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1.
Nat Commun ; 11(1): 5061, 2020 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-33033262

RESUMO

The interplay between the Yamanaka factors (OCT4, SOX2, KLF4 and c-MYC) and transcriptional/epigenetic co-regulators in somatic cell reprogramming is incompletely understood. Here, we demonstrate that the histone H3 lysine 27 trimethylation (H3K27me3) demethylase JMJD3 plays conflicting roles in mouse reprogramming. On one side, JMJD3 induces the pro-senescence factor Ink4a and degrades the pluripotency regulator PHF20 in a reprogramming factor-independent manner. On the other side, JMJD3 is specifically recruited by KLF4 to reduce H3K27me3 at both enhancers and promoters of epithelial and pluripotency genes. JMJD3 also promotes enhancer-promoter looping through the cohesin loading factor NIPBL and ultimately transcriptional elongation. This competition of forces can be shifted towards improved reprogramming by using early passage fibroblasts or boosting JMJD3's catalytic activity with vitamin C. Our work, thus, establishes a multifaceted role for JMJD3, placing it as a key partner of KLF4 and a scaffold that assists chromatin interactions and activates gene transcription.


Assuntos
Reprogramação Celular , Histona Desmetilases com o Domínio Jumonji/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Animais , Catálise , Proliferação de Células , Senescência Celular , Desmetilação , Elementos Facilitadores Genéticos/genética , Células Epiteliais/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Genoma , Histonas/metabolismo , Lisina/metabolismo , Camundongos , Modelos Biológicos , Regiões Promotoras Genéticas , Ativação Transcricional/genética
2.
Nat Commun ; 11(1): 4544, 2020 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-32917861

RESUMO

Stratification of enhancers by signal strength in ChIP-seq assays has resulted in the establishment of super-enhancers as a widespread and useful tool for identifying cell type-specific, highly expressed genes and associated pathways. We examine a distinct method of stratification that focuses on peak breadth, termed hyperacetylated chromatin domains (HCDs), which classifies broad regions exhibiting histone modifications associated with gene activation. We find that this analysis serves to identify genes that are both more highly expressed and more closely aligned to cell identity than super-enhancer analysis does using multiple data sets. Moreover, genetic manipulations of selected gene loci suggest that some enhancers located within HCDs work at least in part via a distinct mechanism involving the modulation of histone modifications across domains and that this activity can be imported into a heterologous gene locus. In addition, such genetic dissection reveals that the super-enhancer concept can obscure important functions of constituent elements.


Assuntos
Cromatina/metabolismo , Elementos Facilitadores Genéticos/genética , Loci Gênicos/genética , Ativação Transcricional , Acetilação , Animais , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Sequenciamento de Cromatina por Imunoprecipitação , Conjuntos de Dados como Assunto , Embrião de Mamíferos , Eritroblastos , Feminino , Feto , Código das Histonas/genética , Histonas/genética , Histonas/metabolismo , Humanos , Camundongos , Regiões Promotoras Genéticas/genética , RNA-Seq
3.
Nat Commun ; 11(1): 4903, 2020 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-32994412

RESUMO

The CRISPR-Cas9 system has increased the speed and precision of genetic editing in cells and animals. However, model generation for drug development is still expensive and time-consuming, demanding more target flexibility and faster turnaround times with high reproducibility. The generation of a tightly controlled ObLiGaRe doxycycline inducible SpCas9 (ODInCas9) transgene and its use in targeted ObLiGaRe results in functional integration into both human and mouse cells culminating in the generation of the ODInCas9 mouse. Genomic editing can be performed in cells of various tissue origins without any detectable gene editing in the absence of doxycycline. Somatic in vivo editing can model non-small cell lung cancer (NSCLC) adenocarcinomas, enabling treatment studies to validate the efficacy of candidate drugs. The ODInCas9 mouse allows robust and tunable genome editing granting flexibility, speed and uniformity at less cost, leading to high throughput and practical preclinical in vivo therapeutic testing.


