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1.
Nat Genet ; 51(10): 1494-1505, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31570894

RESUMO

A hallmark of the immune system is the interplay among specialized cell types transitioning between resting and stimulated states. The gene regulatory landscape of this dynamic system has not been fully characterized in human cells. Here we collected assay for transposase-accessible chromatin using sequencing (ATAC-seq) and RNA sequencing data under resting and stimulated conditions for up to 32 immune cell populations. Stimulation caused widespread chromatin remodeling, including response elements shared between stimulated B and T cells. Furthermore, several autoimmune traits showed significant heritability in stimulation-responsive elements from distinct cell types, highlighting the importance of these cell states in autoimmunity. Allele-specific read mapping identified variants that alter chromatin accessibility in particular conditions, allowing us to observe evidence of function for a candidate causal variant that is undetected by existing large-scale studies in resting cells. Our results provide a resource of chromatin dynamics and highlight the need to characterize the effects of genetic variation in stimulated cells.


Assuntos
Linfócitos B/imunologia , Cromatina/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Elementos de Resposta/genética , Linfócitos T/imunologia , Desequilíbrio Alélico , Linfócitos B/efeitos dos fármacos , Linfócitos B/metabolismo , Células Cultivadas , Cromatina/efeitos dos fármacos , Cromatina/imunologia , Epigênese Genética , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/imunologia , Humanos , Interleucina-2/farmacologia , Interleucina-4/farmacologia , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/metabolismo , Polissacarídeos/farmacologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Transcriptoma
2.
Nucleic Acids Res ; 47(19): 10212-10234, 2019 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-31538203

RESUMO

Chronic hypoxia is associated with a variety of physiological conditions such as rheumatoid arthritis, ischemia/reperfusion injury, stroke, diabetic vasculopathy, epilepsy and cancer. At the molecular level, hypoxia manifests its effects via activation of HIF-dependent transcription. On the other hand, an important transcription factor p53, which controls a myriad of biological functions, is rendered transcriptionally inactive under hypoxic conditions. p53 and HIF-1α are known to share a mysterious relationship and play an ambiguous role in the regulation of hypoxia-induced cellular changes. Here we demonstrate a novel pathway where HIF-1α transcriptionally upregulates both WT and MT p53 by binding to five response elements in p53 promoter. In hypoxic cells, this HIF-1α-induced p53 is transcriptionally inefficient but is abundantly available for protein-protein interactions. Further, both WT and MT p53 proteins bind and chaperone HIF-1α to stabilize its binding at its downstream DNA response elements. This p53-induced chaperoning of HIF-1α increases synthesis of HIF-regulated genes and thus the efficiency of hypoxia-induced molecular changes. This basic biology finding has important implications not only in the design of anti-cancer strategies but also for other physiological conditions where hypoxia results in disease manifestation.


Assuntos
Hipóxia Celular/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Mapas de Interação de Proteínas/genética , Proteína Supressora de Tumor p53/genética , Regulação da Expressão Gênica , Humanos , Chaperonas Moleculares/genética , Regiões Promotoras Genéticas/genética , Elementos de Resposta/genética , Transdução de Sinais/genética
3.
Nucleic Acids Res ; 47(19): 9967-9989, 2019 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-31501881

RESUMO

The NF-κB family of dimeric transcription factors regulates transcription by selectively binding to DNA response elements present within promoters or enhancers of target genes. The DNA response elements, collectively known as κB sites or κB DNA, share the consensus 5'-GGGRNNNYCC-3' (where R, Y and N are purine, pyrimidine and any nucleotide base, respectively). In addition, several DNA sequences that deviate significantly from the consensus have been shown to accommodate binding by NF-κB dimers. X-ray crystal structures of NF-κB in complex with diverse κB DNA have helped elucidate the chemical principles that underlie target selection in vitro. However, NF-κB dimers encounter additional impediments to selective DNA binding in vivo. Work carried out during the past decades has identified some of the barriers to sequence selective DNA target binding within the context of chromatin and suggests possible mechanisms by which NF-κB might overcome these obstacles. In this review, we first highlight structural features of NF-κB:DNA complexes and how distinctive features of NF-κB proteins and DNA sequences contribute to specific complex formation. We then discuss how native NF-κB dimers identify DNA binding targets in the nucleus with support from additional factors and how post-translational modifications enable NF-κB to selectively bind κB sites in vivo.


