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1.
Nat Commun ; 12(1): 1987, 2021 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-33790284

RESUMO

A widely regarded model for glucocorticoid receptor (GR) action postulates that dimeric binding to DNA regulates unfavorable metabolic pathways while monomeric receptor binding promotes repressive gene responses related to its anti-inflammatory effects. This model has been built upon the characterization of the GRdim mutant, reported to be incapable of DNA binding and dimerization. Although quantitative live-cell imaging data shows GRdim as mostly dimeric, genomic studies based on recovery of enriched half-site response elements suggest monomeric engagement on DNA. Here, we perform genome-wide studies on GRdim and a constitutively monomeric mutant. Our results show that impairing dimerization affects binding even to open chromatin. We also find that GRdim does not exclusively bind half-response elements. Our results do not support a physiological role for monomeric GR and are consistent with a common mode of receptor binding via higher order structures that drives both the activating and repressive actions of glucocorticoids.


Assuntos
DNA/metabolismo , Estudo de Associação Genômica Ampla/métodos , Multimerização Proteica , Receptores de Glucocorticoides/química , Receptores de Glucocorticoides/metabolismo , Animais , Cromatina/genética , Cromatina/metabolismo , DNA/genética , Regulação da Expressão Gênica , Glucocorticoides/metabolismo , Humanos , Camundongos , Mutação , Ligação Proteica , Receptores de Glucocorticoides/genética , Elementos de Resposta/genética , Transdução de Sinais/genética
2.
Int J Mol Sci ; 22(5)2021 Mar 09.
Artigo em Inglês | MEDLINE | ID: mdl-33803193

RESUMO

The SCN5A gene encodes the α-subunit of the voltage-gated cardiac sodium channel (NaV1.5), a key player in cardiac action potential depolarization. Genetic variants in protein-coding regions of the human SCN5A have been largely associated with inherited cardiac arrhythmias. Increasing evidence also suggests that aberrant expression of the SCN5A gene could increase susceptibility to arrhythmogenic diseases, but the mechanisms governing SCN5A expression are not yet well understood. To gain insights into the molecular basis of SCN5A gene regulation, we used rat gastrocnemius muscle four days following denervation, a process well known to stimulate Scn5a expression. Our results show that denervation of rat skeletal muscle induces the expression of the adult cardiac Scn5a isoform. RNA-seq experiments reveal that denervation leads to significant changes in the transcriptome, with Scn5a amongst the fifty top upregulated genes. Consistent with this increase in expression, ChIP-qPCR assays show enrichment of H3K27ac and H3K4me3 and binding of the transcription factor Gata4 near the Scn5a promoter region. Also, Gata4 mRNA levels are significantly induced upon denervation. Genome-wide analysis of H3K27ac by ChIP-seq suggest that a super enhancer recently described to regulate Scn5a in cardiac tissue is activated in response to denervation. Altogether, our experiments reveal that similar mechanisms regulate the expression of Scn5a in denervated muscle and cardiac tissue, suggesting a conserved pathway for SCN5A expression among striated muscles.


Assuntos
Epigênese Genética , Denervação Muscular , Músculo Esquelético/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.5/biossíntese , Elementos de Resposta , Transcriptoma , Animais , Fator de Transcrição GATA4/genética , Fator de Transcrição GATA4/metabolismo , Masculino , Músculo Esquelético/inervação , Músculo Esquelético/patologia , RNA-Seq , Ratos , Ratos Sprague-Dawley
3.
Plant Mol Biol ; 106(1-2): 49-65, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33625643

RESUMO

KEY MESSAGE: Three novel transcription factors were successfully identified and shown to interact with the trichome-specific THCAS promoter regulatory region. Cannabinoids are important secondary metabolites present in Cannabis sativa L. (cannabis). One cannabinoid that has received considerable attention, 9-tetrahydrocannabinol (THC), is derived from Delta-9-Tetrahydrocannabinolic acid (THCA) and responsible for the mood-altering and pain-relieving effects of cannabis. A detailed understanding of transcriptional control of THCA synthase (THCAS) is currently lacking. The primary site of cannabinoid biosynthesis is the glandular trichomes that form on female flowers. Transcription factors (TFs) have been shown to play an important role in secondary-metabolite biosynthesis and glandular trichome formation in Artemisia annua, Solanum lycopersicum and Humulus lupulus. However, analogous information is not available for cannabis. Here, we characterize a 548 bp fragment of the THCAS promoter and regulatory region that drives trichome-specific expression. Using this promoter fragment in a yeast-one-hybrid screen, we identified 3 novel TFs (CsAP2L1, CsWRKY1 and CsMYB1) and provided evidence that these 3 TFs regulate the THCAS promoter in planta. The O-Box element within the proximal region of the THCAS promoter is necessary for CsAP2L1-induced transcriptional activation of THCAS promoter. Similar to THCAS, the genes for all three TFs have trichome-specific expression, and subcellular localization of the TFs indicates that all three proteins are in the nucleus. CsAP2L1 and THCAS exhibit a similar temporal, spatial and strain-specific gene expression profiles, while those expression patterns of CsWRKY1 and CsMYB1 are opposite from THCAS. Our results identify CsAP2L1 playing a positive role in the regulation of THCAS expression, while CsWRKY1 and CsMYB1 may serve as negative regulators of THCAS expression.