Assuntos
Sistemas CRISPR-Cas/genética , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Descoberta de Drogas/métodos , Edição de Genes/métodos , Neoplasias Pulmonares/tratamento farmacológico , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Proteína 9 Associada à CRISPR/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Linhagem Celular Tumoral , Doxiciclina/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Feminino , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Vetores Genéticos/genética , Células HEK293 , Ensaios de Triagem em Larga Escala/métodos , Humanos , Neoplasias Pulmonares/genética , Masculino , Camundongos , Camundongos Transgênicos , RNA Guia/genética , Recombinação Genética/efeitos dos fármacos , Reprodutibilidade dos Testes , Ativação Transcricional/efeitos dos fármacos , Transfecção/métodos , Transgenes/genética
4.
Ecotoxicol Environ Saf ; 205: 111089, 2020 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-32810645

RESUMO

Early molecular events after the exposure of heavy metals, such as aberrant DNA methylation, suggest that DNA methylation was important in regulating physiological processes for animals and accordingly could be used as environmental biomarkers. In the present study, we found that copper (Cu) exposure increased lipid content and induced the DNA hypermethylation at the whole genome level. Especially, Cu induced hypermethylation of glucose-regulated protein 78 (grp78) and peroxisome proliferator-activated receptor gamma coactivator-1α (pgc1α). CCAAT/enhancer binding protein α (C/EBPα) could bind to the methylated sequence of grp78, whereas C/EBPß could not bind to the methylated sequence of grp78. These synergistically influenced grp78 expression and increased lipogenesis. In contrast, DNA methylation of PGC1α blocked the specific protein 1 (SP1) binding and interfered mitochondrial function. Moreover, Cu increased reactive oxygen species (ROS) production, activated endoplasmic reticulum (ER) stress and damaged mitochondrial function, and accordingly increased lipid deposition. Notably, we found a new toxicological mechanism for Cu-induced lipid deposition at DNA methylation level. The measurement of DNA methylation facilitated the use of these epigenetic biomarkers for the evaluation of environmental risk.


Assuntos
Carpas/fisiologia , Cobre/toxicidade , Poluentes Químicos da Água/toxicidade , Animais , Carpas/metabolismo , Cobre/metabolismo , Estresse do Retículo Endoplasmático , Glucose/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Lipídeos , Metilação , Mitocôndrias/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Ativação Transcricional , Regulação para Cima
5.
Mol Cell ; 79(4): 660-676.e8, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32755593

RESUMO

Specific combinations of two transcription factors (Hnf4α plus Foxa1, Foxa2, or Foxa3) can induce direct conversion of mouse fibroblasts into hepatocyte-like cells. However, the molecular mechanisms underlying hepatic reprogramming are largely unknown. Here, we show that the Foxa protein family members and Hnf4α sequentially and cooperatively bind to chromatin to activate liver-specific gene expression. Although all Foxa proteins bind to and open regions of closed chromatin as pioneer factors, Foxa3 has the unique potential of transferring from the distal to proximal regions of the transcription start site of target genes, binding RNA polymerase II, and co-traversing target genes. These distinctive characteristics of Foxa3 are essential for inducing the hepatic fate in fibroblasts. Similar functional coupling of transcription factors to RNA polymerase II may occur in other contexts whereby transcriptional activation can induce cell differentiation.


Assuntos
Fator 3-gama Nuclear de Hepatócito/metabolismo , Fator 4 Nuclear de Hepatócito/metabolismo , Fígado/citologia , Fígado/fisiologia , Ativação Transcricional , Animais , Sítios de Ligação , Células Cultivadas , Reprogramação Celular/fisiologia , Cromatina/metabolismo , DNA Polimerase II/genética , DNA Polimerase II/metabolismo , Fibroblastos/citologia , Fibroblastos/fisiologia , Regulação da Expressão Gênica , Fator 3-gama Nuclear de Hepatócito/genética , Fator 4 Nuclear de Hepatócito/genética , Camundongos Endogâmicos C57BL , Domínios Proteicos , Sítio de Iniciação de Transcrição
6.
Yakugaku Zasshi ; 140(8): 993-1000, 2020.
Artigo em Japonês | MEDLINE | ID: mdl-32741873

RESUMO

The human genome consists of more than 20000 genes and is essential for all biological phenomena. To understand these biological phenomena, including diseases, and to be able to modify them, approaches that enable optical control of the genome may be useful. Recently, we developed an optogenetic tool, named photoactivatable Cas9 (PA-Cas9). We divided Cas9 nuclease from the CRISPR-Cas9 system into two fragments and connected photo-inducible dimerization proteins, named Magnet system, to the fragments, leading to the development of PA-Cas9 of which nuclease activity is switchable with light. PA-Cas9 allows direct editing of DNA sequences by light stimulation. Additionally, we developed a light-inducible, RNA-guided programmable system for endogenous gene activation based on the CRISPR-Cas9 system. We demonstrated that this optogenetic tool allows rapid and reversible targeted gene activation by light. Using this tool, we exemplified optical control of neuronal differentiation of human induced pluripotent stem cells (iPSCs). The CRISPR-Cas9-based, photoactivatable transcription system offers a simple and versatile approach to precise gene activation. In addition to the CRISPR-Cas9-based optogenetic tools, we developed a photoactivatable Cre-loxP system. This tool allows optical control of DNA recombination reaction in an internal organ even by external, noninvasive illumination using LED light source. To date, genome engineering technology and optogenetics technology have emerged separately as different applications. Our studies described above merge these emerging research fields together.


