RESUMO
RNA interference (RNAi) is a powerful tool for sequence-specific gene knockdown in therapeutic and research applications. However, spatiotemporal control of RNAi is required to decrease nonspecific targeting, potential toxicity, and allow targeting of essential genes. Herein we describe a class of de-novo-designed RNA switches that enable sequence-specific regulation of RNAi in mammalian cells. Using cis-repressing RNA elements, we engineer RNA devices that only initiate microRNA biogenesis when binding with cognate trigger RNAs. We demonstrate that this conditional RNAi system, termed Orthogonal RNA Interference induced by Trigger RNA (ORIENTR), provides up to 14-fold increases in artificial miRNA biogenesis upon activation in orthogonal libraries. We show that integration of ORIENTR triggers with dCas13d enhances dynamic range to up to 31-fold. We further demonstrate that ORIENTR can be applied to detect endogenous RNA signals and to conditionally knockdown endogenous genes, thus enabling regulatory possibilities including cell-type-specific RNAi and rewiring of transcriptional networks via RNA profile.
Assuntos
MicroRNAs , Interferência de RNA , Ativação Transcricional , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Células HEK293 , Animais , Técnicas de Silenciamento de Genes , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , RNA/metabolismo , RNA/genéticaRESUMO
Hepatocellular carcinoma (HCC) is a significant global health challenge. The activation of autophagy plays an essential role in promoting the proliferation and survival of cancer cells. However, the upstream regulatory network and mechanisms governing autophagy in HCC remain unclear. This study demonstrated that histone deacetylase 2 (HDAC2) regulates autophagy in HCC. Its expression was elevated in HCC tissues, and high HDAC2 expression was strongly associated with poor prognosis in individuals with HCC. Integrated in vitro and in vivo investigations confirmed that HDAC2 promotes autophagy and autophagy-related malignant progression in HCC. Mechanistically, HDAC2 bound specifically to the lysosome-associated protein transmembrane 4-ß (LAPTM4B) promoter at four distinct binding sites, enhancing its transcriptional activation and driving autophagy-related malignant progression in HCC. These findings establish LAPTM4B as a direct target gene of HDAC2. Furthermore, the selective inhibitor of HDAC2 effectively alleviated the malignant development of HCC. In addition, multivariate Cox regression analysis of 105 human HCC samples revealed that HDAC2 expression is an independent predictor of HCC prognosis. This study underscores the crucial role of the HDAC2-LAPTM4B axis in regulating autophagy in the malignant evolution of HCC and highlights the potential of targeting HDAC2 to prevent and halt the malignant progression of HCC.
Assuntos
Autofagia , Carcinoma Hepatocelular , Progressão da Doença , Histona Desacetilase 2 , Neoplasias Hepáticas , Proteínas de Membrana , Humanos , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Histona Desacetilase 2/metabolismo , Histona Desacetilase 2/genética , Autofagia/genética , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Masculino , Animais , Regulação Neoplásica da Expressão Gênica , Feminino , Linhagem Celular Tumoral , Camundongos , Camundongos Nus , Ativação Transcricional/genética , Pessoa de Meia-Idade , Camundongos Endogâmicos BALB C , Prognóstico , Proliferação de Células/genética , Regiões Promotoras Genéticas/genética , Proteínas OncogênicasRESUMO
The double-stranded DNA (dsDNA) sensor STING has been increasingly implicated in responses to "sterile" endogenous threats and pathogens without nominal DNA or cyclic di-nucleotide stimuli. Previous work showed an endoplasmic reticulum (ER) stress response, known as the unfolded protein response (UPR), activates STING. Herein, we sought to determine if ER stress generated a STING ligand, and to identify the UPR pathways involved. Induction of IFN-ß expression following stimulation with the UPR inducer thapsigargin (TPG) or oxygen glucose deprivation required both STING and the dsDNA-sensing cyclic GMP-AMP synthase (cGAS). Furthermore, TPG increased cytosolic mitochondrial DNA, and immunofluorescence visualized dsDNA punctae in murine and human cells, providing a cGAS stimulus. N-acetylcysteine decreased IFN-ß induction by TPG, implicating reactive oxygen species (ROS). However, mitoTEMPO, a mitochondrial oxidative stress inhibitor did not impact TPG-induced IFN. On the other hand, inhibiting the inositol requiring enzyme 1 (IRE1) ER stress sensor and its target transcription factor XBP1 decreased the generation of cytosolic dsDNA. iNOS upregulation was XBP1-dependent, and an iNOS inhibitor decreased cytosolic dsDNA and IFN-ß, implicating ROS downstream of the IRE1-XBP1 pathway. Inhibition of the PKR-like ER kinase (PERK) pathway also attenuated cytoplasmic dsDNA release. The PERK-regulated apoptotic factor Bim was required for both dsDNA release and IFN-ß mRNA induction. Finally, XBP1 and PERK pathways contributed to cytosolic dsDNA release and IFN-induction by the RNA virus, Vesicular Stomatitis Virus (VSV). Together, our findings suggest that ER stressors, including viral pathogens without nominal STING or cGAS ligands such as RNA viruses, trigger multiple canonical UPR pathways that cooperate to activate STING and downstream IFN-ß via mitochondrial dsDNA release.