Assuntos
DNA/genética , Genoma Humano/genética , NF-kappa B/genética , Elementos de Resposta/genética , Cromatina/genética , Cristalografia por Raios X , DNA/química , Humanos , Modelos Moleculares , NF-kappa B/química , Regiões Promotoras Genéticas/genética , Fatores de Transcrição/genética
4.
PLoS Genet ; 15(8): e1007877, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31425502

RESUMO

Patterned expression of many developmental genes is specified by transcription factor gene expression, but is thought to be refined by chromatin-mediated repression. Regulatory DNA sequences called Polycomb Response Elements (PREs) are required to repress some developmental target genes, and are widespread in genomes, suggesting that they broadly affect developmental programs. While PREs in transgenes can nucleate trimethylation on lysine 27 of the histone H3 tail (H3K27me3), none have been demonstrated to be necessary at endogenous chromatin domains. This failure is thought to be due to the fact that most endogenous H3K27me3 domains contain many PREs, and individual PREs may be redundant. In contrast to these ideas, we show here that PREs near the wing selector gene vestigial have distinctive roles at their endogenous locus, even though both PREs are repressors in transgenes. First, a PRE near the promoter is required for vestigial activation and not for repression. Second, only the distal PRE contributes to H3K27me3, but even removal of both PREs does not eliminate H3K27me3 across the vestigial domain. Thus, endogenous chromatin domains appear to be intrinsically marked by H3K27me3, and PREs appear required to enhance this chromatin modification to high levels at inactive genes.


Assuntos
Cromatina/genética , Proteínas de Drosophila/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Nucleares/genética , Proteínas do Grupo Polycomb/metabolismo , Elementos de Resposta/genética , Animais , Animais Geneticamente Modificados , Metilação de DNA , Drosophila melanogaster/fisiologia , Feminino , Histonas/genética , Masculino , Mutagênese Sítio-Dirigida , Ativação Transcricional/genética , Asas de Animais/crescimento & desenvolvimento
5.
J Mol Model ; 25(8): 246, 2019 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-31342181

RESUMO

It is well known that the DNA-binding specificity of transcription factors (TFs) is influenced by protein-protein interactions (PPIs). However, the underlying molecular mechanisms remain largely unknown. In this work, we adopted the cAMP-response element-binding protein (CREB) of the basic leucine zipper (bZIP) TF family as a model system, and a workflow of combined bioinformatics and molecular modeling analysis of protein-DNA interaction was tested. First, the multiple sequence alignment and SDPsite method were used to find potential bZIP family binding specificity determining positions (SDPs) within the protein-protein interaction region. Second, the mutation system was analyzed using molecular dynamics simulation. Molecular mechanics Poisson-Boltzmann surface area (MM/PBSA) free energy calculations confirmed the enhancement of the binding affinity of the mutation, which was in agreement with experimental results. The root mean square fluctuation (RMSF) and hydrogen bonding changes suggested an open and close protein dimerization process after the system was mutated, which resulted in the change of the hydrogen bonding of the protein-DNA interface and a slight conformational change. We believe that this work will contribute to understanding the protein-protein interaction-regulated binding specificity of bZIP transcription factors.


Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Mapeamento de Interação de Proteínas , Sequência de Aminoácidos , Fatores de Transcrição de Zíper de Leucina Básica/química , AMP Cíclico/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/química , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , DNA/química , DNA/metabolismo , Ligações de Hidrogênio , Simulação de Dinâmica Molecular , Mutação/genética , Filogenia , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Elementos de Resposta/genética , Termodinâmica
6.
PLoS One ; 14(4): e0215911, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31039173

RESUMO

For most complex traits, the majority of SNPs identified through genome-wide association studies (GWAS) reside within noncoding regions that have no known function. However, these regions are enriched for the regulatory enhancers specific to the cells relevant to the specific trait. Indeed, many of the GWAS loci that have been functionally characterized lie within enhancers that regulate expression levels of key genes. In order to identify polymorphisms with potential allele-specific regulatory effects, we developed a bioinformatics pipeline that harnesses epigenetic signatures as well as transcription factor (TF) binding motifs to identify putative enhancers containing a SNP with potential allele-specific TF binding in linkage disequilibrium (LD) with a GWAS-identified SNP. We applied the approach to GWAS findings for blood lipids, revealing 7 putative enhancers harboring associated SNPs, 3 of which lie within the introns of LCAT and ABCA1, genes that play crucial roles in cholesterol biogenesis and lipoprotein metabolism. All 3 enhancers demonstrated allele-specific in vitro regulatory activity in liver-derived cell lines. We demonstrated that these putative enhancers are in close physical proximity to the promoters of their respective genes, in situ, likely through chromatin looping. In addition, the associated alleles altered the likelihood of transcription activator STAT3 binding. Our results demonstrate that through our approach, the LD blocks that contain GWAS signals, often hundreds of kilobases in size with multiple SNPs serving as statistical proxies to the true functional site, can provide an experimentally testable hypothesis for the underlying regulatory mechanism linking genetic variants to complex traits.


Assuntos
Transportador 1 de Cassete de Ligação de ATP/genética , Alelos , HDL-Colesterol/metabolismo , Elementos Facilitadores Genéticos , Fosfatidilcolina-Esterol O-Aciltransferase/genética , Sequência de Bases , Linhagem Celular , Cromatina/metabolismo , Estudo de Associação Genômica Ampla , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , Fígado/metabolismo , Polimorfismo de Nucleotídeo Único/genética , Regiões Promotoras Genéticas , Ligação Proteica , Elementos de Resposta/genética , Fator de Transcrição STAT3/metabolismo
7.
Endocrinology ; 160(5): 1044-1056, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30980716

RESUMO

Mineralocorticoid and glucocorticoid receptors (MRs and GRs) constitute a functionally important dual receptor system detecting and transmitting circulating corticosteroid signals. High expression of MRs and GRs occurs in the same cells in the limbic system, the primary site of glucocorticoid action on cognition, behavior, and mood; however, modes of interaction between the receptors are poorly characterized. We used chromatin immunoprecipitation with nucleotide resolution using exonuclease digestion, unique barcode, and single ligation (ChIP-nexus) for high-resolution genome-wide characterization of MR and GR DNA binding profiles in neuroblastoma cells and demonstrate recruitment to highly similar DNA binding sites. Expressed MR or GR showed differential regulation of endogenous gene targets, including Syt2 and Ddc, whereas coexpression produced augmented transcriptional responses even when MRs were unable to bind DNA (MR-XDBD). ChIP confirmed that MR-XDBD could be tethered to chromatin by GR. Our data demonstrate that MR can interact at individual genomic DNA sites in multiple modes and suggest a role for MR in increasing the transcriptional response to glucocorticoids.