Assuntos
Proteína 9 Associada à CRISPR , Sistemas CRISPR-Cas , Engenharia Genética , Luz , Optogenética , Ativação Transcricional , Animais , Diferenciação Celular , DNA/genética , Edição de Genes , Genoma Humano , Humanos , Células-Tronco Pluripotentes Induzidas/fisiologia , Camundongos , Recombinação Genética
7.
Nat Commun ; 11(1): 4267, 2020 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-32848148

RESUMO

While footprinting analysis of ATAC-seq data can theoretically enable investigation of transcription factor (TF) binding, the lack of a computational tool able to conduct different levels of footprinting analysis has so-far hindered the widespread application of this method. Here we present TOBIAS, a comprehensive, accurate, and fast footprinting framework enabling genome-wide investigation of TF binding dynamics for hundreds of TFs simultaneously. We validate TOBIAS using paired ATAC-seq and ChIP-seq data, and find that TOBIAS outperforms existing methods for bias correction and footprinting. As a proof-of-concept, we illustrate how TOBIAS can unveil complex TF dynamics during zygotic genome activation in both humans and mice, and propose how zygotic Dux activates cascades of TFs, binds to repeat elements and induces expression of novel genetic elements.


Assuntos
Sequenciamento de Cromatina por Imunoprecipitação/métodos , Fatores de Transcrição/metabolismo , Ativação Transcricional , Zigoto/metabolismo , Animais , Sítios de Ligação/genética , Desenvolvimento Embrionário/genética , Epigênese Genética , Feminino , Genoma Humano , Proteínas de Homeodomínio/metabolismo , Humanos , Cinética , Camundongos , Regiões Promotoras Genéticas , Estudo de Prova de Conceito , Ligação Proteica/genética , Especificidade da Espécie
8.
Ecotoxicol Environ Saf ; 204: 111063, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32791358

RESUMO

The lipocalins genes have been assigned for involving in the responses of organisms to various stress factors. The function of lipocalins under PCB18 stress was addressed by pathway complementation in the Oryza sativa L. OsTIL-silenced mutant. The growth of wild type (WT) and OsTIL-silenced mutant (MT) callus were suppressed by PCB18, and MT varieties were inhibited more seriously than WT varieties. Meanwhile, only WT varieties showed "Hormesis" effect. Compared with WT (3 day > 90.0%, 6 day ≤45.5%), MT varieties kept high removing efficiency by HPLC analysis. Varied gene transcription after OsTIL silencing was demonstrated between two varieties, especially obvious under PCB stress. Silenced OsTIL induced more protective gene transcriptions by qPCR analysis, OsVDE at 3 day, OsCHL, OsZEP1, OsZEP2 and OsUN at 6 day and OsZEP2 at 9 day. PCB18 stress further irritated these genes transcription in MT varieties. The defense stagy in WT varieties was that the transcriptions of lipocalins were inhibited to reduce PCB18 accumulation and toxicity. OsTIL could effectively limit PCB18 accumulation and toxicity. After TIL lacking, OsCHL, OsZEP1, OsZEP2 and OsUN in mutant were strongly evoked to against PCB stress. Remarkably, OsUN and OsZEP2 gene expressions were responded to PCB18 stress in both two varieties.


Assuntos
Poluentes Ambientais/toxicidade , Lipocalinas/genética , Oryza/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Bifenilos Policlorados/toxicidade , Ativação Transcricional/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas , Inativação Gênica , Genes de Plantas , Oryza/genética , Oryza/crescimento & desenvolvimento , Estresse Oxidativo/genética
9.
PLoS Genet ; 16(8): e1008505, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32776934