Assuntos
Citosol , Estresse do Retículo Endoplasmático , Interferon beta , Proteínas de Membrana , Nucleotidiltransferases , Resposta a Proteínas não Dobradas , Humanos , Animais , Camundongos , Nucleotidiltransferases/metabolismo , Citosol/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Interferon beta/metabolismo , DNA/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , eIF-2 Quinase/metabolismo , Endorribonucleases/metabolismo , Proteína 1 de Ligação a X-Box/metabolismo , Proteína 1 de Ligação a X-Box/genética , Tapsigargina/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Ativação Transcricional , DNA Mitocondrial/metabolismoRESUMO
Whilst western diet and sedentary lifestyles heavily contribute to the global obesity epidemic, it is likely that chemical exposure may also contribute. A substantial body of literature implicates a variety of suspected environmental chemicals in metabolic disruption and obesogenic mechanisms. Chemically induced obesogenic metabolic disruption is not yet considered in regulatory testing paradigms or regulations, but this is an internationally recognised human health regulatory development need. An early step in the development of relevant regulatory test methods is to derive appropriate minimum chemical selection lists for the target endpoint and its key mechanisms, such that the test method can be suitably optimised and validated. Independently collated and reviewed reference and proficiency chemicals relevant for the regulatory chemical universe that they are intended to serve, assist regulatory test method development and validation, particularly in relation to the OECD Test Guidelines Programme. To address obesogenic mechanisms and modes of action for chemical hazard assessment, key initiating mechanisms include molecular-level Peroxisome Proliferator-Activated Receptor (PPAR) α and γ agonism and the tissue/organ-level key event of perturbation of the adipogenesis process that may lead to excess white adipose tissue. Here we present a critical literature review, analysis and evaluation of chemicals suitable for the development, optimisation and validation of human PPARα and PPARγ agonism and human white adipose tissue adipogenesis test methods. The chemical lists have been derived with consideration of essential criteria needed for understanding the strengths and limitations of the test methods. With a weight of evidence approach, this has been combined with practical and applied aspects required for the integration and combination of relevant candidate test methods into test batteries, as part of an Integrated Approach to Testing and Assessment for metabolic disruption. The proposed proficiency and reference chemical list includes a long list of negatives and positives (20 chemicals for PPARα, 21 for PPARγ, and 11 for adipogenesis) from which a (pre-)validation proficiency chemicals list has been derived.
Assuntos
Adipogenia , Obesidade , PPAR alfa , PPAR gama , Humanos , PPAR alfa/metabolismo , PPAR alfa/genética , PPAR gama/metabolismo , PPAR gama/genética , Adipogenia/efeitos dos fármacos , Obesidade/metabolismo , Obesidade/induzido quimicamente , Ativação Transcricional/efeitos dos fármacosRESUMO
During implantation, embryos undergo an unpolarized-to-polarized transition to initiate postimplantation morphogenesis. However, the underlying molecular mechanism is unknown. Here, we identify a transient transcriptional activation governing embryonic morphogenesis and pluripotency transition during implantation. In naive pluripotent embryonic stem cells (ESCs), which represent preimplantation embryos, we find that the microprocessor component DGCR8 can recognize stem-loop structures within nascent mRNAs to sequester transcriptional coactivator FLII to suppress transcription directly. When mESCs exit from naive pluripotency, the ERK/RSK/P70S6K pathway rapidly activates, leading to FLII phosphorylation and disruption of DGCR8/FLII interaction. Phosphorylated FLII can bind to transcription factor JUN, activating cell migration-related genes to establish poised pluripotency akin to implanting embryos. Resequestration of FLII by DGCR8 drives poised ESCs into formative pluripotency. In summary, we identify a DGCR8/FLII/JUN-mediated transient transcriptional activation mechanism. Disruption of this mechanism inhibits naive-poised-formative pluripotency transition and the corresponding unpolarized-to-polarized transition during embryo implantation, which are conserved in mice and humans.
