Showing papers on "Psychological repression published in 2016"
TL;DR: This study shows that direct binding by CCA1 in the morning provides strong repression of PRR5, and repression by C CA1 also temporally regulates an evening-expressed gene set that includesPRR5.
Abstract: The circadian clock is a biological timekeeping system that provides organisms with the ability to adapt to day-night cycles. Timing of the expression of four members of the Arabidopsis thaliana PSEUDO-RESPONSE REGULATOR (PRR) family is crucial for proper clock function, and transcriptional control of PRRs remains incompletely defined. Here, we demonstrate that direct regulation of PRR5 by CIRCADIAN CLOCK-ASSOCIATED1 (CCA1) determines the repression state of PRR5 in the morning. Chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) analyses indicated that CCA1 associates with three separate regions upstream of PRR5. CCA1 and its homolog LATE ELONGATED HYPOCOTYL (LHY) suppressed PRR5 promoter activity in a transient assay. The regions bound by CCA1 in the PRR5 promoter gave rhythmic patterns with troughs in the morning, when CCA1 and LHY are at high levels. Furthermore, ChIP-seq revealed that CCA1 associates with at least 449 loci with 863 adjacent genes. Importantly, this gene set contains genes that are repressed but upregulated in cca1 lhy double mutants in the morning. This study shows that direct binding by CCA1 in the morning provides strong repression of PRR5, and repression by CCA1 also temporally regulates an evening-expressed gene set that includes PRR5.
191 citations
TL;DR: It is found that YB-1 directly binds to tiRNAs via its cold shock domain, which is required for packaging of tiRNA-repressed mRNAs into SGs but is dispensable for ti RNA-mediated translational repression.
Abstract: Stress-induced angiogenin (ANG)-mediated tRNA cleavage promotes a cascade of cellular events that starts with production of tRNA-derived stress-induced RNAs (tiRNAs) and culminates with enhanced cell survival. This stress response program relies on a subset tiRNAs that inhibit translation initiation and induce the assembly of stress granules (SGs), cytoplasmic ribonucleoprotein complexes with cytoprotective and pro-survival properties. SG-promoting tiRNAs bear oligoguanine motifs at their 5'-ends, assemble G-quadruplex-like structures and interact with the translational silencer YB-1. We used CRISPR/Cas9-based genetic manipulations and biochemical approaches to examine the role of YB-1 in tiRNA-mediated translational repression and SG assembly. We found that YB-1 directly binds to tiRNAs via its cold shock domain. This interaction is required for packaging of tiRNA-repressed mRNAs into SGs but is dispensable for tiRNA-mediated translational repression. Our studies reveal the functional role of YB-1 in the ANG-mediated stress response program.
172 citations
TL;DR: It is shown that three muscle-specific NETs, NET39, Tmem38A, and WFS1, direct specific myogenic genes to the nuclear periphery to facilitate their repression, unequivocally demonstrating that NET-directed gene repositioning is critical for developmental gene regulation.
155 citations
TL;DR: The results raise the possibilities that pathophysiology-induced activation of specific transcription factor may lead to increased expression of miR-34a gene and miR -34a mediated concurrent repression of its target genes in neural networks may result in dysfunction of synaptic plasticity, energy metabolism and resting state network activity.
126 citations
TL;DR: It is shown that GAPDH–TNF mRNA binding regulates expression of TNF based on cellular metabolic state, and this mechanism has potentially significant implications for treatment of various immunometabolic conditions, including immune paralysis during septic shock.
Abstract: Expression of the inflammatory cytokine TNF is tightly controlled. During endotoxin tolerance, transcription of TNF mRNA is repressed, although not entirely eliminated. Production of TNF cytokine, however, is further controlled by posttranscriptional regulation. In this study, we detail a mechanism of posttranscriptional repression of TNF mRNA by GAPDH binding to the TNF 3' untranslated region. Using RNA immunoprecipitation, we demonstrate that GAPDH-TNF mRNA binding increases when THP-1 monocytes are in a low glycolysis state, and that this binding can be reversed by knocking down GAPDH expression or by increasing glycolysis. We show that reducing glycolysis decreases TNF mRNA association with polysomes. We demonstrate that GAPDH-TNF mRNA binding results in posttranscriptional repression of TNF and that the TNF mRNA 3' untranslated region is sufficient for repression. Finally, after exploring this model in THP-1 cells, we demonstrate this mechanism affects TNF expression in primary human monocytes and macrophages. We conclude that GAPDH-TNF mRNA binding regulates expression of TNF based on cellular metabolic state. We think this mechanism has potentially significant implications for treatment of various immunometabolic conditions, including immune paralysis during septic shock.
