ER/GRHL2-coregulated genes revealing that GRHL2 can both enhance and antagonize E2-mediated ER transcriptional activity. Gene ontology analysis indicated that coregulated genes are involved in cell migration. Accordingly, knockdown of GRHL2, combined with estrogen treatment, resulted in increased cell migration but no change in proliferation. These results support a model in which GRHL2 binds to selected enhancers and facilitates pS118-ER recruitment to chromatin, which then results in differential activation and repression of genes that control estrogen-regulated ER-positive breast cancer cell migration.

The Arabidopsis (Arabidopsis thaliana) BTB-TAZ DOMAIN PROTEIN 2 (BT2) contains an N-terminal BTB domain, a central TAZ zinc-finger protein–protein interaction domain, and a C-terminal calmodulin-binding domain. We previously demonstrated that BT2 regulates telomerase activity and mediates multiple responses to nutrients, hormones, and abiotic stresses in Arabidopsis. Here, we describe the essential role of BT2 in activation of genes by multimerized Cauliflower mosaic virus 35S (35S) enhancers. Loss of BT2 function in several well-characterized 35S enhancer activation-tagged lines resulted in suppression of the activation phenotypes. Suppression of the phenotypes was associated with decreased transcript abundance of the tagged genes. Nuclear run-on assays, mRNA decay studies, and bisulfite sequencing revealed that BT2 is required to maintain the transcriptionally active state of the multimerized 35S enhancers, and lack of BT2 leads to hypermethylation of the 35S enhancers. The TAZ domain and the Ca++/calmodulin-binding domain of BT2 are critical for its function and 35S enhancer activity. We further demonstrate that BT2 requires CULLIN3 and two bromodomain-containing Global Transcription factor group E proteins (GTE9 and GTE11), to regulate 35S enhancer activity. We propose that the BT2-CULLIN3 ubiquitin ligase, through interactions with GTE9 and GTE11, regulates 35S enhancer activity in Arabidopsis., Activation of gene expression by the multimerized Cauliflower mosaic virus 35S enhancers requires the function of a putative BTB-TAZ ubiquitin ligase.

HIV-1 transactivator of transcription (Tat) protein is required for HIV-1 replication, and it has been implicated in the pathogenesis of HIV-1-associated neurocognitive disorder (HAND). HIV-1 Tat can enter cells via receptor-mediated endocytosis where it can reside in endolysosomes; upon its escape from these acidic organelles, HIV-1 Tat can enter the cytosol and nucleus where it activates the HIV-1 LTR promoter. Although it is known that HIV-1 replication is affected by the iron status of people living with HIV-1 (PLWH), very little is known about how iron affects HIV-1 Tat activation of the HIV-1 LTR promoter. Because HIV-1 proteins de-acidify endolysosomes and endolysosome de-acidification affects subcellular levels and actions of iron, we tested the hypothesis that the endolysosome pool of iron is sufficient to affect Tat-induced HIV-1 LTR transactivation. Ferric (Fe(3+)) and ferrous (Fe(2+)) iron both restricted Tat-mediated HIV-1 LTR transactivation. Chelation of endolysosome iron with deferoxamine (DFO) and 2–2 bipyridyl, but not chelation of cytosolic iron with deferiprone and deferasirox, significantly enhanced Tat-mediated HIV-1 LTR transactivation. In the presence of iron, HIV-1 Tat increasingly oligomerized and DFO prevented the oligomerization. DFO also reduced protein expression levels of the HIV-1 restriction agent beta-catenin in the cytosol and nucleus. These findings suggest that DFO increases HIV-1 LTR transactivation by increasing levels of the more active dimeric form of Tat relative to the less active oligomerized form of Tat, increasing the escape of dimeric Tat from endolysosomes, and/or reducing beta-catenin protein expression levels. Thus, intracellular iron might play a significant role in regulating HIV-1 replication, and these findings raise cautionary notes for chelation therapies in PLWH.