Assuntos
Glucocorticoides/farmacologia , Receptores de Glucocorticoides/metabolismo , Receptores de Mineralocorticoides/metabolismo , Transcrição Genética/efeitos dos fármacos , Animais , Sequência de Bases , Sítios de Ligação/genética , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , DNA/genética , DNA/metabolismo , Camundongos , Ligação Proteica , Interferência de RNA , Ratos , Receptores de Glucocorticoides/genética , Receptores de Mineralocorticoides/genética , Elementos de Resposta/genética
8.
BMC Plant Biol ; 19(1): 145, 2019 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-30991965

RESUMO

BACKGROUND: This study aimed to identify the transcription factors of nitrate reductase genes (NIA1 and NIA2) promoters and hypothetical cis-element of NRE2. Based on the constructed cDNA library of Nicotiana tabacum K326, a yeast one-hybrid system was established using the Matchmaker® Gold Yeast One-Hybrid Library Screening System from Clontech. The transcription factors of NIA1 andNIA2 promoters and NRE2 cis-elements were screened. RESULTS: After sequencing and bioinformatics analysis, 15 cDNA sequences were identified: 9 for NIA1 (including XP_016503563.1 and NP_001312236.1), 3 for NIA2 (including XP_016510250.1), and 3 for NRE2 (including XM_016576899.1). XP_016503563.1 was annotated in PREDICTED: CRM-domain containing factor CFM3, and NP_001312236.1chloroplastic/mitochondrial-like in Nicotiana tabacum. NP_001312236.1 was annotated in Sulfite oxidase-like of Nicotiana tabacum. XP_016510250.1 was annotated as PREDICTED: uncharacterized protein LOC107827596 in Nicotiana tabacum. XM_016576899.1 was annotated in PREDICTED: Nicotiana tabacum RING-H2 finger protein ATL16-like (LOC107759033). CONCLUSION: A yeast one-hybrid library was successfully constructed. The identified transcription factors may provide a theoretical basis for the study of plant nitrate reductase.


Assuntos
Genes de Plantas , Nitrato Redutase/genética , Proteínas de Plantas/genética , Regiões Promotoras Genéticas , Elementos de Resposta/genética , Tabaco/genética , Fatores de Transcrição/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Clonagem Molecular , Biblioteca Gênica , Glucuronidase/metabolismo , Nitrato Redutase/metabolismo , Proteínas de Plantas/metabolismo
9.
PLoS One ; 14(4): e0214145, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30947257

RESUMO

Root growth angle (RGA) in response to gravity controlled by auxin is a pertinent target trait for obtainment of higher yield in cereals. But molecular basis of this root architecture trait remain obscure in wheat and barley. We selected four cultivars two each for wheat and barley to unveil the molecular genetic mechanism of Deeper Rooting 1-like gene which controls RGA in rice leading to higher yield under drought imposition. Morphological analyses revealed a deeper and vertically oriented root growth in "NARC 2009" variety of wheat than "Galaxy" and two other barley cultivars "Scarlet" and "ISR42-8". Three new homoeologs designated as TaANDRO1-like, TaBNDRO1-like and TaDNDRO1-like corresponding to A, B and D genomes of wheat could be isolated from "NARC 2009". Due to frameshift and intronization/exonization events the gene structures of these paralogs exhibit variations in size. DRO1-like genes with five distinct domains prevail in diverse plant phyla from mosses to angiosperms but in lower plants their differentiation from LAZY, NGR and TAC1 (root and shoot angle genes) is enigmatic. Instead of IGT as denominator motif of this family, a new C-terminus motif WxxTD in the V-domain is proposed as family specific motif. The EAR-like motif IVLEM at the C-terminus of the TaADRO1-like and TaDDRO1-like that diverged to KLHTLIPNK in TaBDRO1-like and HvDRO1-like is the hallmark of these proteins. Split-YFP and yeast two hybrid assays complemented the interaction of TaDRO1-like with TOPLESS-a repressor of auxin regulated root promoting genes in plants-through IVLEM/KLHTLIPNK motif. Quantitative RT-PCR revealed abundance of DRO1-like RNA in root tips and spikelets while transcript signals were barely detectable in shoot and leaf tissues. Interestingly, wheat exhibited stronger expression of TaBDRO1-like than barley (HvDRO1-like), but TaBDRO1-like was the least expressing among three paralogs. The underlying cause of this expression divergence seems to be the presence of AuxRE motif TGTCTC and core TGTC with a coupling AuxRE-like motif ATTTTCTT proximal to the transcriptional start site in TaBDRO1-like and HvDRO1-like promoters. This is evident from binding of ARF1 to TGTCTC and TGTC motifs of TaBDRO1-like as revealed by yeast one-hybrid assay. Thus, evolution of DRO1-like wheat homoeologs might incorporate the C-terminus mutations as well as gain and loss of AuxREs and other cis-regulatory elements during expression divergence. Since root architecture is an important target trait for wheat crop improvement, therefore DRO1-like genes have potential applications in plant breeding for enhancement of plant productivity by the use of modern genome editing approaches.