Assuntos
Implantação do Embrião , Regulação da Expressão Gênica no Desenvolvimento , Morfogênese , Ativação Transcricional , Animais , Implantação do Embrião/genética , Camundongos , Humanos , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Fosforilação , Células-Tronco Embrionárias Murinas/metabolismo , Células-Tronco Embrionárias Murinas/citologia , Feminino , Proteínas Proto-Oncogênicas c-jun/metabolismo , Proteínas Proto-Oncogênicas c-jun/genética , Transdução de SinaisRESUMO
Pausing of RNA polymerase II (Pol II) at transcription start sites (TSSs) primes target genes for productive elongation. Coincidentally, DNA double-strand breaks (DSBs) enrich at highly transcribed and Pol II-paused genes, although their interplay remains undefined. Using androgen receptor (AR) signaling as a model, we have uncovered AR-interacting protein 4 (ARIP4) helicase as a driver of androgen-dependent transcription induction. Chromatin immunoprecipitation sequencing analysis revealed that ARIP4 preferentially co-occupies TSSs with paused Pol II. Moreover, we found that ARIP4 complexes with topoisomerase II beta and mediates transient DSB formation upon hormone stimulation. Accordingly, ARIP4 deficiency compromised release of paused Pol II and resulted in R-loop accumulation at a panel of highly transcribed AR target genes. Last, we showed that ARIP4 binds and unwinds R-loops in vitro and that its expression positively correlates with prostate cancer progression. We propose that androgen stimulation triggers ARIP4-mediated unwinding of R-loops at TSSs, enforcing Pol II pause release to effectively drive an androgen-dependent expression program.
Assuntos
Androgênios , Neoplasias da Próstata , Estruturas R-Loop , RNA Polimerase II , Receptores Androgênicos , Humanos , Androgênios/metabolismo , Receptores Androgênicos/metabolismo , Receptores Androgênicos/genética , Masculino , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , RNA Polimerase II/metabolismo , RNA Polimerase II/genética , Linhagem Celular Tumoral , DNA Topoisomerases Tipo II/metabolismo , DNA Topoisomerases Tipo II/genética , Transcrição Gênica , Quebras de DNA de Cadeia Dupla , Sítio de Iniciação de Transcrição , Regulação Neoplásica da Expressão Gênica , Ligação Proteica , Ativação TranscricionalRESUMO
bZIP transcription factors can function as homodimers or heterodimers through interactions with their disordered coiled-coil domain. Such dimer assemblies are known to influence DNA-binding specificity and/or the recruitment of binding partners, which can cause a functional switch of a transcription factor from being an activator to a repressor. We recently identified the genomic targets of a bZIP transcription factor called CREB3L1 in rat hypothalamic supraoptic nucleus by ChIP-seq. The objective of this study was to investigate the CREB3L1 protein-to-protein interactome of which little is known. For this approach, we created and screened a rat supraoptic nucleus yeast two-hybrid prey library with the bZIP region of rat CREB3L1 as the bait. Our yeast two-hybrid approach captured five putative CREB3L1 interacting prey proteins in the supraoptic nucleus. One interactor was selected by bioinformatic analyses for more detailed investigation by co-immunoprecipitation, immunofluorescent cellular localisation, and reporter assays in vitro. Here we identify dimerisation hub protein Dynein Light Chain LC8-Type 1 as a CREB3L1 interacting protein that in vitro enhances CREB3L1 activation of target genes.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico , Dineínas do Citoplasma , Proteínas do Tecido Nervoso , Ativação Transcricional , Animais , Humanos , Ratos , Arginina Vasopressina/metabolismo , Arginina Vasopressina/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/genética , Fatores de Transcrição de Zíper de Leucina Básica/química , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Dineínas do Citoplasma/metabolismo , Dineínas do Citoplasma/genética , Multimerização Proteica , Núcleo Supraóptico/metabolismo , Ativação Transcricional/genética , Técnicas do Sistema de Duplo-HíbridoRESUMO
Treatment of fungal infections associated with the filamentous fungus Aspergillus fumigatus is becoming more problematic as this organism is developing resistance to the main chemotherapeutic drug at an increasing rate. Azole drugs represent the current standard-of-care in the treatment of aspergillosis with this drug class acting by inhibiting a key step in the biosynthesis of the fungal sterol ergosterol. Azole compounds block the activity of the lanosterol α-14 demethylase, encoded by the cyp51A gene. A common route of azole resistance involves an increase in transcription of cyp51A. This transcriptional increase requires the function of a Zn2Cys6 DNA-binding domain-containing transcription activator protein called AtrR. AtrR was identified through its action as a positive regulator of expression of an ATP-binding cassette transporter (abcC/cdr1B here called abcG1). Using both deletion and alanine scanning mutagenesis, we demonstrate that a conserved C-terminal domain in A. fumigatus is required for the expression of abcG1 but dispensable for cyp51A transcription. This domain is also found in several other fungal pathogen AtrR homologs consistent with a conserved gene-selective function of this protein segment being conserved. Using RNA sequencing (RNA-seq), we find that this gene-specific transcriptional defect extends to several other membrane transporter-encoding genes including a second ABC transporter locus. Our data reveal that AtrR uses at least two distinct mechanisms to induce gene expression and that normal susceptibility to azole drugs cannot be provided by maintenance of wild-type expression of the ergosterol biosynthetic pathway when ABC transporter expression is reduced. IMPORTANCE: Aspergillus fumigatus is the primary human filamentous fungal pathogen. The principal chemotherapeutic drug used to control infections associated with A. fumigatus is the azole compound. These drugs are well-tolerated and effective, but resistance is emerging at an alarming rate. Most resistance is associated with mutations that lead to overexpression of the azole target enzyme, lanosterol α-14 demethylase, encoded by the cyp51A gene. A key regulator of cyp51A gene expression is the transcription factor AtrR. Very little is known of the molecular mechanisms underlying the effect of AtrR on gene expression. Here, we use deletion and clustered amino acid substitution mutagenesis to map a region of AtrR that confers gene-specific activation on target genes of this transcription factor. This region is highly conserved across AtrR homologs from other pathogenic species arguing that its importance in transcriptional regulation is maintained across evolution.