Complex sphingolipids are important components of the lipid bilayer of budding yeast Saccharomyces cerevisiae, and a defect of the biosynthesis causes widespread cellular dysfunction. In this study, we found that mutations causing upregulation of the cAMP/protein kinase A (PKA) pathway cause hypersensitivity to the defect of complex sphingolipid biosynthesis caused by repression of AUR1 encoding inositol phosphorylceramide synthase, whereas loss of PKA confers resistance to the defect. Loss of PDE2 encoding cAMP phosphodiesterase or PKA did not affect the reduction in complex sphingolipid levels and ceramide accumulation caused by AUR1 repression, suggesting that the change in sensitivity to the AUR1 repression due to the mutation of the cAMP/PKA pathway is not caused by exacerbation or suppression of the abnormal metabolism of sphingolipids. We also identified PBS2 encoding MAPKK in the high-osmolarity glycerol (HOG) pathway as a multicopy suppressor gene that rescues the hypersensitivity to AUR1 repression caused by deletion of IRA2, which causes hyperactivation of the cAMP/PKA pathway. Since the HOG pathway has been identified as one of the rescue systems against the growth defect caused by the impaired biosynthesis of complex sphingolipids, it was assumed that PKA affects activation of the HOG pathway under AUR1-repressive conditions. Under AUR1-repressive conditions, hyperactivation of PKA suppressed the phosphorylation of Hog1, MAPK in the HOG pathway, and transcriptional activation downstream of the HOG pathway. These findings suggested that PKA is possibly involved in the avoidance of excessive activation of the HOG pathway under impaired biosynthesis of complex sphingolipids.

Apoptosis plays a key role in keloids. Growth arrest and DNA damage‐inducible gene 153 (GADD153) is regulated by apoptosis. Botulinum toxin type A (BTXA) can induce apoptosis in keloid fibroblasts. This research aimed to explore the hypothesis that GADD153 mediates apoptosis in keloid fibroblasts exposed to BTXA. BTXA significantly induced GADD153 protein and mRNA expression in keloid fibroblasts. Treatment with c‐Jun N‐terminal kinase (JNK) inhibitor SP600125, JNK small interfering RNA (siRNA) and tumour necrosis factor‐alpha (TNF‐α) antibodies reversed the BTXA‐induced GADD153 expression. BTXA enhanced the transcriptional activity of GADD153, whereas the GADD153 mutant plasmid, JNK siRNA and anti‐TNF‐α antibody treatment abolished the BTXA‐induced transcriptional activity of GADD153. The addition of TNF‐α to keloid fibroblasts markedly increased GADD153 protein expression. The addition of GADD153 siRNA, SP600125 and anti‐TNF‐α antibodies reversed cell death and caspase 3 and 9 activity induced by BTXA.

Key message Expression of Cre recombinase by AtRps5apro or AtDD45pro enabled Cre/lox-mediated recombination at an early embryonic developmental stage upon crossing, activating transgenes in the hybrid cowpea and tobacco. Abstract Genetic engineering ideally results in precise spatiotemporal control of transgene expression. To activate transgenes exclusively in a hybrid upon fertilization, we evaluated a Cre/lox-mediated gene activation system with the Cre recombinase expressed by either AtRps5a or AtDD45 promoters that showed activity in egg cells and young embryos. In crosses between Cre recombinase lines and transgenic lines harboring a lox-excision reporter cassette with ZsGreen driven by the AtUbq3 promoter after Cre/lox-mediated recombination, we observed complete excision of the lox-flanked intervening DNA sequence between the AtUbq3pro and the ZsGreen coding sequence in F1 progeny upon genotyping but no ZsGreen expression in F1 seeds or seedlings. The incapability to observe ZsGreen fluorescence was attributed to the activity of the AtUbq3pro. Strong ZsGreen expression in F1 seeds was observed after recombination when ZsGreen was driven by the AtUbq10 promoter. Using the AtDD45pro to express Cre resulted in more variation in recombination frequencies between transgenic lines and crosses. Regardless of the promoter used to regulate Cre, mosaic F1 progeny were rare, suggesting gene activation at an early embryo-developmental stage. Observation of ZsGreen-expressing tobacco embryos at the globular stage from crosses with the AtRps5aproCre lines pollinated by the AtUbq3prolox line supported the early activation mode.