RESUMO

Dynamic gene expression in neurons shapes fundamental processes in the nervous systems of animals. However, how neuronal activation by different stimuli can lead to distinct transcriptional responses is not well understood. We have been studying how microbial metabolites modulate gene expression in chemosensory neurons of Caenorhabditis elegans. Considering the diverse environmental stimuli that can activate chemosensory neurons of C. elegans, we sought to understand how specific transcriptional responses can be generated in these neurons in response to distinct cues. We have focused on the mechanism of rapid (<6 min) and selective transcriptional induction of daf-7, a gene encoding a TGF-ß ligand, in the ASJ chemosensory neurons in response to the pathogenic bacterium Pseudomonas aeruginosa. DAF-7 is required for the protective behavioral avoidance of P. aeruginosa by C. elegans. Here, we define the involvement of two distinct cyclic GMP (cGMP)-dependent pathways that are required for daf-7 expression in the ASJ neuron pair in response to P. aeruginosa. We show that a calcium-independent pathway dependent on the cGMP-dependent protein kinase G (PKG) EGL-4, and a canonical calcium-dependent signaling pathway dependent on the activity of a cyclic nucleotide-gated channel subunit CNG-2, function in parallel to activate rapid, selective transcription of daf-7 in response to P. aeruginosa metabolites. Our data suggest that fast, selective early transcription of neuronal genes require PKG in shaping responses to distinct microbial stimuli in a pair of C. elegans chemosensory neurons.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Células Quimiorreceptoras/metabolismo , GMP Cíclico/metabolismo , Pseudomonas aeruginosa/metabolismo , Fator de Crescimento Transformador beta/genética , Animais , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/metabolismo , Sinalização do Cálcio , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Canais de Cátion Regulados por Nucleotídeos Cíclicos/metabolismo , Ativação Transcricional , Fator de Crescimento Transformador beta/metabolismo
10.
PLoS Genet ; 16(8): e1009005, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32841236

RESUMO

Azole drugs are the most frequently used antifungal agents. The pathogenic yeast Candida glabrata acquires resistance to azole drugs via single amino acid substitution mutations eliciting a gain-of-function (GOF) hyperactive phenotype in the Pdr1 transcription factor. These GOF mutants constitutively drive high transcription of target genes such as the ATP-binding cassette transporter-encoding CDR1 locus. Previous characterization of Pdr1 has demonstrated that this factor is negatively controlled by the action of a central regulatory domain (CRD) of ~700 amino acids, in which GOF mutations are often found. Our earlier experiments demonstrated that a Pdr1 derivative in which the CRD was deleted gave rise to a transcriptional regulator that could not be maintained as the sole copy of PDR1 in the cell owing to its toxically high activity. Using a set of GOF PDR1 alleles from azole-resistant clinical isolates, we have analyzed the mechanisms acting to repress Pdr1 transcriptional activity. Our data support the view that Pdr1-dependent transactivation is mediated by a complex network of transcriptional coactivators interacting with the extreme C-terminal part of Pdr1. These coactivators include but are not limited to the Mediator component Med15A. Activity of this C-terminal domain is controlled by the CRD and requires multiple regions across the C-terminus for normal function. We also provide genetic evidence for an element within the transactivation domain that mediates the interaction of Pdr1 with coactivators on one hand while restricting Pdr1 activity on the other hand. These data indicate that GOF mutations in PDR1 block nonidentical negative inputs that would otherwise restrain Pdr1 transcriptional activation. The strong C-terminal transactivation domain of Pdr1 uses multiple different protein regions to recruit coactivators.


Assuntos
Candida glabrata/efeitos dos fármacos , Candidíase/tratamento farmacológico , Farmacorresistência Fúngica/genética , Proteínas Fúngicas/genética , Antifúngicos/efeitos adversos , Antifúngicos/farmacologia , Azóis/efeitos adversos , Azóis/farmacologia , Candida glabrata/genética , Candida glabrata/patogenicidade , Candidíase/genética , Candidíase/microbiologia , Proteínas de Ligação a DNA , Farmacorresistência Fúngica/efeitos dos fármacos , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Humanos , Ativação Transcricional/efeitos dos fármacos
11.
Nucleic Acids Res ; 48(15): 8393-8407, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32619221

RESUMO

The glucocorticoid receptor is an important immunosuppressive drug target and metabolic regulator that acts as a ligand-gated transcription factor. Generally, GR's anti-inflammatory effects are attributed to the silencing of inflammatory genes, while its adverse effects are ascribed to the upregulation of metabolic targets. GR binding directly to DNA is proposed to activate, whereas GR tethering to pro-inflammatory transcription factors is thought to repress transcription. Using mice with a point mutation in GR's zinc finger, that still tether via protein-protein interactions while being unable to recognize DNA, we demonstrate that DNA binding is essential for both transcriptional activation and repression. Performing ChIP-Seq, RNA-Seq and proteomics under inflammatory conditions, we show that DNA recognition is required for the assembly of a functional co-regulator complex to mediate glucocorticoid responses. Our findings may contribute to the development of safer immunomodulators with fewer side effects.