Assuntos
Vias Biossintéticas , Canabinoides/biossíntese , Cannabis/metabolismo , Proteínas de Plantas/metabolismo , Fatores de Transcrição/metabolismo , Cannabis/genética , Flores/genética , Flores/fisiologia , Regulação da Expressão Gênica de Plantas , Filogenia , Proteínas de Plantas/genética , Regiões Promotoras Genéticas/genética , Ligação Proteica , Proteínas Repressoras/metabolismo , Elementos de Resposta/genética , Frações Subcelulares/metabolismo , Fatores de Transcrição/genética , Transcrição Genética
4.
Nucleic Acids Res ; 49(3): 1364-1382, 2021 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-33444431

RESUMO

Sequence-specific protein-DNA interactions are at the heart of the response of the tumor-suppressor p53 to numerous physiological and stress-related signals. Large variability has been previously reported in p53 binding to and transactivating from p53 response elements (REs) due, at least in part, to changes in direct (base) and indirect (shape) readouts of p53 REs. Here, we dissect p53 REs to decipher the mechanism by which p53 optimizes this highly regulated variable level of interaction with its DNA binding sites. We show that hemi-specific binding is more prevalent in p53 REs than previously envisioned. We reveal that sequences flanking the REs modulate p53 binding and activity and show that these effects extend to 4-5 bp from the REs. Moreover, we show here that the arrangement of p53 half-sites within its REs, relative to transcription direction, has been fine-tuned by selection pressure to optimize and regulate the response levels from p53 REs. This directionality in the REs arrangement is at least partly encoded in the structural properties of the REs. Furthermore, we show here that in the p21-5' RE the orientation of the half-sites is such that the effect of the flanking sequences is minimized and we discuss its advantages.


Assuntos
Elementos de Resposta , Ativação Transcricional , Proteína Supressora de Tumor p53/metabolismo , Sítios de Ligação , DNA/química , DNA/metabolismo , Humanos , Conformação de Ácido Nucleico , Ligação Proteica , Regulação para Cima
5.
Mol Cell ; 81(3): 584-598.e5, 2021 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-33444546

RESUMO

Severe-acute-respiratory-syndrome-related coronavirus 2 (SARS-CoV-2) is the positive-sense RNA virus that causes coronavirus disease 2019 (COVID-19). The genome of SARS-CoV-2 is unique among viral RNAs in its vast potential to form RNA structures, yet as much as 97% of its 30 kilobases have not been structurally explored. Here, we apply a novel long amplicon strategy to determine the secondary structure of the SARS-CoV-2 RNA genome at single-nucleotide resolution in infected cells. Our in-depth structural analysis reveals networks of well-folded RNA structures throughout Orf1ab and reveals aspects of SARS-CoV-2 genome architecture that distinguish it from other RNA viruses. Evolutionary analysis shows that several features of the SARS-CoV-2 genomic structure are conserved across ß-coronaviruses, and we pinpoint regions of well-folded RNA structure that merit downstream functional analysis. The native, secondary structure of SARS-CoV-2 presented here is a roadmap that will facilitate focused studies on the viral life cycle, facilitate primer design, and guide the identification of RNA drug targets against COVID-19.


Assuntos
Genoma Viral , Conformação de Ácido Nucleico , RNA Viral , Elementos de Resposta , /genética , Linhagem Celular Tumoral , Humanos , RNA Viral/genética , RNA Viral/metabolismo , /metabolismo
6.
Nat Commun ; 12(1): 484, 2021 01 20.
Artigo em Inglês | MEDLINE | ID: mdl-33473123

RESUMO

The tumor suppressor p53 integrates stress response pathways by selectively engaging one of several potential transcriptomes, thereby triggering cell fate decisions (e.g., cell cycle arrest, apoptosis). Foundational to this process is the binding of tetrameric p53 to 20-bp response elements (REs) in the genome (RRRCWWGYYYN0-13RRRCWWGYYY). In general, REs at cell cycle arrest targets (e.g. p21) are of higher affinity than those at apoptosis targets (e.g., BAX). However, the RE sequence code underlying selectivity remains undeciphered. Here, we identify molecular mechanisms mediating p53 binding to high- and low-affinity REs by showing that key determinants of the code are embedded in the DNA shape. We further demonstrate that differences in minor/major groove widths, encoded by G/C or A/T bp content at positions 3, 8, 13, and 18 in the RE, determine distinct p53 DNA-binding modes by inducing different Arg248 and Lys120 conformations and interactions. The predictive capacity of this code was confirmed in vivo using genome editing at the BAX RE to interconvert the DNA-binding modes, transcription pattern, and cell fate outcome.