103 citations
TL;DR: It is concluded that Kr-h1 protein molecules directly bind to the KBS sequence in the BR-C promoter and thereby repress 20E-dependent activation of the pupal specifier,BR-C.
100 citations
TL;DR: It is shown that the distance between an mRNA and overlapping lncRNA promoter determines whether Set2–Rpd3S or Set3C represses, and many previously unreported cryptic ncRNAs induced by specific carbon sources are revealed, showing that cryptic promoters can be environmentally regulated.
Abstract: H3K36 methylation by Set2 targets Rpd3S histone deacetylase to transcribed regions of mRNA genes, repressing internal cryptic promoters and slowing elongation. Here we explore the function of this pathway by analysing transcription in yeast undergoing a series of carbon source shifts. Approximately 80 mRNA genes show increased induction upon SET2 deletion. A majority of these promoters have overlapping lncRNA transcription that targets H3K36me3 and deacetylation by Rpd3S to the mRNA promoter. We previously reported a similar mechanism for H3K4me2-mediated repression via recruitment of the Set3C histone deacetylase. Here we show that the distance between an mRNA and overlapping lncRNA promoter determines whether Set2–Rpd3S or Set3C represses. This analysis also reveals many previously unreported cryptic ncRNAs induced by specific carbon sources, showing that cryptic promoters can be environmentally regulated. Therefore, in addition to repression of cryptic transcription and modulation of elongation, H3K36 methylation maintains optimal expression dynamics of many mRNAs and ncRNAs. H3K36 methylation by Set2 targets Rpd3S histone deacetylase to transcribed mRNA genes, repressing internal cryptic promoters and modulating elongation. Here, the authors provide evidence that the Set2-Rpd3S pathway also regulates dynamic expression of mRNAs and lncRNAs.
90 citations
TL;DR: The mechanism of translational repression by 4E-T is addressed by first identifying and delineating the interacting sites of its major partners by mass spectrometry and western blotting, including DDX6, UNR, unrip, PAT1B, LSM14A and CNOT4.
Abstract: 4E-Transporter binds eIF4E via its consensus sequence YXXXXLΦ, shared with eIF4G, and is a nucleocytoplasmic shuttling protein found enriched in P-(rocessing) bodies. 4E-T inhibits general protein synthesis by reducing available eIF4E levels. Recently, we showed that 4E-T bound to mRNA however represses its translation in an eIF4E-independent manner, and contributes to silencing of mRNAs targeted by miRNAs. Here, we address further the mechanism of translational repression by 4E-T by first identifying and delineating the interacting sites of its major partners by mass spectrometry and western blotting, including DDX6, UNR, unrip, PAT1B, LSM14A and CNOT4. Furthermore, we document novel binding between 4E-T partners including UNR-CNOT4 and unrip-LSM14A, altogether suggesting 4E-T nucleates a complex network of RNA-binding protein interactions. In functional assays, we demonstrate that joint deletion of two short conserved motifs that bind UNR and DDX6 relieves repression of 4E-T-bound mRNA, in part reliant on the 4E-T-DDX6-CNOT1 axis. We also show that the DDX6-4E-T interaction mediates miRNA-dependent translational repression and de novo P-body assembly, implying that translational repression and formation of new P-bodies are coupled processes. Altogether these findings considerably extend our understanding of the role of 4E-T in gene regulation, important in development and neurogenesis.
90 citations
TL;DR: A critical role for NF-κB-dependent upregulation of miR-181a is supported, which represents a new pathway for the repression of PTEN and the promotion of cell proliferation upon IL-1β induction.
87 citations
TL;DR: HuR might act as a ‘miRNA sponge‘ to regulate miRNA-mediated translation regulation under conditions of stress-induced nuclear-cytoplasmic translocation of HuR, which would allow fine-tuned gene expression in complex regulatory environments.