Assuntos
Proteínas de Ligação a DNA/genética , DNA/genética , Inflamação/genética , Receptores de Glucocorticoides/genética , Animais , DNA/metabolismo , Regulação da Expressão Gênica/genética , Glucocorticoides/genética , Glucocorticoides/metabolismo , Humanos , Inflamação/patologia , Camundongos , Domínios e Motivos de Interação entre Proteínas/genética , RNA-Seq , Ativação Transcricional/genética
12.
Nucleic Acids Res ; 48(15): 8332-8348, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32633757

RESUMO

Negative cofactor 2 (NC2), including two subunits NC2α and NC2ß, is a conserved positive/negative regulator of class II gene transcription in eukaryotes. It is known that NC2 functions by regulating the assembly of the transcription preinitiation complex. However, the exact role of NC2 in transcriptional regulation is still unclear. Here, we reveal that, in Neurospora crassa, NC2 activates catalase-3 (cat-3) gene transcription in the form of heterodimer mediated by histone fold (HF) domains of two subunits. Deletion of HF domain in either of two subunits disrupts the NC2α-NC2ß interaction and the binding of intact NC2 heterodimer to cat-3 locus. Loss of NC2 dramatically increases histone variant H2A.Z deposition at cat-3 locus. Further studies show that NC2 recruits chromatin remodeling complex INO80C to remove H2A.Z from the nucleosomes around cat-3 locus, resulting in transcriptional activation of cat-3. Besides HF domains of two subunits, interestingly, C-terminal repression domain of NC2ß is required not only for NC2 binding to cat-3 locus, but also for the recruitment of INO80C to cat-3 locus and removal of H2A.Z from the nucleosomes. Collectively, our findings reveal a novel mechanism of NC2 in transcription activation through recruiting INO80C to remove H2A.Z from special H2A.Z-containing nucleosomes.


Assuntos
Catalase/genética , Fosfoproteínas/genética , Fatores de Transcrição/genética , Transcrição Genética , Núcleo Celular/genética , Montagem e Desmontagem da Cromatina/genética , Regulação da Expressão Gênica/genética , Genes MHC da Classe II/genética , Histonas/genética , Neurospora crassa/genética , Nucleossomos/genética , Nucleossomos/ultraestrutura , Fosfoproteínas/ultraestrutura , Ligação Proteica/genética , Fatores de Transcrição/ultraestrutura , Ativação Transcricional/genética
13.
Nucleic Acids Res ; 48(15): 8408-8430, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32663283

RESUMO

The chromatin remodelers SWI/SNF and RSC function in evicting promoter nucleosomes at highly expressed yeast genes, particularly those activated by transcription factor Gcn4. Ino80 remodeling complex (Ino80C) can establish nucleosome-depleted regions (NDRs) in reconstituted chromatin, and was implicated in removing histone variant H2A.Z from the -1 and +1 nucleosomes flanking NDRs; however, Ino80C's function in transcriptional activation in vivo is not well understood. Analyzing the cohort of Gcn4-induced genes in ino80Δ mutants has uncovered a role for Ino80C on par with SWI/SNF in evicting promoter nucleosomes and transcriptional activation. Compared to SWI/SNF, Ino80C generally functions over a wider region, spanning the -1 and +1 nucleosomes, NDR and proximal genic nucleosomes, at genes highly dependent on its function. Defects in nucleosome eviction in ino80Δ cells are frequently accompanied by reduced promoter occupancies of TBP, and diminished transcription; and Ino80 is enriched at genes requiring its remodeler activity. Importantly, nuclear depletion of Ino80 impairs promoter nucleosome eviction even in a mutant lacking H2A.Z. Thus, Ino80C acts widely in the yeast genome together with RSC and SWI/SNF in evicting promoter nucleosomes and enhancing transcription, all in a manner at least partly independent of H2A.Z editing.