Assuntos
Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Apoptose/genética , Ciclo Celular , Pontos de Checagem do Ciclo Celular , Linhagem Celular , DNA/química , Proteínas de Ligação a DNA , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Modelos Moleculares , Conformação Molecular , Ligação Proteica/genética , Elementos de Resposta
7.
PLoS One ; 15(12): e0244255, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33332446

RESUMO

Reactive oxygen species are bona fide intracellular second messengers that influence cell metabolism and aging by mechanisms that are incompletely resolved. Mitochondria generate superoxide that is dis-mutated to hydrogen peroxide, which in turn oxidises cysteine-based enzymes such as phosphatases, peroxiredoxins and redox-sensitive transcription factors to modulate their activity. Signal Transducer and Activator of Transcription 3 (Stat3) has been shown to participate in an oxidative relay with peroxiredoxin II but the impact of Stat3 oxidation on target gene expression and its biological consequences remain to be established. Thus, we created murine embryonic fibroblasts (MEFs) that express either WT-Stat3 or a redox-insensitive mutant of Stat3 (Stat3-C3S). The Stat3-C3S cells differed from WT-Stat3 cells in morphology, proliferation and resistance to oxidative stress; in response to cytokine stimulation, they displayed elevated Stat3 tyrosine phosphorylation and Socs3 expression, implying that Stat3-C3S is insensitive to oxidative inhibition. Comparative analysis of global gene expression in WT-Stat3 and Stat3-C3S cells revealed differential expression (DE) of genes both under basal conditions and during oxidative stress. Using differential gene regulation pattern analysis, we identified 199 genes clustered into 10 distinct patterns that were selectively responsive to Stat3 oxidation. GO term analysis identified down-regulated genes to be enriched for tissue/organ development and morphogenesis and up-regulated genes to be enriched for cell-cell adhesion, immune responses and transport related processes. Although most DE gene promoters contain consensus Stat3 inducible elements (SIEs), our chromatin immunoprecipitation (ChIP) and ChIP-seq analyses did not detect Stat3 binding at these sites in control or oxidant-stimulated cells, suggesting that oxidised Stat3 regulates these genes indirectly. Our further computational analysis revealed enrichment of hypoxia response elements (HREs) within DE gene promoters, implying a role for Hif-1. Experimental validation revealed that efficient stabilisation of Hif-1α in response to oxidative stress or hypoxia required an oxidation-competent Stat3 and that depletion of Hif-1α suppressed the inducible expression of Kcnb1, a representative DE gene. Our data suggest that Stat3 and Hif-1α cooperate to regulate genes involved in immune functions and developmental processes in response to oxidative stress.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Estresse Oxidativo , Regiões Promotoras Genéticas , Elementos de Resposta , Fator de Transcrição STAT3/química , Fator de Transcrição STAT3/fisiologia , Animais , Fibroblastos/citologia , Fibroblastos/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Camundongos , Camundongos Knockout , Transdução de Sinais , Ativação Transcricional
8.
Sci Rep ; 10(1): 16615, 2020 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-33024203

RESUMO

Middle East Respiratory Syndrome coronavirus (MERS-CoV) is a highly virulent pathogen that causes Middle East Respiratory Syndrome (MERS). Anti-MERS-CoV antibodies play an integral role in the prevention and treatment against MERS-CoV infections. Bioactivity is a key quality attribute of therapeutic antibodies, and high accuracy and precision are required. The major methods for evaluating the antiviral effect of antiviral antibodies include neutralization assays using live viruses or pseudoviruses are highly variable. Recent studies have demonstrated that the antibody-dependent cellular cytotoxicity (ADCC) activity of antiviral antibodies is more consistent with the virus clearance effect in vivo than neutralization activity. However, no reports evaluating the ADCC activity of anti-MERS antibodies have been published to date. Here, we describe the development of a robust and reliable cell-based reporter gene assay for the determination of ADCC activity of anti-MERS antibodies using 293T/MERS cells stably expressing the spike protein of MERS-CoV (MERS-S) as target cells and the engineered Jurkat/NFAT-luc/FcγRIIIa stably expressing FcγRIIIA and NFAT reporter gene as effector cells. According to the ICH-Q2 analytical method guidelines, we carefully optimized the experimental conditions and assessed the performance of our assay. In addition, we found that the ADCC activity of afucosylated anti-MERS antibodies is higher than their fucosylated counterparts. The establishment of this ADCC determination system provides a novel method for evaluating the bioactivity of anti-MERS antibodies and improving ADCC activity through modification of N-glycosylation of the Fc segment.