Abstract: Translation control of proinflammatory genes has a crucial role in regulating the inflammatory response and preventing chronic inflammation, including a transition to cancer. The proinflammatory tumor suppressor protein programmed cell death 4 (PDCD4) is important for maintaining the balance between inflammation and tumorigenesis. PDCD4 messenger RNA translation is inhibited by the oncogenic microRNA, miR-21. AU-rich element-binding protein HuR was found to interact with the PDCD4 3′-untranslated region (UTR) and prevent miR-21-mediated repression of PDCD4 translation. Cells stably expressing miR-21 showed higher proliferation and reduced apoptosis, which was reversed by HuR expression. Inflammatory stimulus caused nuclear-cytoplasmic relocalization of HuR, reversing the translation repression of PDCD4. Unprecedentedly, HuR was also found to bind to miR-21 directly, preventing its interaction with the PDCD4 3′-UTR, thereby preventing the translation repression of PDCD4. This suggests that HuR might act as a ‘miRNA sponge‘ to regulate miRNA-mediated translation regulation under conditions of stress-induced nuclear-cytoplasmic translocation of HuR, which would allow fine-tuned gene expression in complex regulatory environments.
84 citations
TL;DR: It is reported that miR-552 is in the nucleus and cytosol and inhibits human cytochrome P450 (CYP) 2E1 expression at both transcriptional and post-transcriptional levels.
TL;DR: The present study unveils the mechanisms that underlie the GC-induced GR direct transrepression function mediated by the evolutionary conserved inverted repeated negative response element, and demonstrates that HDAC3 is instrumental in IR nGRE-mediated repression.
Abstract: Unique among the nuclear receptor superfamily, the glucocorticoid (GC) receptor (GR) can exert three distinct transcriptional regulatory functions on binding of a single natural (cortisol in human and corticosterone in mice) and synthetic [e.g., dexamethasone (Dex)] hormone. The molecular mechanisms underlying GC-induced positive GC response element [(+)GRE]-mediated activation of transcription are partially understood. In contrast, these mechanisms remain elusive for GC-induced evolutionary conserved inverted repeated negative GC response element (IR nGRE)-mediated direct transrepression and for tethered indirect transrepression that is mediated by DNA-bound NF-κB/activator protein 1 (AP1)/STAT3 activators and instrumental in GC-induced anti-inflammatory activity. We demonstrate here that SUMOylation of lysine K293 (mouse K310) located within an evolutionary conserved sequence in the human GR N-terminal domain allows the formation of a GR-small ubiquitin-related modifiers (SUMOs)-NCoR1/SMRT-HDAC3 repressing complex mandatory for GC-induced IR nGRE-mediated direct repression in vitro, but does not affect transactivation. Importantly, these results were validated in vivo: in K310R mutant mice and in mice ablated selectively for nuclear receptor corepressor 1 (NCoR1)/silencing mediator for retinoid or thyroid-hormone receptors (SMRT) corepressors in skin keratinocytes, Dex-induced direct repression and the formation of repressing complexes on IR nGREs were impaired, whereas transactivation was unaffected. In mice selectively ablated for histone deacetylase 3 (HDAC3) in skin keratinocytes, GC-induced direct repression, but not bindings of GR and of corepressors NCoR1/SMRT, was abolished, indicating that HDAC3 is instrumental in IR nGRE-mediated repression. Moreover, we demonstrate that the binding of HDAC3 to IR nGREs in vivo is mediated through interaction with SMRT/NCoR1. We also show that the GR ligand binding domain (LBD) is not required for SMRT-mediated repression, which can be mediated by a LBD-truncated GR, whereas it is mandatory for NCoR1-mediated repression through an interaction with K579 in the LBD.
TL;DR: This work highlights the function of the conserved tetraproline motifs of TTP and identifies 4EHP-GYF2 as a cofactor in translational repression and mRNA decay by TTP.
Abstract: The zinc finger protein tristetraprolin (TTP) promotes translation repression and degradation of mRNAs containing AU-rich elements (AREs). Although much attention has been directed toward understanding the decay process and machinery involved, the translation repression role of TTP has remained poorly understood. Here we identify the cap-binding translation repression 4EHP-GYF2 complex as a cofactor of TTP. Immunoprecipitation and in vitro pull-down assays demonstrate that TTP associates with the 4EHP-GYF2 complex via direct interaction with GYF2, and mutational analyses show that this interaction occurs via conserved tetraproline motifs of TTP. Mutant TTP with diminished 4EHP-GYF2 binding is impaired in its ability to repress a luciferase reporter ARE-mRNA. 4EHP knockout mouse embryonic fibroblasts (MEFs) display increased induction and slower turnover of TTP-target mRNAs as compared to wild-type MEFs. Our work highlights the function of the conserved tetraproline motifs of TTP and identifies 4EHP-GYF2 as a cofactor in translational repression and mRNA decay by TTP.