Assuntos
Antifúngicos , Aspergillus fumigatus , Proteínas Fúngicas , Regulação Fúngica da Expressão Gênica , Ativação Transcricional , Aspergillus fumigatus/genética , Aspergillus fumigatus/efeitos dos fármacos , Aspergillus fumigatus/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Antifúngicos/farmacologia , Azóis/farmacologia , Sistema Enzimático do Citocromo P-450/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Farmacorresistência Fúngica/genética , Domínios ProteicosRESUMO
Signal-induced transcriptional programs regulate critical biological processes through the precise spatiotemporal activation of Immediate Early Genes (IEGs); however, the mechanisms of transcription induction remain poorly understood. By combining an acute depletion system with several genomics approaches to interrogate synchronized, temporal transcription, we reveal that KAP1/TRIM28 is a first responder that fulfills the temporal and heightened transcriptional demand of IEGs. Acute KAP1 loss triggers an increase in RNA polymerase II elongation kinetics during early stimulation time points. This elongation defect derails the normal progression through the transcriptional cycle during late stimulation time points, ultimately leading to decreased recruitment of the transcription apparatus for re-initiation thereby dampening IEGs transcriptional output. Collectively, KAP1 plays a counterintuitive role by negatively regulating transcription elongation to support full activation across multiple transcription cycles of genes critical for cell physiology and organismal functions.
Assuntos
RNA Polimerase II , Proteína 28 com Motivo Tripartido , Proteína 28 com Motivo Tripartido/metabolismo , Proteína 28 com Motivo Tripartido/genética , RNA Polimerase II/metabolismo , Humanos , Cinética , Elongação da Transcrição Genética , Genes Precoces , Transcrição Gênica , Transdução de Sinais , Ativação Transcricional , AnimaisRESUMO
Gene expression in Arabidopsis is regulated by more than 1,900 transcription factors (TFs), which have been identified genome-wide by the presence of well-conserved DNA-binding domains. Activator TFs contain activation domains (ADs) that recruit coactivator complexes; however, for nearly all Arabidopsis TFs, we lack knowledge about the presence, location and transcriptional strength of their ADs1. To address this gap, here we use a yeast library approach to experimentally identify Arabidopsis ADs on a proteome-wide scale, and find that more than half of the Arabidopsis TFs contain an AD. We annotate 1,553 ADs, the vast majority of which are, to our knowledge, previously unknown. Using the dataset generated, we develop a neural network to accurately predict ADs and to identify sequence features that are necessary to recruit coactivator complexes. We uncover six distinct combinations of sequence features that result in activation activity, providing a framework to interrogate the subfunctionalization of ADs. Furthermore, we identify ADs in the ancient AUXIN RESPONSE FACTOR family of TFs, revealing that AD positioning is conserved in distinct clades. Our findings provide a deep resource for understanding transcriptional activation, a framework for examining function in intrinsically disordered regions and a predictive model of ADs.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Regulação da Expressão Gênica de Plantas , Domínios Proteicos , Fatores de Transcrição , Ativação Transcricional , Arabidopsis/química , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/classificação , Proteínas de Arabidopsis/metabolismo , Sequência Conservada/genética , Conjuntos de Dados como Assunto , Regulação da Expressão Gênica de Plantas/genética , Ácidos Indolacéticos/metabolismo , Proteínas Intrinsicamente Desordenadas , Anotação de Sequência Molecular , Redes Neurais de Computação , Proteoma/química , Proteoma/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/classificação , Fatores de Transcrição/metabolismo , Ativação Transcricional/genéticaRESUMO
Euryhaline fish experience variable osmotic environments requiring physiological adjustments to tolerate elevated salinity. Mozambique tilapia (Oreochromis mossambicus) possess one of the highest salinity tolerance limits of any fish. In tilapia and other euryhaline fish species, the myo-inositol biosynthesis (MIB) pathway enzymes, myo-inositol phosphate synthase (MIPS) and inositol monophosphatase 1 (IMPA1.1), are among the most upregulated mRNAs and proteins indicating the high importance of this pathway for hyperosmotic (HO) stress tolerance. These abundance changes must be precluded by HO perception and signaling mechanism activation to regulate the expression of MIPS and IMPA1.1 genes. In previous work using a O. mossambicus cell line (OmB), a reoccurring osmosensitive enhancer element (OSRE1) in both MIPS and IMPA1.1 was shown to transcriptionally upregulate these enzymes in response to HO stress. The OSRE1 core consensus (5'-GGAAA-3') matches the core binding sequence of the predominant mammalian HO response transcription factor, nuclear factor of activated T-cells (NFAT5). HO-challenged OmB cells showed an increase in NFAT5 mRNA suggesting NFAT5 may contribute to MIB pathway regulation in euryhaline fish. Ectopic expression of wild-type NFAT5 induced an IMPA1.1 