Assuntos
Histonas/genética , Proteínas de Saccharomyces cerevisiae/genética , Transcrição Genética , Ativação Transcricional/genética , Adenosina Trifosfatases/genética , Cromatina/genética , Montagem e Desmontagem da Cromatina/genética , Proteínas de Ligação a DNA/genética , Regulação Fúngica da Expressão Gênica/genética , Nucleossomos/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Fatores de Transcrição/genética
14.
Gene ; 760: 144990, 2020 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-32721476

RESUMO

The MYB transcription factors are involved in the regulation of plant secondary metabolism, cell development and morphogenesis, and stress response. Here, a full-length, 816-bp NtMYB4a cDNA, which encodes a protein comprising 271 amino acids, was isolated from tobacco leaves. Phylogenetic analysis revealed that NtMYB4a is most similar to Nicotiana. attenuata MYB4, followed by Eriobotrya japonica MYB4, and NtMYB4a clustered with transcriptional activators rather than repressors. Subcellular localization assays showed that NtMYB4 localized in the nucleus, membrane, and cytoplasm. Expression analyses revealed differential expression of NtMYB4a among different tissues and organs and between different developmental stages, with most expression occurring in the stems and leaves during the full-bloom stage. Moreover, NtMYB4a expression was induced by cold, NaCl, PEG, abscisic acid, methyl jasmonate, and dark stressors, and the expression patterns and maximum expression levels varied with the type of stress. Overexpression of NtMYB4a upregulated NtPAL, Nt4CL, NtCHS, NtCHI, NtF3H, NtDFR, NtANS, and NtUFGT, which resulted in increased anthocyanin content in the tobacco corolla and darker colors. However, CRISPR/Cas9-mediated knockout of NtMYB4a downregulated NtPAL, NtC4H, Nt4CL, NtCHS, NtCHI, NtF3H, NtANS, and NtUFGT, which resulted in reduced anthocyanin content, and lighter corolla colors. These results indicated that NtMYB4a positively regulates anthocyanin biosynthesis and is involved in abiotic stress responses in tobacco plants.


Assuntos
Tabaco/metabolismo , Fatores de Transcrição/isolamento & purificação , Fatores de Transcrição/metabolismo , Ácido Abscísico/metabolismo , Sequência de Aminoácidos , Antocianinas/biossíntese , Regulação da Expressão Gênica de Plantas/genética , Filogenia , Folhas de Planta/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/genética , Estresse Fisiológico/genética , Tabaco/genética , Fatores de Transcrição/genética , Ativação Transcricional/genética
15.
PLoS One ; 15(7): e0236508, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32726333

RESUMO

Extracellular vesicles (EVs) are cell membrane-derived phospholipid bilayer nanostructures that contain bioactive proteins, enzymes, lipids and polymers of nucleotides. They play a role in intercellular communication and are present in body fluids. EVs can be isolated by methods like ultracentrifugation (UC), polyethylene-glycol-precipitation (PEG) or size exclusion chromatography (SEC). The co-presence of immunoglobulins (Ig) in EV samples isolated from plasma (pEVs) is often reported and this may influence the assessment of the biological function and phenotype of EVs in bio- and immunoassay. Here, we studied the presence of an Ig-based therapeutic (etanercept) in pEV samples isolated from rheumatoid arthritis (RA) patients and improved the isolation method to obtain purer pEVs. From plasma of etanercept (Tumor-necrosis-factor (TNF)-α antibodies)-treated RA patients pEVs were isolated by either UC, PEG or SEC. SEC isolated pEVs showed the highest particle-to-protein ratio. Strong TNF-α inhibition determined in a TNF-α sensitive reporter assay was observed by pEVs isolated by UC and PEG, and to a lesser extent by SEC, suggesting the presence of functional etanercept. SEC isolation of etanercept or labelled immunoglobulin G (IgG) showed co-isolation of these antibodies in the pEV fraction in the presence of plasma or a high protein (albumin) concentration. To minimize the presence of etanercept or immunoglobulins, we extended SEC (eSEC) column length from 56mm to 222mm (total stacking volume unchanged). No effect on the amount of isolated pEVs was observed while protein and IgG content were markedly reduced (90%). Next, from six etanercept- treated RA patients, pEVs were isolated on a eSEC or standard SEC column, in parallel. TNF-α inhibition was again observed in pEVs isolated by conventional SEC but not by eSEC. To confirm the purer pEVs isolated by eSEC the basal IL-8 promoter activation in human monocytes was determined and in 4 out of 5 SEC isolated pEVs activation was observed while eSEC isolated pEVs did not. This study shows that extended SEC columns yielded pEVs without detectable biologicals and with low protein and IgG levels. This isolation method will improve the characterization of pEVs as potential biomarkers and mediators of disease.