TL;DR: In castration-resistant PCa cells, which are dependent on high-level AR expression, this anti-proliferative repression function might be exploited through treatment with androgen in combination with agents that suppress AR-driven metabolic functions or cell cycle progression.
TL;DR: In this article, the authors have generated isogenic mouse embryonic stem cells (ESCs) with a modified H3K27me2/H3k27me3 ratio.
TL;DR: The findings reveal a new mechanism by which SOX2-mediated transcription repression of TIF1γ promotes TGF-β-induced EMT in NSCLC.
Abstract: Repression of TIF1γ by SOX2 promotes TGF-β-induced epithelial–mesenchymal transition in non-small-cell lung cancer
TL;DR: The author discusses how a new class of models with protein sequestration-based repression differs dramatically from those based on Hill-type repression in several fundamental aspects: conditions for rhythm generation, robust network designs and the periods of coupled oscillators.
Abstract: Circadian (∼24 h) clocks are self-sustained endogenous oscillators with which organisms keep track of daily and seasonal time. Circadian clocks frequently rely on interlocked transcriptional-translational feedback loops to generate rhythms that are robust against intrinsic and extrinsic perturbations. To investigate the dynamics and mechanisms of the intracellular feedback loops in circadian clocks, a number of mathematical models have been developed. The majority of the models use Hill functions to describe transcriptional repression in a way that is similar to the Goodwin model. Recently, a new class of models with protein sequestration-based repression has been introduced. Here, the author discusses how this new class of models differs dramatically from those based on Hill-type repression in several fundamental aspects: conditions for rhythm generation, robust network designs and the periods of coupled oscillators. Consistently, these fundamental properties of circadian clocks also differ among Neurospora, Drosophila, and mammals depending on their key transcriptional repression mechanisms (Hill-type repression or protein sequestration). Based on both theoretical and experimental studies, this review highlights the importance of careful modelling of transcriptional repression mechanisms in molecular circadian clocks.
TL;DR: Reverse genetics revealed that val1 mutant seeds accumulated elevated levels of proteins compared with the wild type, suggesting that VAL1 functions as a repressor of seed metabolism; however, in the absence of VAL1, the levels of metabolites, ABA, auxin and jasmonate derivatives did not change significantly in developing embryos.
Abstract: Summary
Developing Arabidopsis seeds accumulate oils and seed storage proteins synthesized by the pathways of primary metabolism. Seed development and metabolism are positively regulated by transcription factors belonging to the LAFL (LEC1, AB13, FUSCA3 and LEC2) regulatory network. The VAL gene family encodes repressors of the seed maturation program in germinating seeds, although they are also expressed during seed maturation. The possible regulatory role of VAL1 in seed development has not been studied to date. Reverse genetics revealed that val1 mutant seeds accumulated elevated levels of proteins compared with the wild type, suggesting that VAL1 functions as a repressor of seed metabolism; however, in the absence of VAL1, the levels of metabolites, ABA, auxin and jasmonate derivatives did not change significantly in developing embryos. Two VAL1 splice variants were identified through RNA sequencing analysis: a full-length form and a truncated form lacking the plant homeodomain-like domain associated with epigenetic repression. None of the transcripts encoding the core LAFL network transcription factors were affected in val1 embryos. Instead, activation of VAL1 by FUSCA3 appears to result in the repression of a subset of seed maturation genes downstream of core LAFL regulators, as 39% of transcripts in the FUSCA3 regulon were derepressed in the val1 mutant. The LEC1 and LEC2 regulons also responded, but to a lesser extent. Additional 832 transcripts that were not LAFL targets were derepressed in val1 mutant embryos. These transcripts are candidate targets of VAL1, acting through epigenetic and/or transcriptional repression.
TL;DR: The authors showed that WRKY45 and WRKY62 positively regulate defense genes, including diterpenoid phytoalexin biosynthetic genes and their transcriptional regulator DPF.