Assuntos
Evolução Molecular , Ácidos Indolacéticos/farmacologia , Mutação/genética , Proteínas de Plantas/química , Proteínas de Plantas/genética , Elementos de Resposta/genética , Homologia de Sequência de Aminoácidos , Triticum/genética , Motivos de Aminoácidos , Sequência de Aminoácidos , Sequência de Bases , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Hordeum/anatomia & histologia , Hordeum/efeitos dos fármacos , Íntrons/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/anatomia & histologia , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/genética , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Triticum/anatomia & histologia , Triticum/efeitos dos fármacos
10.
Int J Mol Sci ; 20(5)2019 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-30813528

RESUMO

Glucocorticoids are used during prostate cancer (PCa) treatment. However, they may also have the potential to drive castration resistant prostate cancer (CRPC) growth via the glucocorticoid receptor (GR). Given the association between inflammation and PCa, and the anti-inflammatory role of heme oxygenase 1 (HO-1), we aimed at identifying the molecular processes governed by the interaction between HO-1 and GR. PCa-derived cell lines were treated with Hemin, Dexamethasone (Dex), or both. We studied GR gene expression by RTqPCR, protein expression by Western Blot, transcriptional activity using reporter assays, and nuclear translocation by confocal microscopy. We also evaluated the expression of HO-1, FKBP51, and FKBP52 by Western Blot. Hemin pre-treatment reduced Dex-induced GR activity in PC3 cells. Protein levels of FKBP51, a cytoplasmic GR-binding immunophilin, were significantly increased in Hemin+Dex treated cells, possibly accounting for lower GR activity. We also evaluated these treatments in vivo using PC3 tumors growing as xenografts. We found non-significant differences in tumor growth among treatments. Immunohistochemistry analyses revealed strong nuclear GR staining in almost all groups. We did not observe HO-1 staining in tumor cells, but high HO-1 reactivity was detected in tumor infiltrating macrophages. Our results suggest an association and crossed modulation between HO-1 and GR pathways.


Assuntos
Heme Oxigenase-1/metabolismo , Neoplasias da Próstata/metabolismo , Receptores de Glucocorticoides/metabolismo , Animais , Linhagem Celular Tumoral , Dexametasona/farmacologia , Intervalo Livre de Doença , Heme Oxigenase-1/genética , Hemina/farmacologia , Humanos , Masculino , Camundongos , Regiões Promotoras Genéticas/genética , Elementos de Resposta/genética , Transdução de Sinais , Proteínas de Ligação a Tacrolimo/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
11.
Int J Mol Med ; 43(5): 1927-1938, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30896855