promoter-driven reporter by 5.1-fold (P < 0.01). Moreover, expression of dominant negative NFAT5 in HO media resulted in a 47% suppression of the reporter signal (P < 0.005). Furthermore, reductions of IMPA1.1 (37-49%) and MIPS (6-37%) mRNA abundance were observed in HO-challenged NFAT5 knockout cells relative to control cells. Collectively, these multiple lines of experimental evidence establish NFAT5 as a tilapia transcription factor contributing to HO-induced activation of the MIB pathway.NEW & NOTEWORTHY In our study, we use a multi-pronged synthetic biology approach to demonstrate that the fish homolog of the predominant mammalian osmotic stress transcription factor nuclear factor of activated T-cells (NFAT5) also contributes to the activation of hyperosmolality inducible genes in cells of extremely euryhaline fish. However, in addition to NFAT5 the presence of other strong osmotically inducible signaling mechanisms is required for full activation of osmoregulated tilapia genes.
Assuntos
Inositol , Mio-Inositol-1-Fosfato Sintase , Pressão Osmótica , Tilápia , Regulação para Cima , Animais , Tilápia/genética , Tilápia/metabolismo , Inositol/metabolismo , Mio-Inositol-1-Fosfato Sintase/genética , Mio-Inositol-1-Fosfato Sintase/metabolismo , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Fatores de Transcrição NFATC/metabolismo , Fatores de Transcrição NFATC/genética , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Linhagem Celular , Transdução de Sinais , Transcrição Gênica , Osmorregulação/genética , Ativação TranscricionalRESUMO
Germline TP53 pathogenic variants can lead to a cancer susceptibility syndrome known as Li-Fraumeni (LFS). Variants affecting its activity can drive tumorigenesis altering p53 pathways and their identification is crucial for assessing individual risk. This study explored the functional impact of TP53 missense variants on its transcription factor activity. We selected seven TP53 missense variants (c.129G > C, c.320A > G, c.417G > T, c.460G > A, c,522G > T, c.589G > A and c.997C > T) identified in Brazilian families at-risk for LFS. Variants were created through site-directed mutagenesis and transfected into SK-OV-3 cells to assess their transcription activation capabilities. Variants K139N and V197M displayed significantly reduced transactivation activity in a TP53-dependent luciferase reporter assay. Additionally, K139N negatively impacted CDKN1A and MDM2 expression and had a limited effect on GADD45A and PMAIP1 upon irradiation-induced DNA damage. Variant V197M demonstrated functional impact in all target genes evaluated and loss of Ser15 phosphorylation. K139N and V197M variants presented a reduction of p21 levels after irradiation. Our data show that K139N and V197M negatively impact p53 functions, supporting their classification as pathogenic variants. This underscores the significance of conducting functional studies on germline TP53 missense variants classified as variants of uncertain significance to ensure proper management of LFS-related cancer risks.
Assuntos
Predisposição Genética para Doença , Mutação em Linhagem Germinativa , Síndrome de Li-Fraumeni , Mutação de Sentido Incorreto , Proteína Supressora de Tumor p53 , Síndrome de Li-Fraumeni/genética , Humanos , Proteína Supressora de Tumor p53/genética , Brasil , Proteínas Proto-Oncogênicas c-mdm2/genética , Feminino , Inibidor de Quinase Dependente de Ciclina p21/genética , Masculino , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Ativação Transcricional/genética , Proteínas GADD45RESUMO
The androgen receptor (AR) is a modular transcription factor which functions as a master regulator of gene expression. AR protein is composed of three functional domains; the ligand-binding domain (LBD); DNA-binding domain (DBD); and the intrinsically disordered N-terminal transactivation domain (TAD). AR is transactivated upon binding to the male sex hormone testosterone and other androgens. While the AR may tolerate loss of its LBD, the TAD contains activation function-1 (AF-1) that is essential for all AR transcriptional activity. AR is frequently over-expressed in most prostate cancer. Currently, androgen deprivation therapy (ADT) in the form of surgical or chemical castration remains the standard of care for patients with high risk localized disease, advanced and metastatic disease, and those patients that experience biochemical relapse following definitive primary treatment. Patients with recurrent disease that receive ADT will ultimately progress to lethal metastatic castration-resistant prostate cancer. In addition to ADT not providing a cure, it is associated with numerous adverse effects including cardiovascular disease, osteoporosis and sexual dysfunction. Recently there has been a renewed interest in investigating the possibility of using antiandrogens which competitively bind the AR-LBD without ADT for patients with hormone sensitive, non-metastatic prostate cancer. Here we describe a class of compounds termed AR transactivation domain inhibitors (ARTADI) and their mechanism of action. These compounds bind to the AR-TAD to inhibit AR transcriptional activity in the absence and presence of androgens. Thus these inhibitors may have utility in preventing prostate cancer growth in the non-castrate setting.