Abstract: WRKY62 is a transcriptional repressor regulated downstream of WRKY45, a central transcription factor of the salicylic acid signaling pathway in rice. Previously, WRKY62 was reported to regulate defense negatively. However, our expressional analysis using WRKY62-knockdown rice indicated that WRKY62 positively regulates defense genes, including diterpenoid phytoalexin biosynthetic genes and their transcriptional regulator DPF. Blast and leaf blight resistance tests also showed that WRKY62 is a positive defense regulator. Yeast two-hybrid, co-immunoprecipitation and gel-shift assays showed that WRKY45 and WRKY62 can form a heterodimer, as well as homodimers, that bind to W-boxes in the DPF promoter. In transient assays in rice sheaths, the simultaneous introduction of WRKY45 and WRKY62 as effectors resulted in a strong activation of the DPF promoter:hrLUC reporter gene, whereas the activity declined with excessive WRKY62. Thus, the WRKY45-WRKY62 heterodimer acts as a strong activator, while the WRKY62 homodimer acts as a repressor. While benzothiadiazole induced equivalent numbers of WRKY45 and WRKY62 transcripts, consistent with heterodimer formation and DPF activation, submergence and nitrogen replacement induced only WRKY62 transcripts, consistent with WRKY62 homodimer formation and DPF repression. Moreover, WRKY62 positively regulated hypoxia genes, implying a role forWRKY62 in the modulation of the 'trade-off' between defense and hypoxia responses.
TL;DR: Cell-type-specific exploitation of RUNX gene super-enhancers by multiple EBV TFs via the Notch pathway to fine tune RUNX3 and RUNX1 expression and manipulate B-cell growth is revealed.
Abstract: In B cells infected by the cancer-associated Epstein-Barr virus (EBV), RUNX3 and RUNX1 transcription is manipulated to control cell growth. The EBV-encoded EBNA2 transcription factor (TF) activates RUNX3 transcription leading to RUNX3-mediated repression of the RUNX1 promoter and the relief of RUNX1-directed growth repression. We show that EBNA2 activates RUNX3 through a specific element within a −97 kb super-enhancer in a manner dependent on the expression of the Notch DNA-binding partner RBP-J. We also reveal that the EBV TFs EBNA3B and EBNA3C contribute to RUNX3 activation in EBV-infected cells by targeting the same element. Uncovering a counter-regulatory feed-forward step, we demonstrate EBNA2 activation of a RUNX1 super-enhancer (−139 to −250 kb) that results in low-level RUNX1 expression in cells refractory to RUNX1-mediated growth inhibition. EBNA2 activation of the RUNX1 super-enhancer is also dependent on RBP-J. Consistent with the context-dependent roles of EBNA3B and EBNA3C as activators or repressors, we find that these proteins negatively regulate the RUNX1 super-enhancer, curbing EBNA2 activation. Taken together our results reveal cell-type-specific exploitation of RUNX gene super-enhancers by multiple EBV TFs via the Notch pathway to fine tune RUNX3 and RUNX1 expression and manipulate B-cell growth.
TL;DR: A pathway by which RAS and PIK3CA oncogenes induce EMT through regulation of ∆Np63α is suggested, which suggests that targeting specific genes within this Ras-to-EMT axis may be useful as a therapy to block breast cancer progression.
Abstract: The p53-related transcription factor p63 is required for maintenance of epithelial cell differentiation. We found that activated forms of the Harvey Rat Sarcoma Virus GTPase (H-RAS) and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) oncogenes strongly repress expression of ∆Np63α, the predominant p63 isoform in basal mammary epithelial cells. This regulation occurs at the transcriptional level, and a short region of the ∆Np63 promoter is sufficient for repression induced by H-RasV12. The suppression of ∆Np63α expression by these oncogenes concomitantly leads to an epithelial-to-mesenchymal transition (EMT). In addition, the depletion of ∆Np63α alone is sufficient to induce EMT. Both H-RasV12 expression and ∆Np63α depletion induce individual cell invasion in a 3D collagen gel in vitro system, thereby demonstrating how Ras can drive the mammary epithelial cell state toward greater invasive ability. Together, these results suggest a pathway by which RAS and PIK3CA oncogenes induce EMT through regulation of ∆Np63α.
TL;DR: Four new genes are defined as direct targets of E. coli SgrS, which encode transcription factors or enzymes of diverse metabolic pathways, including aspartate semialdehyde dehydrogenase, arginine decarboxylase gene activator, GTP cyclohydrolase I and a repressor of purine biosynthesis, respectively.