Assuntos
Anticorpos Antivirais/análise , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Infecções por Coronavirus/imunologia , Testes Imunológicos de Citotoxicidade/métodos , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Coronavírus da Síndrome Respiratória do Oriente Médio/metabolismo , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Infecções por Coronavirus/virologia , Genes Reporter , Células HEK293 , Humanos , Células Jurkat , Luciferases/genética , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Fatores de Transcrição NFATC/genética , Receptores de IgG/genética , Receptores de IgG/imunologia , Elementos de Resposta , Glicoproteína da Espícula de Coronavírus/metabolismo , Transfecção
9.
Nucleic Acids Res ; 48(19): 10768-10784, 2020 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-32986841

RESUMO

Estrogen receptor alpha (ERα) signaling pathway is essential for ERα-positive breast cancer progression and endocrine therapy resistance. Bromodomain PHD Finger Transcription Factor (BPTF) associated protein of 18kDa (BAP18) has been recognized as a crucial H3K4me3 reader. However, the whole genomic occupation of BAP18 and its biological function in breast cancer is still elusive. Here, we found that higher expression of BAP18 in ERα-positive breast cancer is positively correlated with poor prognosis. ChIP-seq analysis further demonstrated that the half estrogen response elements (EREs) and the CCCTC binding factor (CTCF) binding sites are the significant enrichment sites found in estrogen-induced BAP18 binding sites. Also, we provide the evidence to demonstrate that BAP18 as a novel co-activator of ERα is required for the recruitment of COMPASS-like core subunits to the cis-regulatory element of ERα target genes in breast cancer cells. BAP18 is recruited to the promoter regions of estrogen-induced genes, accompanied with the enrichment of the lysine 4-trimethylated histone H3 tail (H3K4me3) in the presence of E2. Furthermore, BAP18 promotes cell growth and associates the sensitivity of antiestrogen in ERα-positive breast cancer. Our data suggest that BAP18 facilitates the association between ERα and COMPASS-like core subunits, which might be an essential epigenetic therapeutic target for breast cancer.


Assuntos
Biomarcadores Tumorais/genética , Neoplasias da Mama/genética , Proteínas de Ligação a DNA/genética , Resistencia a Medicamentos Antineoplásicos , Receptor alfa de Estrogênio/genética , Código das Histonas , Animais , Antineoplásicos Hormonais/farmacologia , Antineoplásicos Hormonais/uso terapêutico , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proteínas de Ligação a DNA/metabolismo , Moduladores de Receptor Estrogênico/farmacologia , Moduladores de Receptor Estrogênico/uso terapêutico , Receptor alfa de Estrogênio/antagonistas & inibidores , Receptor alfa de Estrogênio/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Células MCF-7 , Neoplasias Mamárias Experimentais/tratamento farmacológico , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Elementos de Resposta
10.
Nucleic Acids Res ; 48(17): 9969-9985, 2020 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-32974652

RESUMO

Retinoic acid receptors (RARs) as a functional heterodimer with retinoid X receptors (RXRs), bind a diverse series of RA-response elements (RAREs) in regulated genes. Among them, the non-canonical DR0 elements are bound by RXR-RAR with comparable affinities to DR5 elements but DR0 elements do not act transcriptionally as independent RAREs. In this work, we present structural insights for the recognition of DR5 and DR0 elements by RXR-RAR heterodimer using x-ray crystallography, small angle x-ray scattering, and hydrogen/deuterium exchange coupled to mass spectrometry. We solved the crystal structures of RXR-RAR DNA-binding domain in complex with the Rarb2 DR5 and RXR-RXR DNA-binding domain in complex with Hoxb13 DR0. While cooperative binding was observed on DR5, the two molecules bound non-cooperatively on DR0 on opposite sides of the DNA. In addition, our data unveil the structural organization and dynamics of the multi-domain RXR-RAR DNA complexes providing evidence for DNA-dependent allosteric communication between domains. Differential binding modes between DR0 and DR5 were observed leading to differences in conformation and structural dynamics of the multi-domain RXR-RAR DNA complexes. These results reveal that the topological organization of the RAR binding element confer regulatory information by modulating the overall topology and structural dynamics of the RXR-RAR heterodimers.


Assuntos
Sítio Alostérico , Elementos de Resposta , Receptores X Retinoide/química , Regulação Alostérica , DNA/química , DNA/metabolismo , Proteínas de Homeodomínio/genética , Humanos , Simulação de Acoplamento Molecular , Ligação Proteica , Receptores X Retinoide/metabolismo
11.
Nat Commun ; 11(1): 4782, 2020 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-32963223

RESUMO

Polycomb and Trithorax group proteins maintain stable epigenetic memory of gene expression states for some genes, but many targets show highly dynamic regulation. Here we combine experiment and theory to examine the mechanistic basis of these different modes of regulation. We present a mathematical model comprising a Polycomb/Trithorax response element (PRE/TRE) coupled to a promoter and including Drosophila developmental timing. The model accurately recapitulates published studies of PRE/TRE mediated epigenetic memory of both silencing and activation. With minimal parameter changes, the same model can also recapitulate experimental data for a different PRE/TRE that allows dynamic regulation of its target gene. The model predicts that both cell cycle length and PRE/TRE identity are critical for determining whether the system gives stable memory or dynamic regulation. Our work provides a simple unifying framework for a rich repertoire of PRE/TRE functions, and thus provides insights into  genome-wide Polycomb/Trithorax regulation.