Assuntos
Neoplasias da Próstata , Receptores Androgênicos , Ativação Transcricional , Masculino , Humanos , Receptores Androgênicos/metabolismo , Receptores Androgênicos/química , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Ativação Transcricional/efeitos dos fármacos , Antagonistas de Receptores de Andrógenos/farmacologia , Antagonistas de Receptores de Andrógenos/química , Animais , Domínios ProteicosRESUMO
BACKGROUND/AIM: How tumors regulate the genes of the coagulome is crucial for cancer-associated thrombosis and the occurrence of venous thromboembolic complications in patients with cancer. We have previously reported potent yet complex effects of glucocorticoids (GC) on the expression of three genes that play a key role in the regulation of thrombin/plasmin activation (F3, PLAU, and SERPINE1). This study aimed to extend the investigation of GC effects to the whole tumor coagulome and assess the resulting impact on the ability of cancer cells to activate thrombin and plasmin. MATERIALS AND METHODS: Cancer RNA expression data were retrieved from various sources. Additionally, oral squamous cell carcinoma (OSCC) cells exposed to GC in vitro were analyzed using QPCR, enzymatic assays measuring thrombin and urokinase-type Plasminogen Activator (uPA) activity, and D-dimer concentrations. RESULTS: Our findings highlight the potent and specific stimulatory effect of GC on SERPINE1 expression across different types of cancer. Consistently, GC were found to inhibit uPA proteolytic activity and reduce the concentrations of D-dimers in OSCC in vitro. CONCLUSION: Fibrinolysis inhibition is a key consequence of cancer cell exposure to GC, possibly leading to the stabilization of the fibrin clot in cancer.
Assuntos
Fibrinólise , Glucocorticoides , Inibidor 1 de Ativador de Plasminogênio , Humanos , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Inibidor 1 de Ativador de Plasminogênio/genética , Fibrinólise/efeitos dos fármacos , Glucocorticoides/farmacologia , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/genética , Trombina/metabolismo , Trombina/farmacologia , Produtos de Degradação da Fibrina e do Fibrinogênio/metabolismo , Ativação Transcricional/efeitos dos fármacos , Neoplasias Bucais/genética , Neoplasias Bucais/metabolismo , Neoplasias Bucais/patologia , Neoplasias Bucais/tratamento farmacológico , Neoplasias/genética , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/patologia , Coagulação Sanguínea/efeitos dos fármacosRESUMO
Zinc finger protein 180 (ZNF180) is a multifunctional protein that interacts with nucleic acids and regulates various cellular processes; however, the function of ZNF180 in colorectal cancer (CRC) remains unclear. The present study investigated the role and function of ZNF180 in CRC, and aimed to reveal the underlying molecular mechanism. The results revealed that ZNF180 was downregulated in CRC tissues and was associated with a good prognosis in patients with CRC. Additionally, the expression of ZNF180 was downregulated by methylation in CRC. In vivo and in vitro experiments revealed that ZNF180 overexpression was functionally associated with the inhibition of cell proliferation and the induction of apoptosis. Mechanistically, chromatin immunoprecipitationPCR and luciferase assays demonstrated that ZNF180 markedly regulated the transcriptional activity of methyltransferase 14, N6adenosinemethyltransferase noncatalytic subunit (METTL14) by directly binding to and activating its promoter region. Simultaneous overexpression of ZNF180 and knockdown of METTL14 indicated that the reduction of METTL14 could suppress the effects of ZNF180 on the induction of apoptosis. Clinically, the present study observed a significant positive correlation between ZNF180 and METTL14 expression levels, and low expression of ZNF180 and METTL14 predicted a poor prognosis in CRC. Overall, these findings revealed a novel mechanism by which the ZNF180/METTL14 axis may modulate apoptosis and cell proliferation in CRC. This evidence suggests that this axis may serve as a prognostic biomarker and therapeutic target in patients with CRC.