Abstract: The Escherichia coli small RNA SgrS controls a metabolic stress response that occurs upon accumulation of certain glycolytic intermediates. SgrS base pairs with and represses translation of ptsG and manXYZ mRNAs, which encode sugar transporters, and activates translation of yigL mRNA, encoding a sugar phosphatase. This study defines four new genes as direct targets of E. coli SgrS. These new targets, asd, adiY, folE and purR, encode transcription factors or enzymes of diverse metabolic pathways, including aspartate semialdehyde dehydrogenase, arginine decarboxylase gene activator, GTP cyclohydrolase I and a repressor of purine biosynthesis, respectively. SgrS represses translation of each of the four target mRNAs via distinct mechanisms. SgrS binding sites overlapping the Shine-Dalgarno sequences of adiY and folE mRNAs suggest that SgrS pairing with these targets directly occludes ribosome binding and prevents translation initiation. SgrS binding within the purR coding sequence recruits the RNA chaperone Hfq to directly repress purR translation. Two separate SgrS binding sites were found on asd mRNA, and both are required for full translational repression. Ectopic overexpression of asd, adiY and folE is specifically detrimental to cells experiencing glucose-phosphate stress, suggesting that SgrS-dependent repression of the metabolic functions encoded by these targets promotes recovery from glucose-phosphate stress.
TL;DR: MiR-124, a novel tumor suppressor miRNA that is epigenetically silenced in EC, can reverse EMT and the invasive properties, by attenuating the expression of the IQGAP1 oncogene.
Abstract: Overexpression of IQGAP1 and microRNA (miRNA) dysregulation are frequent in human tumors, but little is known about the role of IQGAP1 and its relationship to miRNA in endometrial carcinogenesis. We demonstrate that IQGAP1 activates the epithelial–mesenchymal transition (EMT) program and that miR-124 directly represses IQGAP1 expression in endometrial cancer (EC) cells. The overexpression of IQGAP1 stimulates EMT features and enhances migration, invasion and proliferation of EC cells, whereas knocking down IQGAP1 expression reverses EMT and inhibits these malignant properties. Using miRNA microarray profiling, we identified 29 miRNAs (let-7b, let-7f, miR-10b, miR-15b, miR-23a, miR-24, miR-25, miR-27a, miR-29b, miR-30a-5p, miR-34a, miR-124, miR-127, miR-130b, miR-148a, miR-155, miR-191*, miR-194, miR-224, miR-362, miR-409-3p, miR-422b, miR-424, miR-453, miR-497, miR-518d, miR-518f*, miR-526a and miR-656) that are significantly down-regulated in an in vitro-selected highly invasive derivative cell line (HEC-50-HI) relative to the parental HEC-50 cells. We further identified miR-124 as a direct regulator of IQGAP1 in EC cells. Enforced expression of miR-124 suppresses EC cell invasion and proliferation. The expression of IQGAP1 mRNA was significantly elevated in EC tissues, while the expression of miR-124 was decreased. The downregulation of miR-124 correlates with a poor survival outcome for patients with EC. Treating EC cells with the demethylating agent 5-aza-2′-deoxycytidine increased miR-124 expression and down-regulated IQGAP1 levels. Our data suggest that IQGAP1 promotes EMT, migration and invasion of EC cells. MiR-124, a novel tumor suppressor miRNA that is epigenetically silenced in EC, can reverse EMT and the invasive properties, by attenuating the expression of the IQGAP1 oncogene.
TL;DR: This work finds that binding site selection plays a key role in NF-κΒ-mediated transcriptional activation and repression, and highlights the involvement of topology in cis-regulatory element function during acute transcriptional responses, where primary DNA sequence and its higher-order structure constitute a regulatory context leading to either gene activation or repression.
Abstract: Mammalian cells have developed intricate mechanisms to interpret, integrate, and respond to extracellular stimuli. For example, tumor necrosis factor (TNF) rapidly activates proinflammatory genes, but our understanding of how this occurs against the ongoing transcriptional program of the cell is far from complete. Here, we monitor the early phase of this cascade at high spatiotemporal resolution in TNF-stimulated human endothelial cells. NF-κB, the transcription factor complex driving the response, interferes with the regulatory machinery by binding active enhancers already in interaction with gene promoters. Notably, >50% of these enhancers do not encode canonical NF-κB binding motifs. Using a combination of genomics tools, we find that binding site selection plays a key role in NF-κB-mediated transcriptional activation and repression. We demonstrate the latter by describing the synergy between NF-κB and the corepressor JDP2. Finally, detailed analysis of a 2.8-Mbp locus using sub-kbp-resolution targeted chromatin conformation capture and genome editing uncovers how NF-κB that has just entered the nucleus exploits pre-existing chromatin looping to exert its multimodal role. This work highlights the involvement of topology in cis-regulatory element function during acute transcriptional responses, where primary DNA sequence and its higher-order structure constitute a regulatory context leading to either gene activation or repression.