Assuntos
Produtos Biológicos/sangue , Proteínas Sanguíneas/análise , Vesículas Extracelulares/metabolismo , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/patologia , Cromatografia em Gel , Etanercepte/sangue , Etanercepte/uso terapêutico , Vesículas Extracelulares/química , Humanos , Imunoglobulina G/análise , Interleucina-8/genética , Regiões Promotoras Genéticas , Ativação Transcricional , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/metabolismo
16.
Proc Natl Acad Sci U S A ; 117(28): 16292-16301, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32601208

RESUMO

Notch pathway signaling is implicated in several human cancers. Aberrant activation and mutations of Notch signaling components are linked to tumor initiation, maintenance, and resistance to cancer therapy. Several strategies, such as monoclonal antibodies against Notch ligands and receptors, as well as small-molecule γ-secretase inhibitors (GSIs), have been developed to interfere with Notch receptor activation at proximal points in the pathway. However, the use of drug-like small molecules to target the downstream mediators of Notch signaling, the Notch transcription activation complex, remains largely unexplored. Here, we report the discovery of an orally active small-molecule inhibitor (termed CB-103) of the Notch transcription activation complex. We show that CB-103 inhibits Notch signaling in primary human T cell acute lymphoblastic leukemia and other Notch-dependent human tumor cell lines, and concomitantly induces cell cycle arrest and apoptosis, thereby impairing proliferation, including in GSI-resistant human tumor cell lines with chromosomal translocations and rearrangements in Notch genes. CB-103 produces Notch loss-of-function phenotypes in flies and mice and inhibits the growth of human breast cancer and leukemia xenografts, notably without causing the dose-limiting intestinal toxicity associated with other Notch inhibitors. Thus, we describe a pharmacological strategy that interferes with Notch signaling by disrupting the Notch transcription complex and shows therapeutic potential for treating Notch-driven cancers.


Assuntos
Receptores Notch/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Ativação Transcricional/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Sítios de Ligação , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Drosophila , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Células HeLa , Humanos , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/química , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/genética , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/metabolismo , Intestino Delgado/efeitos dos fármacos , Intestino Delgado/metabolismo , Camundongos , Mutação , Fenótipo , Multimerização Proteica , Transdução de Sinais/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/uso terapêutico
17.
Anticancer Res ; 40(8): 4687-4694, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32727793

RESUMO

BACKGROUND/AIM: The Japanese apricot "Prunus mume" is a traditional Japanese medicine. MK615, a compound extract from Prunus mume has been reported to have anti-tumor effects. Herein, we used 3D floating (3DF) culture to evaluate the anticancer effects of MK615 against human colorectal cancer (CRC) cells that contain mutant (mt) KRAS. MATERIALS AND METHODS: HKe3 cells exogenously expressing mtKRAS (HKe3-mtKRAS) were treated with MK615 in 3DF cultures. The protein levels of hypoxia-inducible factor 1 (HIF-1) and E-cadherin were quantified by western blotting. RESULTS: MtKRAS enhanced hypoxia tolerance via up-regulation of HIF-1. The expression of HIF-1 protein was suppressed by constitutive overexpression of E-cadherin in CRC HCT116 spheroids. MK615 increased the expression of E-cadherin and decreased the expression of HIF-1 in HKe3-mtKRAS. These results suggest that MK615 suppresses hypoxia tolerance by up-regulation of E-cadherin in CRC cells with mtKRAS. CONCLUSION: MK615 exhibits properties useful for the potential treatment of CRC patients with mtKRAS.


Assuntos
Antígenos CD/metabolismo , Caderinas/metabolismo , Hipóxia Celular/fisiologia , Neoplasias do Colo/metabolismo , Neoplasias Colorretais/metabolismo , Extratos Vegetais/farmacologia , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Regulação para Cima/efeitos dos fármacos , Hipóxia Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/patologia , Células HCT116 , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Prunus/química , Ativação Transcricional/efeitos dos fármacos
18.
Gene ; 758: 144954, 2020 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-32683079

RESUMO

Teosinte branched1/cycloidea/proliferating cell factor1 (TCP) is a plant-specific protein family member involved in plant growth and development. However, the functions of most members of the cotton TCP family are unknown. In this study, the GbTCP5 gene encodes a sea-island cotton class II TCP CIN subclass transcription factor. The GbTCP5 transcription factor is located in the nucleus, has transcriptional activation activity, and can bind to TCP II cis-acting elements. GbTCP5 was widely expressed in tissues with the highest transcript level in the calyx. GbTCP5 is expressed at different developmental stages of the fiber and has significantly high transcriptional level expression in the fibers at 20, 30 and 35 days post anthesis (DPA). Heterologous overexpression of the GbTCP5 gene increased root hair length, root hair and stem trichome density, and stem lignin content in transgenic Arabidopsis compared to the wild type (WT). GbTCP5 binds the promoters of the GL3, EGL3, CPC, MYB46, LBD30, CesA4, VND7, CCOMT1, and CAD5 genes to upregulate their expression. Moreover, the homologous genes of these genes are expressed in the fibers of different developmental stages of the sea-island cotton fiber. These results indicate that GbTCP5 regulates root hair development and secondary wall formation in Arabidopsis and may be a candidate gene for improving cotton fiber quality.