Assuntos
Apoptose , Proliferação de Células , Neoplasias Colorretais , Regulação Neoplásica da Expressão Gênica , Metiltransferases , Humanos , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Neoplasias Colorretais/metabolismo , Metiltransferases/genética , Metiltransferases/metabolismo , Apoptose/genética , Proliferação de Células/genética , Masculino , Feminino , Prognóstico , Pessoa de Meia-Idade , Linhagem Celular Tumoral , Animais , Ativação Transcricional , Camundongos , Regiões Promotoras Genéticas , Idoso , Regulação para Baixo , Metilação de DNARESUMO
Understanding the impact of splicing and nonsense variants on RNA is crucial for the resolution of variant classification as well as their suitability for precision medicine interventions. This is primarily enabled through RNA studies involving transcriptomics followed by targeted assays using RNA isolated from clinically accessible tissues (CATs) such as blood or skin of affected individuals. Insufficient disease gene expression in CATs does however pose a major barrier to RNA based investigations, which we show is relevant to 1,436 Mendelian disease genes. We term these "silent" Mendelian genes (SMGs), the largest portion (36%) of which are associated with neurological disorders. We developed two approaches to induce SMG expression in human dermal fibroblasts (HDFs) to overcome this limitation, including CRISPR-activation-based gene transactivation and fibroblast-to-neuron transdifferentiation. Initial transactivation screens involving 40 SMGs stimulated our development of a highly multiplexed transactivation system culminating in the 6- to 90,000-fold induction of expression of 20/20 (100%) SMGs tested in HDFs. Transdifferentiation of HDFs directly to neurons led to expression of 193/516 (37.4%) of SMGs implicated in neurological disease. The magnitude and isoform diversity of SMG expression following either transactivation or transdifferentiation was comparable to clinically relevant tissues. We apply transdifferentiation and/or gene transactivation combined with short- and long-read RNA sequencing to investigate the impact that variants in USH2A, SCN1A, DMD, and PAK3 have on RNA using HDFs derived from affected individuals. Transactivation and transdifferentiation represent rapid, scalable functional genomic solutions to investigate variants impacting SMGs in the patient cell and genomic context.
Assuntos
Transdiferenciação Celular , Fibroblastos , Neurônios , Ativação Transcricional , Humanos , Transdiferenciação Celular/genética , Fibroblastos/metabolismo , Fibroblastos/citologia , Neurônios/metabolismo , Neurônios/citologia , RNA/genética , RNA/metabolismo , Sistemas CRISPR-CasRESUMO
Triple-negative breast cancer (TNBC) is more aggressive and has a higher metastasis rate compared with other subtypes of breast cancer. Due to the lack of drug-targetable receptors, chemotherapy is now the only available systemic treatment for TNBC. However, some patients might still develop drug resistance and have poor prognosis. Therefore, novel molecular biomarkers and new treatment targets are urgently needed for patients with TNBC. To provide molecular insights into TNBC progression, we investigated the function and the underlying mechanism of Defective in cullin neddylation 1 domain containing 5 (DCUN1D5) in the regulation of TNBC. By TCGA dataset and surgical specimens with immunohistochemical (IHC) staining method, DCUN1D5 was identified to be significantly upregulated in TNBC tumor tissues and negatively associated with prognosis. A series of in vitro and in vivo experiments were performed to confirm the oncogenic role of DCUN1D5 in TNBC. Overexpression of FN1 or PI3K/AKT activator IGF-1 could restore the proliferative and invasive ability induced by DCUN1D5 knockdown and DCUN1D5 could act as a novel transcriptional target of transcription factor Yin Yang 1 (YY1). In conclusion, YY1-enhanced DCUN1D5 expression could promote TNBC progression by FN1/PI3K/AKT pathway and DCUN1D5 might be a potential prognostic biomarker and therapeutic target for TNBC treatment.
Assuntos
Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas c-akt , Neoplasias de Mama Triplo Negativas , Fator de Transcrição YY1 , Animais , Feminino , Humanos , Camundongos , Linhagem Celular Tumoral , Progressão da Doença , Fibronectinas , Regulação Neoplásica da Expressão Gênica , Camundongos Nus , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Transdução de Sinais , Ativação Transcricional , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismo , Fator de Transcrição YY1/metabolismo , Fator de Transcrição YY1/genéticaRESUMO
Familial platelet disorder with associated myeloid malignancies (FPDMM) is an autosomal dominant disease caused by heterozygous germline mutations in RUNX1. It is characterized by thrombocytopenia, platelet dysfunction, and a predisposition to hematological malignancies. Although FPDMM is a precursor for diseases involving abnormal DNA methylation, the DNA methylation status in FPDMM remains unknown, largely due to a lack of animal models and challenges in obtaining patient-derived samples. Here, using genome editing techniques, we established two lines of human induced pluripotent stem cells (iPSCs) with different FPDMM-mimicking heterozygous RUNX1 mutations. These iPSCs showed defective differentiation of hematopoietic progenitor cells (HPCs) and megakaryocytes (Mks), consistent with FPDMM. The FPDMM-mimicking HPCs showed DNA methylation patterns distinct from those of wild-type HPCs, with hypermethylated regions showing the enrichment of ETS transcription factor (TF) motifs. We found that the expression of FLI1, an ETS family member, was significantly downregulated in FPDMM-mimicking HPCs with a RUNX1 transactivation domain (TAD) mutation. We demonstrated that FLI1 promoted binding-site-directed DNA demethylation, and that overexpression of FLI1 restored their megakaryocytic differentiation efficiency and hypermethylation status. These findings suggest that FLI1 plays a crucial role in regulating DNA methylation and correcting defective megakaryocytic differentiation in FPDMM-mimicking HPCs with a RUNX1 TAD mutation.