TL;DR: It is demonstrated that miR-486-5p acts as a tumor suppressor in HCC through the repression of essential members of the IGF-axis, including IGF-1R and its downstream mediators mTOR, STAT3 and c-Myc.
Abstract: The insulin-like growth factor (IGF)-axis has been paradigmatically involved in hepatocellular carcinoma (HCC) tumor initiation, progression and drug resistance. Consequently, members of the IGF-axis and most importantly, IGF-1 receptor (IGF-1R) have been considered as intriguing targets for HCC therapy. Few miRNAs have been recently reported to be associated with IGF-1R regulation. The present study aimed to investigate the role of microRNA (miRNA/miR)-486-5p in the regulation of IGF-1R and its downstream signaling cascades. miR-486-5p was markedly downregulated in hepatitis C virus-induced HCC tissues and Huh-7 cells. Forcing the expression of miR-486-5p in Huh-7 cells resulted in the repression of IGF-1R, mammalian target of rapamycin (mTOR), signal transducer and activator of transcription 3 (STAT3) and c-Myc mRNA levels. Ectopic expression of miR-486-5p in Huh-7 cells markedly repressed cellular viability, proliferation, migration and clonogenicity in a similar pattern to IGF-1R small interfering RNAs, and were evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, BrdU incorporation, wound healing and colony forming assays, respectively. Overall, the study findings demonstrated that miR-486-5p acts as a tumor suppressor in HCC through the repression of essential members of the IGF-axis, including IGF-1R and its downstream mediators mTOR, STAT3 and c-Myc.
TL;DR: The growth assay on starch agar plates showed qualitatively significant repression of amylase activity in C. beijerinckii transformant with CRISPR-dCas9 compared to the control strain.
Abstract: CRISPR-Cas9 has been explored as a powerful tool for genome engineering for many organisms. Meanwhile, dCas9 which lacks endonuclease activity but can still bind to target loci has been engineered for efficient gene transcription repression. Clostridium beijerinckii, an industrially significant species capable of biosolvent production, is generally difficult to metabolically engineer. Recently, we reported our work in developing customized CRISPR-Cas9 system for genome engineering in C. beijerinckii. However, in many cases, gene expression repression (rather than actual DNA mutation) is more desirable for various biotechnological applications. Here, we further demonstrated gene transcription repression in C. beijerinckii using CRISPR-dCas9. A small RNA promoter was employed to drive the expression of the single chimeric guide RNA targeting on the promoter region of amylase gene, while a constitutive thiolase promoter was used to drive Streptococcus pyogenes dCas9 expression. The growth assay on starch agar plates showed qualitatively significant repression of amylase activity in C. beijerinckii transformant with CRISPR-dCas9 compared to the control strain. Further amylase activity quantification demonstrated consistent repression (65-97% through the fermentation process) on the activity in the transformant with CRISPR-dCas9 versus in the control. Our results provided essential references for engineering CRISPR-dCas9 as an effective tool for tunable gene transcription repression in diverse microorganisms. Biotechnol. Bioeng. 2016;113: 2739-2743. © 2016 Wiley Periodicals, Inc.
TL;DR: Host-adapted ZF-AAV constructs displayed a reduced toxicity and a non-viral pNSE promoter improved long-term ZF protein expression and target gene repression, and have potential for treating Huntington’s disease.