Assuntos
Proteínas Cromossômicas não Histona/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Epigenômica , Regulação da Expressão Gênica no Desenvolvimento/genética , Modelos Teóricos , Complexo Repressor Polycomb 1/genética , Animais , Divisão Celular , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/fisiologia , Epigênese Genética , Feminino , Inativação Gênica , Complexo Repressor Polycomb 1/metabolismo , Proteínas do Grupo Polycomb/metabolismo , Regiões Promotoras Genéticas , Elementos de Resposta
12.
Nucleic Acids Res ; 48(18): 10542-10554, 2020 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-32870271

RESUMO

hnRNPA2 is a major component of mRNA transport granules in oligodendrocytes and neurons. However, the structural details of how hnRNPA2 binds the A2 recognition element (A2RE) and if this sequence stimulates granule formation by enhancing phase separation of hnRNPA2 has not yet been studied. Using solution NMR and biophysical studies, we find that each of the two individual RRMs retain the domain structure observed in complex with RNA but are not rigidly confined (i.e. they move independently) in solution in the absence of RNA. hnRNPA2 RRMs bind the minimal rA2RE11 weakly but at least, and most likely, two hnRNPA2 molecules are able to simultaneously bind the longer 21mer myelin basic protein A2RE. Upon binding of the RNA, NMR chemical shift deviations are observed in both RRMs, suggesting both play a role in binding the A2RE11. Interestingly, addition of short A2RE RNAs or longer RNAs containing this sequence completely prevents in vitro phase separation of full-length hnRNPA2 and aggregation of the disease-associated mutants. These findings suggest that RRM interactions with specific recognition sequences alone do not account for nucleating granule formation, consistent with models where multivalent protein:RNA and protein:protein contacts form across many sites in granule proteins and long RNA transcripts.


Assuntos
Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/genética , Motivo de Reconhecimento de RNA/genética , Proteínas de Ligação a RNA/genética , Elementos de Resposta/genética , Sítios de Ligação/genética , Fenômenos Biofísicos , Humanos , Extração Líquido-Líquido , Espectroscopia de Ressonância Magnética , Neurônios/metabolismo , Oligodendroglia/metabolismo , Agregados Proteicos/genética , Ligação Proteica/genética , RNA/genética
13.
Am J Pathol ; 190(11): 2226-2236, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32798443

RESUMO

In a condition of dysfunctional visceral fat depots, as in the case of obesity, alterations in adipokine levels may be detrimental for the cardiovascular system. The proinflammatory leptin and resistin adipokines have been described as possible links between obesity and atherosclerosis. The present study was aimed at evaluating whether proprotein convertase subtilisin/kexin type 9 (PCSK9), a key regulator of low-density lipoprotein metabolism, is induced by leptin and resistin through the involvement of the inflammatory pathway of STAT3. In HepG2 cells, leptin and resistin up-regulated PCSK9 gene and protein expression, as well as the phosphorylation of STAT3. Upon STAT3 silencing, leptin and resistin lost their ability to activate PCSK9. The knockdown of STAT3 did not affect the expression of leptin and resistin receptors or that of PCSK9. The analysis of the human PCSK9 promoter region showed that the two adipokines raised PCSK9 promoter activity via the involvement of a sterol regulatory element motif. In healthy males, a positive association between circulating leptin and PCSK9 levels was found only when the body mass index was <25 kg/m2. In conclusion, this study identified STAT3 as one of the molecular regulators of leptin- and resistin-mediated transcriptional induction of PCSK9.


Assuntos
Regulação Enzimológica da Expressão Gênica , Leptina/metabolismo , Pró-Proteína Convertase 9/biossíntese , Resistina/metabolismo , Fator de Transcrição STAT3/metabolismo , Regulação para Cima , Aterosclerose/genética , Aterosclerose/metabolismo , Aterosclerose/patologia , Células Hep G2 , Humanos , Leptina/genética , Obesidade/genética , Obesidade/metabolismo , Obesidade/patologia , Pró-Proteína Convertase 9/genética , Resistina/genética , Elementos de Resposta , Fator de Transcrição STAT3/genética
14.
PLoS One ; 15(8): e0236881, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32745107