Peroxisome proliferator-activated receptor gamma (PPARγ) is a master transcription factor in adipocyte differentiation, while distal-less homeobox 5 (Dlx5) is essential for initiating osteoblast differentiation by driving Runt-related transcription factor 2 expression. Considering that adipocytes and osteoblasts share common progenitors, there is a reciprocal correlation between bone and fat formation. However, the mechanism by which Dlx5 controls PPARγ remains unclear. We elucidated that Dlx5 physically binds to PPARγ during immunoprecipitation; in particular, the ligand-binding and DNA-binding domains of PPARγ were involved in the interaction. Transcriptional activity of PPARγ was significantly decreased by Dlx5 overexpression, whereas the opposite results were detected with Dlx5 knockdown. Rosiglitazone, a PPARγ agonist, further enhanced the PPARγ-induced transcriptional activity; however, Dlx5 overexpression effectively repressed the rosiglitazone-mediated increase in activity. Finally, DNA-binding affinity assay revealed that Dlx5 interrupts the interaction of PPARγ with the PPARγ response element promoter. In conclusion, our findings indicate that Dlx5 impedes PPARγ-induced activity, and it may be useful for managing diabetes drug-mediated obesity.

Transcriptional regulation, a pivotal biological process by which cells adapt to environmental fluctuations, is achieved by the binding of transcription factors to target sequences in a sequence-specific manner. However, how transcription factors recognize the correct target from amongst the numerous candidates in a genome has not been fully elucidated. We here show that, in the fission-yeast fbp1 gene, when transcription factors bind to target sequences in close proximity, their binding is reciprocally stabilized, thereby integrating distinct signal transduction pathways. The fbp1 gene is massively induced upon glucose starvation by the activation of two transcription factors, Atf1 and Rst2, mediated via distinct signal transduction pathways. Atf1 and Rst2 bind to the upstream-activating sequence 1 region, carrying two binding sites located 45 bp apart. Their binding is reciprocally stabilized due to the close proximity of the two target sites, which destabilizes the independent binding of Atf1 or Rst2. Tup11/12 (Tup-family co-repressors) suppress independent binding. These data demonstrate a previously unappreciated mechanism by which two transcription-factor binding sites, in close proximity, integrate two independent-signal pathways, thereby behaving as a hub for signal integration.

Intermittent hypoxia (IH) is a hallmark manifestation of obstructive sleep apnea (OSA). Rodents treated with IH exhibit hypertension. Hypoxia-inducible factor (HIF)-1-dependent transcriptional activation of NADPH oxidases ( Nox) and the resulting increase in reactive oxygen species (ROS) levels is a major molecular mechanism underlying IH/OSA-induced hypertension. Jumanji C (JmjC)-containing histone lysine demethylases (JmjC-KDMs) are coactivators of HIF-1-dependent transcriptional activation. In the present study, we tested the hypothesis that JmjC-KDMs are required for IH-evoked HIF-1 transcriptional activation of Nox4 and the ensuing hypertension. Studies were performed on pheochromocytoma (PC)12 cells and rats. IH increased KDM6B protein and enzyme activity in PC12 cells in an HIF-1-independent manner as evidenced by unaltered KDM6B activation by IH in HIF-1α shRNA-treated cells. Cells treated with IH showed increased HIF-1-dependent Nox4 transcription as indicated by increased HIF-1α binding to hypoxia-responsive element (HRE) sequence of the Nox4 gene promoter demonstrated by chromatin immunoprecipitation (ChiP) assay. Pharmacological blockade of KDM6B with GSKJ4, a specific KDM6 inhibitor, or genetic silencing of KDM6B with shRNA abolished IH-induced Nox4 transcriptional activation by blocking HIF-1α binding to the promoter of the Nox4 gene. Treating IH-exposed rats with GSKJ4 showed: 1) absence of KDM6B activation and HIF-1-dependent Nox4 transcription in the adrenal medullae, and 2) absence of elevated plasma catecholamines and hypertension. Collectively, these findings indicate that KDM6B functions as a coactivator of HIF-1-mediated Nox4 transactivation and demonstrates a hitherto uncharacterized role for KDMs in IH-induced hypertension by HIF-1.

The PBAF complex, a member of SWI/SNF family of chromatin remodelers, plays an essential role in transcriptional regulation. We revealed a disease progression associated elevation of PHF10 subunit of PBAF in clinical melanoma samples. In melanoma cell lines, PHF10 interacts with MYC and facilitates the recruitment of PBAF complex to target gene promoters, therefore, augmenting MYC transcriptional activation of genes involved in the cell cycle progression. Depletion of either PHF10 or MYC induced G1 accumulation and a senescence-like phenotype. Our data identify PHF10 as a pro-oncogenic mechanism and an essential novel link between chromatin remodeling and MYC-dependent gene transcription.