RESUMO

The farnesoid X receptor (FXR) is known to regulate the gene expression of SR­BI, which mediates plasma high­density lipoprotein (HDL)­cholesterol uptake. Our previous study demonstrated that the activation of FXR by obeticholic acid (OCA) lowered plasma HDL­cholesterol levels and increased the hepatic mRNA and protein expression levels of SR­BI in hypercholesterolemic hamsters, but not in normolipidemic hamsters, suggesting that dietary cholesterol may be involved in the OCA­induced transcription of SR­BI. In the present study, a functional 90­base­pair regulatory region was identified in the first intron of the SR­BI gene of hamster and mouse that contains a FXR response element (IR­1) and an adjacent liver X receptor (LXR) response element (LXRE). By in vitro DNA binding and luciferase reporter gene assays, it was demonstrated that FXR and LXR bind to their recognition sequences within this intronic region and transactivate the SR­BI reporter gene in a synergistic manner. It was also shown that mutations at either the IR­1 site or the LXRE site eliminated OCA­mediated gene transcription. Utilizing chow­fed hamsters as an in vivo model, it was demonstrated that treating normolipidemic hamsters with OCA or GW3965 alone did not effectively induce levels of SR­BI, whereas their combined treatment significantly increased the mRNA and protein levels of SR­BI in the liver. The study further investigated effects of FXR and LXR coactivation on the gene expression of SR­BI in human liver cells. The intronic FXRE and LXRE regulatory region was not conserved in the human SR­BI genomic sequence, however, higher mRNA expression levels of SR­BI were observed in human primary hepatocytes and HepG2 cells exposed to combined treatments of FXR and LXR agonists, compared with those in cells exposed to individual ligand treatment. Therefore, these results suggest that human SR­BI gene transcription may also be subject to concerted activation by FXR and LXR, mediated via currently unidentified regulatory sequences.


Assuntos
Antígenos CD36/genética , Ácido Quenodesoxicólico/análogos & derivados , Regulação da Expressão Gênica/efeitos dos fármacos , Receptores X do Fígado/metabolismo , Fígado/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Transcrição Genética/efeitos dos fármacos , Animais , Sequência de Bases , Benzoatos/farmacologia , Benzilaminas/farmacologia , Antígenos CD36/metabolismo , Ácido Quenodesoxicólico/farmacologia , Genes Reporter , Células Hep G2 , Humanos , Íntrons/genética , Fígado/efeitos dos fármacos , Masculino , Mesocricetus , Motivos de Nucleotídeos/genética , Elementos de Resposta/genética
12.
Cells ; 8(3)2019 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-30841579

RESUMO

Epigenetic regulation has been considered an important mechanism for influencing stem cell differentiation. In particular, histone deacetylases (HDACs) have been shown to play a role in the osteoblast differentiation of mesenchymal stem cells (MSCs). In this study, the effect of the HDAC inhibitor, valproic acid (VPA), on bone formation in vivo by MSCs was determined. Surprisingly, VPA treatment, unlike other HDAC inhibitors, produced a well-organized lamellar bone tissue when MSCs⁻collagen sponge constructs were implanted subcutaneously into nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice, although a decrease of osteocalcin (OC) expression was observed. Consequently, we decided to investigate the molecular mechanisms by which VPA exerts such effects on MSCs. We identified the glucocorticoid receptor (GR) as being responsible for that downregulation, and suggested a correlation between GR and HDAC2 inhibition after VPA treatment, as evidenced by HDAC2 knockdown. Furthermore, using co-immunoprecipitation analysis, we showed for the first time in the cytoplasm, binding between GR and HDAC2. Additionally, chromatin immunoprecipitation (ChIP) assays confirmed the role of GR in OC downregulation, showing recruitment of GR to the nGRE element in the OC promoter. In conclusion, our results highlight the existence of a cross-talk between GR and HDAC2, providing a mechanistic explanation for the influence of the HDAC inhibitor (namely VPA) on osteogenic differentiation in MSCs. Our findings open new directions in targeted therapies, and offer new insights into the regulation of MSC fate determination.