RESUMO

PIERCE1, p53 induced expression 1 in Rb null cells, is a novel p53 target involved in the DNA damage response and cell cycle in mice. These facts prompted us to study the function of PIERCE1 with respect to p53-associated pathophysiology of cancer in humans. Unexpectedly, PIERCE1 did not respond to overexpression and activation of p53 in humans. In this study, we swapped p53 protein expression in human and mouse cells to find the clue of this difference between species. Human p53 expression in mouse cells upregulated PIERCE1 expression, suggesting that p53-responsive elements on the PIERCE1 promoter are crucial, but not the p53 protein itself. Indeed, in silico analyses of PIERCE1 promoters revealed that p53-responsive elements identified in mice are not conserved in humans. Consistently, chromatin immunoprecipitation-sequencing (ChIP-seq) analyses confirmed p53 enrichment against the PIERCE1 promoter region in mice, not in human cells. To complement the p53 study in mice, further promoter analyses suggested that the human PIERCE1 promoter is more similar to guinea pigs, lemurs, and dogs than to rodents. Taken together, our results confirm the differential responsiveness of PIERCE1 expression to p53 due to species differences in PIERCE1 promoters. The results also show partial dissimilarity after p53 induction between mice and humans.


Assuntos
Proteínas de Ciclo Celular , Elementos de Resposta/genética , Proteína Supressora de Tumor p53/metabolismo , Animais , Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Dano ao DNA/genética , Humanos , Camundongos , Regiões Promotoras Genéticas , Transcrição Genética/fisiologia
15.
J Virol ; 94(18)2020 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-32641480

RESUMO

We previously reported that the cellular transcription factor hypoxia-inducible factor 1α (HIF-1α) binds a hypoxia response element (HRE) located within the promoter of Epstein-Barr virus's (EBV's) latent-lytic switch BZLF1 gene, Zp, inducing viral reactivation. In this study, EBV-infected cell lines derived from gastric cancers and Burkitt lymphomas were incubated with HIF-1α-stabilizing drugs: the iron chelator deferoxamine (Desferal [DFO]), a neddylation inhibitor (pevonedistat [MLN-4924]), and a prolyl hydroxylase inhibitor (roxadustat [FG-4592]). DFO and MLN-4924, but not FG-4592, induced accumulation of both lytic EBV proteins and phosphorylated p53 in cell lines that contain a wild-type p53 gene. FG-4592 also failed to activate transcription from Zp in a reporter assay despite inducing accumulation of HIF-1α and transcription from another HRE-containing promoter. Unexpectedly, DFO failed to induce EBV reactivation in cell lines that express mutant or no p53 or when p53 expression was knocked down with short hairpin RNAs (shRNAs). Likewise, HIF-1α failed to activate transcription from Zp when p53 was knocked out by CRISPR-Cas9. Importantly, DFO induced binding of p53 as well as HIF-1α to Zp in chromatin immunoprecipitation (ChIP) assays, but only when the HRE was present. Nutlin-3, a drug known to induce accumulation of phosphorylated p53, synergized with DFO and MLN-4924 in inducing EBV reactivation. Conversely, KU-55933, a drug that inhibits ataxia telangiectasia mutated, thereby preventing p53 phosphorylation, inhibited DFO-induced EBV reactivation. Lastly, activation of Zp transcription by DFO and MLN-4924 mapped to its HRE. Thus, we conclude that induction of BZLF1 gene expression by HIF-1α requires phosphorylated, wild-type p53 as a coactivator, with HIF-1α binding recruiting p53 to Zp.IMPORTANCE EBV, a human herpesvirus, is latently present in most nasopharyngeal carcinomas, Burkitt lymphomas, and some gastric cancers. To develop a lytic-induction therapy for treating patients with EBV-associated cancers, we need a way to efficiently reactivate EBV into lytic replication. EBV's BZLF1 gene product, Zta, usually controls this reactivation switch. We previously showed that HIF-1α binds the BZLF1 gene promoter, inducing Zta synthesis, and HIF-1α-stabilizing drugs can induce EBV reactivation. In this study, we determined which EBV-positive cell lines are reactivated by classes of HIF-1α-stabilizing drugs. We found, unexpectedly, that HIF-1α-stabilizing drugs only induce reactivation when they also induce accumulation of phosphorylated, wild-type p53. Fortunately, p53 phosphorylation can also be provided by drugs such as nutlin-3, leading to synergistic reactivation of EBV. These findings indicate that some HIF-1α-stabilizing drugs may be helpful as part of a lytic-induction therapy for treating patients with EBV-positive malignancies that contain wild-type p53.