Background: eNOS (endothelial nitric oxide synthase) is an endothelial cell (EC)–specific gene predominantly expressed in medium- to large-sized arteries where ECs experience atheroprotective laminar flow with high shear stress. Disturbed flow with lower average shear stress decreases eNOS transcription, which leads to the development of atherosclerosis, especially at bifurcations and curvatures of arteries. This prototypic arterial EC gene contains 2 distinct flow-responsive cis-DNA elements in the promoter, the shear stress response element (SSRE) and the KLF (Krüppel-like factor) element. Previous in vitro studies suggested their positive regulatory functions on flow-induced transcription of EC genes including eNOS. However, the in vivo function of these cis-DNA elements remains unknown. Methods: Insertional transgenic mice with a mutation at each flow-responsive cis-DNA element were generated using a murine eNOS promoter–β-galactosidase reporter by linker-scanning mutagenesis and compared with episomal-based mutations in vitro. DNA methylation at the eNOS proximal promoter in mouse ECs was assessed by bisulfite sequencing or pyrosequencing. Results: Wild type mice with a functional eNOS promoter–reporter transgene exhibited reduced endothelial reporter expression in the atheroprone regions of disturbed flow (n=5). It is surprising that the SSRE mutation abrogated reporter expression in ECs and was associated with aberrant hypermethylation at the eNOS proximal promoter (n=7). Reporter gene silencing was independent of transgene copy number and integration position, indicating that the SSRE is a critical cis-element necessary for eNOS transcription in vivo. The KLF mutation demonstrated an integration site–specific decrease in eNOS transcription, again with marked promoter methylation (n=8), suggesting that the SSRE alone is not sufficient for eNOS transcription in vivo. In wild type mice, the native eNOS promoter was significantly hypermethylated in ECs from the atheroprone regions where eNOS expression was markedly repressed by chronic disturbed flow, demonstrating that eNOS expression is regulated by flow-dependent DNA methylation that is region-specific in the arterial endothelium in vivo. Conclusions: We report, for the first time, that the SSRE and KLF elements are critical flow sensors necessary for a transcriptionally permissive, hypomethylated eNOS promoter in ECs under chronic shear stress in vivo. Moreover, eNOS expression is regulated by flow-dependent epigenetic mechanisms, which offers novel mechanistic insight on eNOS gene regulation in atherogenesis.

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.

Epigenome editing methods enable the precise manipulation of epigenetic modifications, such as histone posttranscriptional modifications (PTMs), for uncovering their biological functions. While histone PTMs have been correlated with certain gene expression status, the causalities remain elusive. Histone H3 Lysine 27 acetylation (H3K27ac) and histone H3 Lysine 4 trimethylation (H3K4me3) are both associated with active genes, and located at active promoters and enhancers or around transcriptional start sites (TSSs). Although crosstalk between histone lysine acetylation and H3K4me3 has been reported, relationships between specific epigenetic marks during transcriptional activation remain largely unclear. Here, using clustered regularly interspaced short palindromic repeats (CRISPR)/dCas-based epigenome editing methods, we discovered that the ectopic introduction of H3K27ac in the promoter region lead to H3K4me3 enrichment around TSS and transcriptional activation, while H3K4me3 installation at the promoter cannot induce H3K27ac increase and failed to activate gene expression. Blocking the reading of H3K27ac by BRD proteins using inhibitor JQ1 abolished H3K27ac-induced H3K4me3 installation and downstream gene activation. Furthermore, we uncovered that BRD2, not BRD4, mediated H3K4me3 installation and gene activation upon H3K27ac writing. Our studies revealed the relationships between H3K27ac and H3K4me3 in gene activation process and demonstrated the application of CRISPR/dCas-based epigenome editing methods in elucidating the crosstalk between epigenetic mechanisms.