Abstract: Synthetic zinc finger (ZF) proteins can be targeted to desired DNA sequences and are useful tools for gene therapy. We recently developed a ZF transcription repressor (ZF-KOX1) able to bind to expanded DNA CAG-repeats in the huntingtin (HTT) gene, which are found in Huntington’s disease (HD). This ZF acutely repressed mutant HTT expression in a mouse model of HD and delayed neurological symptoms (clasping) for up to 3 weeks. In the present work, we sought to develop a long-term single-injection gene therapy approach in the brain. Since non-self proteins can elicit immune and inflammatory responses, we designed a host-matched analogue of ZF-KOX1 (called mZF-KRAB), to treat mice more safely in combination with rAAV vector delivery. We also tested a neuron-specific enolase promoter (pNSE), which has been reported as enabling long-term transgene expression, to see whether HTT repression could be observed for up to 6 months after AAV injection in the brain. After rAAV vector delivery, we found that non-self proteins induce significant inflammatory responses in the brain, in agreement with previous studies. Specifically, microglial cells were activated at 4 and 6 weeks after treatment with non-host-matched ZF-KOX1 or GFP, respectively, and this was accompanied by a moderate neuronal loss. In contrast, the host-matched mZF-KRAB did not provoke these effects. Nonetheless, we found that using a pCAG promoter (CMV early enhancer element and the chicken β-actin promoter) led to a strong reduction in ZF expression by 6 weeks after injection. We therefore tested a new non-viral promoter to see whether the host-adapted ZF expression could be sustained for a longer time. Vectorising mZF-KRAB with a promoter-enhancer from neuron-specific enolase (Eno2, rat) resulted in up to 77 % repression of mutant HTT in whole brain, 3 weeks after bilateral intraventricular injection of 1010 virions. Importantly, repressions of 48 % and 23 % were still detected after 12 and 24 weeks, respectively, indicating that longer term effects are possible. Host-adapted ZF-AAV constructs displayed a reduced toxicity and a non-viral pNSE promoter improved long-term ZF protein expression and target gene repression. The optimized constructs presented here have potential for treating HD.
TL;DR: It is shown that a highly conserved Distal-less regulatory element (DCRE) that controls gene expression in leg precursor cells recruits multiple Hox, Extradenticle and Homothorax complexes to mediate dual outputs: thoracic activation and abdominal repression.
Abstract: cis-regulatory modules (CRMs) generate precise expression patterns by integrating numerous transcription factors (TFs). Surprisingly, CRMs that control essential gene patterns can differ greatly in conservation, suggesting distinct constraints on TF binding sites. Here, we show that a highly conserved Distal-less regulatory element (DCRE) that controls gene expression in leg precursor cells recruits multiple Hox, Extradenticle (Exd) and Homothorax (Hth) complexes to mediate dual outputs: thoracic activation and abdominal repression. Using reporter assays, we found that abdominal repression is particularly robust, as neither individual binding site mutations nor a DNA binding deficient Hth protein abolished cooperative DNA binding and in vivo repression. Moreover, a re-engineered DCRE containing a distinct configuration of Hox, Exd, and Hth sites also mediated abdominal Hox repression. However, the re-engineered DCRE failed to perform additional segment-specific functions such as thoracic activation. These findings are consistent with two emerging concepts in gene regulation: First, the abdominal Hox/Exd/Hth factors utilize protein-protein and protein-DNA interactions to form repression complexes on flexible combinations of sites, consistent with the TF collective model of CRM organization. Second, the conserved DCRE mediates multiple cell-type specific outputs, consistent with recent findings that pleiotropic CRMs are associated with conserved TF binding and added evolutionary constraints.
TL;DR: A protective role of miR-214 signaling in erythroid cells against oxidative stress, and also shed new light on Nrf2-mediated cytoprotective machinery are revealed.
TL;DR: The results together suggest that the repression of many developmental genes may involve both DRM2-mediated non-CG methylation and PRC2- mediated H3K27me3 and that the two marks are not generally mutually exclusive but may cooperate in repression of developmentally regulated genes in rice.
Abstract: H3K27me3 is a repressive chromatin mark of genes and is catalyzed by homologs of Enhancer of zeste [E(z)], a component of Polycomb-repressive complex 2 (PRC2), while DNA methylation that occurs in CG and non-CG (CHG and CHH, where H is A, C, or T) contexts is a hallmark of transposon silencing in plants. However, the relationship between H3K27me3 and DNA methylation in gene repression remains unclear. In addition, the mechanism of PRC2 recruitment to specific genes is not known in plants. Here, we show that SDG711, a rice (Oryza sativa) E(z) homolog, is required to maintain H3K27me3 of many developmental genes after shoot meristem to leaf transition and that many H3K27me3-marked developmental genes are also methylated at non-CG sites in the body regions. SDG711-binding and SDG711-mediated ectopic H3K27me3 also target genes methylated at non-CG sites. Conversely, mutation of OsDRM2, a major rice CHH methyltransferase, resulted in loss of SDG711-binding and H3K27me3 from many genes and their de-repression. Furthermore, we show that SDG711 physically interacts with OsDRM2 and a putative CHG methylation-binding protein. These results together suggest that the repression of many developmental genes may involve both DRM2-mediated non-CG methylation and PRC2-mediated H3K27me3 and that the two marks are not generally mutually exclusive but may cooperate in repression of developmentally regulated genes in rice.