Assuntos
Herpesvirus Humano 4/genética , Interações Hospedeiro-Patógeno/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Transativadores/genética , Proteína Supressora de Tumor p53/genética , Linhagem Celular Tumoral , Ciclopentanos/farmacologia , Desferroxamina/farmacologia , Inibidores Enzimáticos/farmacologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/virologia , Regulação da Expressão Gênica , Glicina/análogos & derivados , Glicina/farmacologia , Herpesvirus Humano 4/efeitos dos fármacos , Herpesvirus Humano 4/crescimento & desenvolvimento , Herpesvirus Humano 4/metabolismo , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/agonistas , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Imidazóis/farmacologia , Quelantes de Ferro/farmacologia , Isoquinolinas/farmacologia , Linfócitos/efeitos dos fármacos , Linfócitos/metabolismo , Linfócitos/virologia , Morfolinas/farmacologia , Piperazinas/farmacologia , Inibidores de Prolil-Hidrolase/farmacologia , Regiões Promotoras Genéticas , Ligação Proteica/efeitos dos fármacos , Pirimidinas/farmacologia , Pironas/farmacologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Elementos de Resposta , Transdução de Sinais , Transativadores/metabolismo , Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína Supressora de Tumor p53/metabolismo , Ativação Viral/efeitos dos fármacos
16.
J Virol ; 94(18)2020 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-32641483

RESUMO

The Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded open reading frame 50 (ORF50) protein is the key transactivator responsible for the latent-to-lytic switch. Here, we investigated the transcriptional activation of the ORF56 gene (encoding a primase protein) by ORF50 and successfully identified an ORF50-responsive element located in the promoter region between positions -97 and -44 (designated 56p-RE). This 56p-RE element contains a noncanonical RBP-Jκ-binding sequence and a nonconsensus Sp1/Sp3-binding sequence. Electrophoretic mobility shift assays revealed that RBP-Jκ, Sp3, and ORF50 could form stable complexes on the 56p-RE element. Importantly, transient-reporter analysis showed that Sp3, but not RBP-Jκ or Sp1, acts in synergy with ORF50 to activate the 56p-RE-containing reporter construct, and the synergy mainly depends on the Sp1/Sp3-binding region of the 56p-RE element. Sequence similarity searches revealed that the promoters for ORF21 (thymidine kinase), ORF60 (ribonucleotide reductase, small subunit), and cellular interleukin-10 (IL-10) contain a sequence motif similar to the Sp1/Sp3-binding region of the 56p-RE element, and we found that these promoters could also be synergistically activated by ORF50 and Sp3 via the conserved motifs. Noteworthily, the conversion of the Sp1/Sp3-binding sequence of the 56p-RE element into a consensus high-affinity Sp-binding sequence completely lost the synergistic response to ORF50 and Sp3. Moreover, transcriptional synergy could not be detected through other ORF50-responsive elements from the viral PAN, K12, ORF57, and K6 promoters. Collectively, the results of our study demonstrate that ORF50 and Sp3 can act in synergy on the transcription of specific gene promoters, and we find a novel conserved cis-acting motif in these promoters essential for transcriptional synergy.IMPORTANCE Despite the critical role of ORF50 in the KSHV latent-to-lytic switch, the molecular mechanism by which ORF50 activates its downstream target genes, especially those that encode the viral DNA replication enzymes, is not yet fully understood. Here, we find that ORF50 can cooperate with Sp3 to synergistically activate promoters of the viral ORF56 (primase), ORF21 (thymidine kinase), and ORF60 (ribonucleotide reductase) genes via similar Sp1/Sp3-binding motifs. Additionally, the same synergistic effect can be seen on the promoter of the cellular IL-10 gene. Overall, our data reveal an important role for Sp3 in ORF50-mediated transactivation, and we propose a new subclass of ORF50-responsive elements in viral and cellular promoters.


Assuntos
Herpesvirus Humano 8/genética , Proteínas Imediatamente Precoces/genética , Regiões Promotoras Genéticas , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp3/genética , Transativadores/genética , Transcrição Genética , Animais , Sítios de Ligação , Linhagem Celular , Linhagem Celular Tumoral , Células Clonais , Fibroblastos/virologia , Regulação da Expressão Gênica , Células HEK293 , Herpesvirus Humano 8/metabolismo , Interações Hospedeiro-Patógeno/genética , Humanos , Proteínas Imediatamente Precoces/metabolismo , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/genética , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/metabolismo , Interleucina-10/genética , Interleucina-10/metabolismo , Linfócitos/virologia , Camundongos , Ligação Proteica , Elementos de Resposta , Transdução de Sinais , Fator de Transcrição Sp1/metabolismo , Fator de Transcrição Sp3/metabolismo , Transativadores/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , Proteínas Virais Reguladoras e Acessórias/genética , Proteínas Virais Reguladoras e Acessórias/metabolismo
17.
Nucleic Acids Res ; 48(14): 8178-8187, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32619241