Long non-coding RNAs (lncRNAs) are essential drivers or suppressors in human hepatocellular carcinoma (HCC) by participating in controlling transcription, translation, mRNA stability, and protein degradation protein-protein interaction. TM4SF1-AS1 is recently identified as a tumor-promoting factor in lung cancer. Nevertheless, its function in HCC and related molecular mechanisms remain unknown. Here, our data indicated that either hypoxia or hypoxia-inducible factor (HIF) prolyl hydroxylase inhibitor (DMOG) induced the upregulation of TM4SF1-AS1 in HCC cells. HIF-1α knockdown rather than HIF-2α silencing remarkably abrogated hypoxia-upregulated TM4SF1-AS1 expression. Furthermore, we confirmed the elevated expression of TM4SF1-AS1 in HCC tissue samples and cell lines. The silencing of TM4SF1-AS1 prominently inhibited the proliferative, migratory, and invasive abilities of HCC cells. TM4SF1-AS1 depletion significantly blocked hypoxia-enhanced Hep3B cell proliferation and mobility. Interfering TM4SF1-AS1 remarkably reduced TM4SF1 mRNA and protein levels in HCC cells. But TM4SF1-AS1 knockdown did not impact the stability of TM4SF1 mRNA. Hypoxia enhanced the expression of TM4SF1 mRNA, which was subsequently decreased by TM4SF1-AS1 knockdown in HCC cells. We confirmed the positive correlation between TM4SF1 mRNA and TM4SF1-AS1 expression in HCC specimens. Finally, TM4SF1 prominently reversed the inhibitory role of TM4SF1-AS1 depletion in Hep3B cells. In summary, hypoxia-responsive TM4SF1-AS1 was overexpressed in human HCC and contributed to the malignant behaviors of tumor cells by enhancing TM4SF1-AS1 expression.

Poor response of tumors to radiotherapy is a major clinical obstacle. Because of the dynamic characteristics of the epigenome, identification of possible epigenetic modifiers may be beneficial to confer radio-sensitivity. This research was set to examine the modulation of ectodermal-neural cortex 1 (ENC1) in radio-resistance in breast carcinoma (BC). In silico identification and immunohistochemical staining revealed that overexpression of ENC1 promoted BC metastasis to the bone and brain. Moreover, its overexpression promoted the translocation of YAP1/TAZ into the nucleus and enhanced expression of GLI1, CTGF, and FGF1 through the Hippo pathway. ENC1 expression was controlled by a ~10-kb long SE. ENC1-SEdistal deletion reduced ENC1 expression and inhibited the malignant behavior of BC cells and their resistance to radiotherapy. The binding sites on the ENC1-SE region enriched the shared sequence between TCF4 and ENC1 promoter. Knocking-down TCF4 inhibited luciferase activity and H3K27ac-enriched binding of the ENC1-SE region. Additionally, SE-driven ENC1 overexpression mediated by TCF4 may have clinical implications in radio-resistance in BC patients. Our findings indicated that ENC1 overexpression is mediated by SE and the downstream TCF4 to potentiate the Hippo/YAP1/TAZ pathway. Targeting this axis might be a therapeutic strategy for overcoming BC radio-resistance.

Uncontrolled proliferation is the hallmark of cancer cells. Previous studies mainly focused on the role of protein-coding genes in cancer cell proliferation. Emerging evidence showed that long non-coding RNAs (lncRNAs) also play critical roles in cancer cell proliferation and growth. LncRNA KCNQ1OT1 is found to contribute to carcinogenesis, but its role in acute promyelocytic leukemia (APL) is unclear. In this study, by analyzing data from Gene Expression Omnibus, The Cancer Genome Atlas database and our clinical samples, we found that KCNQ1OT1 was selectively highly expressed in APL. Functional assays demonstrated that knockdown of KCNQ1OT1 reduced APL cell proliferation and increased apoptosis. Further evidence showed that KCNQ1OT1 was mainly located in the cytoplasm of APL patient-derived NB4 cells and APL patient bone marrow samples. Mechanistically, KCNQ1OT1 bound to RNA binding protein FUS, and silencing either KCNQ1OT1 or FUS reduced the expression level and stability of MAP3K1 mRNA. Whereas KCNQ1OT1 and FUS did not affect each other. Importantly, knockdown of MAP3K1 impaired APL cell proliferation. Finally, c-Myc transactivated KCNQ1OT1 in APL cells through binding to its promoter while knockdown of c-Myc decreased KCNQ1OT1 expression. Our results not only revealed that c-Myc transactivated KCNQ1OT1 and upregulated KCNQ1OT1 promoted APL cell proliferation, but also demonstrated that KCNQ1OT1 bound to FUS to synergistically stabilize MAP3K1 mRNA, thus facilitating APL cell proliferation. This study established a previously unidentified role of KCNQ1OT1 in the development of APL, and KCNQ1OT1 may serve as a potential therapeutic target for APL.