Assuntos
Diferenciação Celular , Subunidade alfa 2 de Fator de Ligação ao Core , Metilação de DNA , Células-Tronco Pluripotentes Induzidas , Megacariócitos , Mutação , Proteína Proto-Oncogênica c-fli-1 , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Humanos , Megacariócitos/metabolismo , Proteína Proto-Oncogênica c-fli-1/genética , Proteína Proto-Oncogênica c-fli-1/metabolismo , Diferenciação Celular/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Transtornos Plaquetários/genética , Transtornos Plaquetários/metabolismo , Transtornos Plaquetários/patologia , Ativação Transcricional , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/citologia , Leucemia Mieloide Aguda , Transtornos Herdados da Coagulação SanguíneaRESUMO
The WRKY transcription factor (TF) genes form a large family in higher plants, with 72 members in Arabidopsis (Arabidopsis thaliana). The gaseous phytohormone ethylene (ET) regulates multiple physiological processes in plants. It is known that 1-aminocyclopropane-1-carboxylic acid (ACC) synthases (ACSs, EC 4.4.1.14) limit the enzymatic reaction rate of ethylene synthesis. However, whether WRKY TFs regulate the expression of ACSs and/or ACC oxidases (ACOs, EC 1.14.17.4) remains largely elusive. Here, we demonstrated that Arabidopsis WRKY22 positively regulated the expression of a few ACS and ACO genes, thus promoting ethylene production. Inducible overexpression of WRKY22 caused shorter hypocotyls without ACC treatment. A qRT-PCR screening demonstrated that overexpression of WRKY22 activates the expression of several ACS and ACO genes. The promoter regions of ACS5, ACS11, and ACO5 were also activated by WRKY22, which was revealed by a dual luciferase reporter assay. A follow-up chromatin immunoprecipitation coupled with quantitative PCR (ChIP-qPCR) and electrophoretic mobility shift assay (EMSA) showed that the promoter regions of ACS5 and ACO5 could be bound by WRKY22 directly. Moreover, wrky22 mutants had longer primary roots and more lateral roots than wild type, while WRKY22-overexpressing lines showed the opposite phenotype. In conclusion, this study revealed that WRKY22 acts as a novel TF activating, at least, the expression of ACS5 and ACO5 to increase ethylene synthesis and modulate root development.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Etilenos , Regulação da Expressão Gênica de Plantas , Liases , Raízes de Plantas , Fatores de Transcrição , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Etilenos/metabolismo , Etilenos/biossíntese , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Liases/genética , Liases/metabolismo , Aminoácido Oxirredutases/genética , Aminoácido Oxirredutases/metabolismo , Regiões Promotoras Genéticas/genética , Carbono-Carbono Liases/metabolismo , Carbono-Carbono Liases/genética , Ativação Transcricional/genéticaRESUMO
Gene Transfer Agents (GTAs) are phage-like particles that cannot self-multiply and be infectious. Caulobacter crescentus, a bacterium best known as a model organism to study bacterial cell biology and cell cycle regulation, has recently been demonstrated to produce bona fide GTA particles (CcGTA). Since C. crescentus ultimately die to release GTA particles, the production of GTA particles must be tightly regulated and integrated with the host physiology to prevent a collapse in cell population. Two direct activators of the CcGTA biosynthetic gene cluster, GafY and GafZ, have been identified, however, it is unknown how GafYZ controls transcription or how they coordinate gene expression of the CcGTA gene cluster with other accessory genes elsewhere on the genome for complete CcGTA production. Here, we show that the CcGTA gene cluster is transcriptionally co-activated by GafY, integration host factor (IHF), and by GafZ-mediated transcription anti-termination. We present evidence that GafZ is a transcription anti-terminator that likely forms an anti-termination complex with RNA polymerase, NusA, NusG, and NusE to bypass transcription terminators within the 14 kb CcGTA cluster. Overall, we reveal a two-tier regulation that coordinates the synthesis of GTA particles in C. crescentus.