RESUMO

The application of gene-editing technology is currently limited by the lack of safe and efficient methods to deliver RNA-guided endonucleases to target cells. We engineered lentivirus-based nanoparticles to co-package the U6-sgRNA template and the CRISPR-associated protein 9 (Cas9) fused with a virion-targeted protein Vpr (Vpr.Prot.Cas9), for simultaneous delivery to cells. Equal spatiotemporal control of the vpr.prot.cas9 and gag/pol gene expression (the presence of Rev responsive element, RRE) greatly enhanced the encapsidation of the fusion protein and resulted in the production of highly efficient lentivector nanoparticles. Transduction of the unconcentrated, Vpr.Prot.Cas9-containing vectors led to >98% disruption of the EGFP gene in reporter HEK293-EGFP cells with minimal cytotoxicity. Furthermore, we detected indels in the targeted endogenous loci at frequencies of up to 100% in cell lines derived from lymphocytes and monocytes and up to 15% in primary CD4+ T cells by high-throughput sequencing. This approach may provide a platform for the efficient, dose-controlled and tissue-specific delivery of genome editing enzymes to cells and it may be suitable for simultaneous endogenous gene disruption and a transgene delivery.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas , Edição de Genes/métodos , Elementos de Resposta , Proteína 9 Associada à CRISPR/genética , Vetores Genéticos/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Lentivirus/genética , Nanopartículas/química , Células THP-1 , Transdução Genética/métodos
18.
J Cancer Res Clin Oncol ; 146(10): 2509-2517, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32620986

RESUMO

BACKGROUND: Colorectal cancer (CRC) is now a major human cancer, and B-cell translocation gene 3 (BTG3) has been reported as a tumor-suppressor in CRC, but its upstream regulator has not been identified. METHODS: Endogenous expression levels of BTG3 were compared between normal colorectal cell line CCD-18Co and two CRC cell lines SW480 and HT29, as well as between CRC patient tumor and adjacent normal tissues. Analysis of BTG3 genomic region was performed which identified a putative hypoxia response element (HRE). Effects of hypoxia condition, BTG3 overexpression, and their combination on the radiation sensitivity of CRC cell lines were assessed. RESULTS: BTG3 was downregulated in CRC cell lines and patient tumor samples, via the HRE in its promoter region. Hypoxia and BTG3 overexpression could both induce radiation resistance in CRC cells. Combining hypoxia with BTG3 overexpression effectively rendered the resistance of CRC cells to radiation to a level lower than hypoxia alone and higher than normoxia alone, indicating the essential role of BTG3 in hypoxia-induced radiation resistance of CRC cells. CONCLUSION: We therefore propose a novel signaling cascade involving hypoxia/BTG3 to be a potential risk factor for CRC patients undergoing radiation therapy, which could possibly serve as therapeutic targets among CRC patients with acquired radiotherapy resistance.


Assuntos
Proteínas de Ciclo Celular/genética , Neoplasias Colorretais/genética , Neoplasias Colorretais/radioterapia , Idoso , Apoptose/efeitos da radiação , Proteínas de Ciclo Celular/biossíntese , Hipóxia Celular/fisiologia , Linhagem Celular Tumoral , Neoplasias Colorretais/metabolismo , Regulação para Baixo , Feminino , Regulação Neoplásica da Expressão Gênica , Células HT29 , Humanos , Masculino , Pessoa de Meia-Idade , Regiões Promotoras Genéticas , Tolerância a Radiação , Elementos de Resposta
19.
Food Chem ; 333: 127529, 2020 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-32679419

RESUMO

A solid-phase extraction (SPE) method for enriching and purifying estrogenic disrupting compounds (EDCs) based on the estrogen response element was established. The estrogen receptor was used for molecular recognition, as it specifically binds EDCs. An estrogen response element was used to maintain the activity of the estrogen receptor. High-performance liquid chromatography (HPLC) was used to quantify the EDCs. This method combined with HPLC was applied to detect three kinds of EDCs, such as bisphenol A (BPA), 17ß-estradiol, and diethylstilbestrol in a liquid milk matrix, with recoveries of 84.1 ± 8.2% to 113.6 ± 2.9%. The limits of detection and quantification of the established method were 1 × 10-6 mg·mL-1 and 5 × 10-6 mg·mL-1. The method was further applied to analyze market samples, including liquid milk, fermented milk, and milk powder. Only BPA was detected from one brand of liquid milk and it was below the regulatory level.


Assuntos
Disruptores Endócrinos/isolamento & purificação , Estrogênios/metabolismo , Elementos de Resposta , Extração em Fase Sólida/métodos , Animais , Cromatografia Líquida de Alta Pressão , Disruptores Endócrinos/análise , Leite/química , Poluentes Químicos da Água/análise , Poluentes Químicos da Água/isolamento & purificação
20.
Cell Syst ; 11(1): 2-4, 2020 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-32702318

RESUMO

One snapshot of the peer review process for "Dissection of c-AMP Response Element Architecture by Using Genomic and Episomal Massively Parallel Reporter Assays" (Davis et al., 2020).


Assuntos
Regulação da Expressão Gênica , Elementos de Resposta , Monofosfato de Adenosina , Genômica , Plasmídeos , Elementos de Resposta/genética
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