Extreme hypoxia is among the most prominent pathogenic features of pancreatic cancer (PC). Both the long non-coding RNA (lncRNA) plasmacytoma variant translocation 1 (PVT1) and hypoxic inducible factor-1α (HIF-1α) are highly expressed in PC patients and play a crucial role in disease progression. Reciprocal regulation involving PVT1 and HIF-1α in PC, however, is poorly understood. Here, we report that PVT1 binds to the HIF-1α promoter and activates its transcription. In addition, we found that PVT1 could bind to HIF-1α and increases HIF-1α post-translationally. Our findings suggest that the PVT1‒HIF-1α positive feedback loop is a potential therapeutic target in the treatment of PC.

OBJECTIVE Systemic sclerosis (SSc) is characterized by widespread fibrosis and vascular complications. This study was undertaken to examine the chromatin landscape and transcription factor footprints in SSc, using an assay for genome-wide chromatin accessibility. METHODS Dermal endothelial cells (ECs) and fibroblasts were isolated from healthy controls and patients with diffuse cutaneous SSc (dcSSc). Assay for transposase-accessible chromatin with sequencing (ATAC-seq) was performed to assess genome-wide chromatin accessibility at a read depth of ~150 million reads per sample. Transcription factor footprinting and motif binding analysis were performed, followed by functional experiments. RESULTS Chromatin accessibility was significantly reduced in dcSSc patients compared to healthy controls. Differentially accessible chromatin loci were enriched in pathways and gene ontologies involved in the nervous system, cell membrane projections and cilia motility, nuclear and steroid receptors, and nitric oxide. In addition, chromatin binding of transcription factors SNAI2, ETV2, and ELF1 was significantly increased in dcSSc ECs, while recruitment of RUNX1 and RUNX2 was enriched in dcSSc fibroblasts. We found significant down-regulation of the neuronal gene NRXN1 and up-regulation of SNAI2 and ETV2 in dcSSc ECs. In dcSSc fibroblasts, down-regulation of the neuronal gene ENTPD1 and up-regulation of RUNX2 were confirmed. Further functional analysis revealed that ETV2 and NRXN1 dysregulation affected angiogenesis in ECs, while ENTPD1 enhanced profibrotic properties in dcSSc fibroblasts. CONCLUSION Our data identify the chromatin blueprint of dcSSc, and suggest that neuronal-related characteristics of SSc ECs and fibroblasts could be a culprit for dysregulated angiogenesis and enhanced fibrosis. Targeting the key pathways and transcription factors identified might present novel therapeutic approaches in SSc.

Meningiomas are the most common benign brain tumors. Mutations of the E3 ubiquitin ligase TRAF7 occur in 25% of meningiomas and commonly cooccur with mutations in KLF4, yet the functional link between TRAF7 and KLF4 mutations remains unclear. By generating an in vitro meningioma model derived from primary meningeal cells, we elucidated the cooperative interactions that promote meningioma development. By integrating TRAF7-driven ubiquitinome and proteome alterations in meningeal cells and the TRAF7 interactome, we identified TRAF7 as a proteostatic regulator of RAS-related small GTPases. Meningioma-associated TRAF7 mutations disrupted either its catalytic activity or its interaction with RAS GTPases. TRAF7 loss in meningeal cells altered actin dynamics and promoted anchorage-independent growth by inducing CDC42 and RAS signaling. TRAF deficiency–driven activation of the RAS/MAPK pathway promoted KLF4-dependent transcription that led to upregulation of the tumor-suppressive Semaphorin pathway, a negative regulator of small GTPases. KLF4 loss of function disrupted this negative feedback loop and enhanced mutant TRAF7-mediated cell transformation. Overall, this study provides new mechanistic insights into meningioma development, which could lead to novel treatment strategies. Significance: The intricate molecular cross-talk between the ubiquitin ligase TRAF7 and the transcription factor KLF4 provides a first step toward the identification of new therapies for patients with meningioma.