Assuntos
Arabidopsis/genética , Gossypium/genética , Lignina/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Fatores de Transcrição/genética , Tricomas/crescimento & desenvolvimento , Arabidopsis/crescimento & desenvolvimento , Fibra de Algodão/análise , Proteínas de Ligação a DNA/genética , Raízes de Plantas/fisiologia , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Regiões Promotoras Genéticas/genética , Ativação Transcricional/genética
19.
Nat Commun ; 11(1): 3420, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32647127

RESUMO

Remyelination of the peripheral and central nervous systems (PNS and CNS, respectively) is a prerequisite for functional recovery after lesion. However, this process is not always optimal and becomes inefficient in the course of multiple sclerosis. Here we show that, when acetylated, eukaryotic elongation factor 1A1 (eEF1A1) negatively regulates PNS and CNS remyelination. Acetylated eEF1A1 (Ac-eEF1A1) translocates into the nucleus of myelinating cells where it binds to Sox10, a key transcription factor for PNS and CNS myelination and remyelination, to drag Sox10 out of the nucleus. We show that the lysine acetyltransferase Tip60 acetylates eEF1A1, whereas the histone deacetylase HDAC2 deacetylates eEF1A1. Promoting eEF1A1 deacetylation maintains the activation of Sox10 target genes and increases PNS and CNS remyelination efficiency. Taken together, these data identify a major mechanism of Sox10 regulation, which appears promising for future translational studies on PNS and CNS remyelination.


Assuntos
Fator 1 de Elongação de Peptídeos/metabolismo , Remielinização/genética , Ativação Transcricional/genética , Acetilação , Envelhecimento/metabolismo , Animais , Desdiferenciação Celular/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Histona Desacetilase 1/metabolismo , Histona Desacetilase 2/metabolismo , Lisina Acetiltransferase 5/metabolismo , Camundongos , Modelos Biológicos , Oligodendroglia/efeitos dos fármacos , Oligodendroglia/metabolismo , Sistema Nervoso Periférico/efeitos dos fármacos , Sistema Nervoso Periférico/fisiologia , Recuperação de Função Fisiológica/efeitos dos fármacos , Remielinização/efeitos dos fármacos , Fatores de Transcrição SOXE/metabolismo , Fator de Transcrição STAT3/metabolismo , Células de Schwann/efeitos dos fármacos , Células de Schwann/metabolismo , Teofilina/farmacologia , Transativadores/metabolismo , Ativação Transcricional/efeitos dos fármacos
20.
Nat Commun ; 11(1): 3501, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32647108

RESUMO

While most monogenic diseases are caused by loss or reduction of protein function, the need for technologies that can selectively increase levels of protein in native tissues remains. Here we demonstrate that antisense-mediated modulation of pre-mRNA splicing can increase endogenous expression of full-length protein by preventing naturally occurring non-productive alternative splicing and promoting generation of productive mRNA. Bioinformatics analysis of RNA sequencing data identifies non-productive splicing events in 7,757 protein-coding human genes, of which 1,246 are disease-associated. Antisense oligonucleotides targeting multiple types of non-productive splicing events lead to increases in productive mRNA and protein in a dose-dependent manner in vitro. Moreover, intracerebroventricular injection of two antisense oligonucleotides in wild-type mice leads to a dose-dependent increase in productive mRNA and protein in the brain. The targeting of natural non-productive alternative splicing to upregulate expression from wild-type or hypomorphic alleles provides a unique approach to treating genetic diseases.


Assuntos
Processamento Alternativo , Regulação da Expressão Gênica , Oligonucleotídeos Antissenso/farmacologia , Alelos , Animais , Animais Recém-Nascidos , Encéfalo/metabolismo , Biologia Computacional , Éxons , Feminino , Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Íntrons , Masculino , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/metabolismo , Ativação Transcricional/efeitos dos fármacos , Regulação para Cima
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