Assuntos
Citoplasma/metabolismo , Histona Desacetilase 2/metabolismo , Células-Tronco Mesenquimais/metabolismo , Osteocalcina/metabolismo , Receptores de Glucocorticoides/metabolismo , Ácido Valproico/farmacologia , Adulto , Biomarcadores/metabolismo , Citoplasma/efeitos dos fármacos , Polpa Dentária/citologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Humanos , Masculino , Células-Tronco Mesenquimais/efeitos dos fármacos , Mifepristona/farmacologia , Osteocalcina/genética , Osteogênese/efeitos dos fármacos , Osteogênese/genética , Implantação de Prótese , Ligação Proteica/efeitos dos fármacos , Elementos de Resposta/genética , Adulto Jovem
13.
Nat Commun ; 10(1): 542, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30710088

RESUMO

The suprachiasmatic nucleus (SCN) co-ordinates circadian behaviour and physiology in mammals. Its cell-autonomous circadian oscillations pivot around a well characterised transcriptional/translational feedback loop (TTFL), whilst the SCN circuit as a whole is synchronised to solar time by its retinorecipient cells that express and release vasoactive intestinal peptide (VIP). The cell-autonomous and circuit-level mechanisms whereby VIP synchronises the SCN are poorly understood. We show that SCN slices in organotypic culture demonstrate rapid and sustained circuit-level circadian responses to VIP that are mediated at a cell-autonomous level. This is accompanied by changes across a broad transcriptional network and by significant VIP-directed plasticity in the internal phasing of the cell-autonomous TTFL. Signalling via ERK1/2 and tuning by its negative regulator DUSP4 are critical elements of the VIP-directed circadian re-programming. In summary, we provide detailed mechanistic insight into VIP signal transduction in the SCN at the level of genes, cells and neural circuit.


Assuntos
Relógios Circadianos/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteínas Tirosina Fosfatases/metabolismo , Núcleo Supraquiasmático/fisiologia , Peptídeo Intestinal Vasoativo/farmacologia , Animais , Sistemas CRISPR-Cas , Relógios Circadianos/genética , Relógios Circadianos/efeitos da radiação , AMP Cíclico/metabolismo , Retroalimentação Fisiológica/efeitos dos fármacos , Retroalimentação Fisiológica/efeitos da radiação , Redes Reguladoras de Genes/efeitos dos fármacos , Redes Reguladoras de Genes/efeitos da radiação , Luz , Sistema de Sinalização das MAP Quinases/efeitos da radiação , Camundongos Knockout , Biossíntese de Proteínas/efeitos dos fármacos , Biossíntese de Proteínas/efeitos da radiação , Elementos de Resposta/genética , Núcleo Supraquiasmático/citologia , Núcleo Supraquiasmático/efeitos dos fármacos , Núcleo Supraquiasmático/efeitos da radiação , Transcrição Genética/efeitos dos fármacos , Transcrição Genética/efeitos da radiação
14.
Aquat Toxicol ; 208: 157-167, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30677711

RESUMO

Environmental estrogens are a serious concern worldwide due to their ubiquity and adverse ecotoxicological and health effects. Chemical structure of these substances is highly diverse, therefore estrogenicity cannot be predicted on the basis of molecular structure. Furthermore, estimation of estrogenicity of environmental samples based on chemical analytics of suspects is difficult given the complex interaction of chemicals and the impact on estrogenicity. The full estrogenic impact of an environmental sample can thus only be revealed by a series of sensitive in vitro and in vivo ecotoxicological tests. Herein we describe a vitellogenin reporter transgenic zebrafish line (Tg(vtg1:mCherry)) that enables the detection of estrogenicity in the environmentally relevant, low concentration ranges in embryonic tests that are in accordance with 3Rs and relevant animal welfare regulations. The transgene construct used for the development of Tg(vtg1:mCherry) carried a long (3.4 kbp) natural vitellogenin-1 promoter sequence with a high number of ERE sites. A test protocol was developed based on our finding that the endogenous vitellogenin and the reporter show similar spatial expression pattern and both endogenous and vitellogenin reporter is only produced in the left hepatic lobe of 5 dpf zebrafish embryos. Seven generations of Tg(vtg1:mCherry) have been established, and the estrogen responsiveness was tested with different estrogenic substances and wastewater samples. Embryos were exposed from 3 to 5 days post fertilization (dpf). Fluorescence in embryos could be detected upon treatment with 17-ß-estradiol from a concentration of 100 ng/L, 17-α-ethynilestradiol from 1 ng/L, zearalenone from 100 ng/L and bisphenol-A from 1 mg/L. In the adult stage transgene activity appeared to be more sensitive to estrogen treatment, with detectable transgene activity from 5 ng/L 17-ß-estradiol concentration. The transgenic line Tg(vtg1:mCherry) was also suitable for the direct measurement of estrogenicity in wastewater samples without sample extraction. The detection of estrogenic activity using the reporter line was confirmed by the bioluminescent yeast estrogen screen.