Biofilm and nitrogen fixation are two competitive strategies used by many plant-associated bacteria; however, the mechanisms underlying the formation of nitrogen-fixing biofilms remain largely unknown. Here, we examined the roles of multiple signalling systems in the regulation of biofilm formation by root-associated diazotrophic P. stutzeri A1501. Physiological analysis, construction of mutant strains and microscale thermophoresis experiments showed that RpoN is a regulatory hub coupling nitrogen fixation and biofilm formation by directly activating the transcription of pslA, a major gene involved in the synthesis of the Psl exopolysaccharide component of the biofilm matrix and nifA, the transcriptional activator of nif gene expression. Genetic complementation studies and determination of the copy number of transcripts by droplet digital PCR confirmed that the regulatory ncRNA RsmZ serves as a signal amplifier to trigger biofilm formation by sequestering the translational repressor protein RsmA away from pslA and sadC mRNAs, the latter of which encodes a diguanylate cyclase that synthesises c-di-GMP. Moreover, RpoS exerts a braking effect on biofilm formation by transcriptionally downregulating RsmZ expression, while RpoS expression is repressed posttranscriptionally by RsmA. These findings provide mechanistic insights into how the Rpo/Gac/Rsm regulatory networks fine-tune nitrogen-fixing biofilm formation in response to the availability of nutrients.

Background: Esophageal cancer is the sixth-most common fatal malignant tumor worldwide. Little is known regarding the genetic drivers that influence targeted therapy outcomes in patients with esophageal cancer. Exploring the pathogenesis of this lethal tumor could provide clues for developing appropriate therapeutic drugs. Ubiquitin-protein ligase E3A (UBE3A) reportedly promotes or suppresses various types of malignant tumors. However, the cancer-related role of UBE3A in esophageal cancer remains unclear. Methods: The relationship of UBE3A with the clinicopathological features of pancreatic tumors was bioinformatically investigated in the TCGA dataset. The protein levels of UBE3A and ZNF185 were assessed by Western blot and immunohistochemistry. The role of UBE3A and ZNF185 in esophageal cancer growth was assessed by MTS assays, colony formation assays, and experiments in mouse xenograft models. The interaction between UBE3A and ZNF185 was investigated by co-immunoprecipitation. The relationship between UBE3A, ZNF185, and NOTCH signaling pathway was explored by Western blot and quantitative real-time PCR. Results: We found that UBE3A was upregulated in patients with esophageal cancer and enhanced the cellular progression of esophageal cancer. Moreover, we found that UBE3A degraded ZNF185 in esophageal cancer. Additionally, ZNF185 suppressed the progression of esophageal cancer by inactivating the NOTCH pathway. Conclusions: These data demonstrated that aberrant expression of UBE3A led to enhanced progression of esophageal cancer through the ZNF185/NOTCH signaling axis. Therefore, UBE3A might be an ideal therapeutic candidate for esophageal cancer.

IL-1β and TNF-α are canonical immune response mediators that play key regulatory roles in a wide range of inflammatory responses to both chronic and acute conditions. Here we employ an automated microscopy platform for the analysis of messenger RNA (mRNA) expression of IL-1β and TNF-α at the single-cell level. The amount of IL-1β and TNF-α mRNA expressed in a human monocytic leukemia cell line (THP-1) is visualized and counted using single-molecule fluorescent in-situ hybridization (smFISH) following exposure of the cells to lipopolysaccharide (LPS), an outer-membrane component of Gram-negative bacteria. We show that the small molecule inhibitors MG132 (a 26S proteasome inhibitor used to block NF-κB signaling) and U0126 (a MAPK Kinase inhibitor used to block CCAAT-enhancer-binding proteins C/EBP) successfully block IL-1β and TNF-α mRNA expression. Based upon this single-cell mRNA expression data, we screened 36 different mathematical models of gene expression, and found two similar models that capture the effects by which the drugs U0126 and MG132 affect the rates at which the genes transition into highly activated states. When their parameters were informed by the action of each drug independently, both models were able to predict the effects of the combined drug treatment. From our data and models, we postulate that IL-1β is activated by both NF-κB and C/EBP, while TNF-α is predominantly activated by NF-κB. Our combined single-cell experimental and modeling efforts show the interconnection between these two genes and demonstrates how the single-cell responses, including the distribution shapes, mean expression, and kinetics of gene expression, change with inhibition.

Angiogenesis is considered as an important process in tumor growth, metastasis of hepatocellular carcinoma (HCC) and associated with cancer progression, suggesting that an important research and development field of clinical molecular targeted drugs for HCC. However, the molecular mechanisms underlying tumor angiogenesis in HCC remains elusive. In the current study, we demonstrate that upregulation of AMYB proto-oncogene-like 1 (MYBL1) was associated with high endothelial vessel (EV) density and contributed to poor prognosis of HCC patient. Functionally, MYBL1 overexpressing enhanced the capacity of HCC cells to induce tube formation, migration of HUVECs, neovascularization in CAMs, finally, enhanced HCC cells metastasis, while silencing MYBL1 had the converse effect. Furthermore, HCC cells with high MYBL1 expression were more resistance to sorafenib treatment. We observed that CD31 staining was significantly increased in tumors formed by MYBL1-overexpressing cells but decreased in MYBL1-silenced tumors. Mechanistically, MYBL1 binds to the ANGPT2 promoter and transcriptionally upregulate ANGPT2 mRNA expression. Strikingly, treatment with monoclonal antibody against ANGPT2 significantly inhibited the growth of MYBL1-overexpressing tumors and efficiently impaired angiogenesis. Furthermore, the histone post-translational factors: protein arginine methyltransferase 5 (PRMT5), MEP50, and WDR5 were required for MYBL1-mediated ANGPT2 upregulation. Importantly, we confirmed the correlation between MYBL1 and ANGPT2 expression in a large cohort of clinical HCC samples and several published datasets in pancreatic cancer, esophageal carcinoma, stomach adenocarcinoma, and colon cancer. Our results demonstrate that MYBL1 upregulated the ANGPT2 expression, then induced angiogenesis and confer sorafenib resistance to HCC cells, and MYBL1 may represent a novel prognostic biomarker and therapeutic target for patients with HCC.

The ribosomal protein uL11 is located at the basis of the ribosome P-stalk and plays a paramount role in translational efficiency. In addition, no mutant for uL11 is available suggesting that this gene is haplo-insufficient as many other Ribosomal Protein Genes (RPGs). We have previously shown that overexpression of Drosophila melanogaster uL11 enhances the transcription of many RPGs and Ribosomal Biogenesis genes (RiBis) suggesting that uL11 might globally regulate the level of translation through its transcriptional activity. Moreover, uL11 trimethylated on lysine 3 (uL11K3me3) interacts with the chromodomain of the Enhancer of Polycomb and Trithorax Corto, and both proteins co- localize with RNA Polymerase II at many sites on polytene chromosomes. These data have led to the hypothesis that the N-terminal end of uL11, and more particularly the trimethylation of lysine 3, supports the extra-ribosomal activity of uL11 in transcription. To address this question, we mutated the lysine 3 codon using a CRISPR/Cas9 strategy and obtained several lysine 3 mutants. We describe here the first mutants of D. melanogaster uL11. Unexpectedly, the uL11K3A mutant, in which the lysine 3 codon is replaced by an alanine, displays a genuine Minute phenotype known to be characteristic of RPG deletions (longer development, low fertility, high lethality, thin and short bristles) whereas the uL11K3Y mutant, in which the lysine 3 codon is replaced by a tyrosine, is unaffected. In agreement, the rate of translation decreases in uL11K3A but not in uL11K3Y. Co-immunoprecipitation experiments show that the interaction between uL11 and the Corto chromodomain is impaired by both mutations. RNA- seq analysis from wing imaginal discs shows enrichment in the GO categories “glutathione metabolism” for up-regulated genes in both uL11K3A and uL11K3Y mutants and “regulation of transcription” for down-regulated genes in uL11K3A only. Analysis of the cis-regulatory sequences of these genes suggests that uL11 might regulate transcription of target genes in concert with the couple of transcription factors Mad/Med that mediate response to the Bone Morphogenetic Protein (BMP) signaling pathway.