Assuntos
Estrogênios/análise , Fígado/metabolismo , Vitelogeninas/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Biomarcadores/metabolismo , Embrião não Mamífero/metabolismo , Estradiol/metabolismo , Fluorescência , Heterozigoto , Homozigoto , Fígado/efeitos dos fármacos , Masculino , Elementos de Resposta/genética , Transgenes , Águas Residuárias/química , Poluentes Químicos da Água/análise , Peixe-Zebra/embriologia
15.
J Virol ; 93(6)2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30602606

RESUMO

Following acute infection, herpes simplex virus 1 (HSV-1) establishes lifelong latency in neurons. Physical, emotional, and chemical stresses are linked to increasing the incidence of reactivation from latency, but the mechanism of action is not well understood. In general, stress increases corticosteroid levels, leading to activation of the glucocorticoid receptor (GR), a pioneer transcription factor. Consequently, we hypothesized that stress-mediated activation of the GR can stimulate productive infection and viral gene expression. New studies demonstrated that the GR-specific antagonist (CORT-108297) significantly reduced HSV-1 productive infection in mouse neuroblastoma cells (Neuro-2A). Additional studies demonstrated that the activated GR and Krüppel-like transcription factor 15 (KLF15) cooperatively transactivated the infected cell protein 0 (ICP0) promoter, a crucial viral regulatory protein. Interestingly, the synthetic corticosteroid dexamethasone and GR or KLF15 alone had little effect on ICP0 promoter activity in transfected Neuro-2A or Vero cells. Chromatin immunoprecipitation (ChIP) studies revealed that the GR and KLF15 occupied ICP0 promoter sequences important for transactivation at 2 and 4 h after infection; however, binding was not readily detected at 6 h after infection. Similar results were obtained for cells transfected with the full-length ICP0 promoter. ICP0 promoter sequences lack a consensus "whole" GR response element (GRE) but contain putative half-GREs that were important for dexamethasone induced promoter activity. The activated GR stimulates expression of, and interacts with, KLF15; consequently, these data suggest KLF15 and the GR form a feed-forward loop that activates viral gene expression and productive infection following stressful stimuli.IMPORTANCE The ability of herpes simplex virus 1 (HSV-1) to periodically reactivate from latency results in virus transmission and recurrent disease. The incidence of reactivation from latency is increased by chronic or acute stress. Stress increases the levels of corticosteroids, which bind and activate the glucocorticoid receptor (GR). Since GR activation is an immediate early response to stress, we tested whether the GR influences productive infection and the promoter that drives infected cell protein 0 (ICP0) expression. Pretreatment of cells with a GR-specific antagonist (CORT-108297) significantly reduced virus replication. Although the GR had little effect on ICP0 promoter activity alone, the Krüppel-like transcription factor 15 (KLF15) cooperated with the GR to stimulate promoter activity in transfected cells. In transfected or infected cells, the GR and KLF15 occupied ICP0 sequences important for transactivation. Collectively, these studies provide insight into how stress can directly stimulate productive infection and viral gene expression.


Assuntos
Herpesvirus Humano 1/patogenicidade , Proteínas Imediatamente Precoces/genética , Fatores de Transcrição Kruppel-Like/genética , Regiões Promotoras Genéticas/genética , Receptores de Glucocorticoides/metabolismo , Ativação Transcricional/genética , Ubiquitina-Proteína Ligases/genética , Animais , Sítios de Ligação/genética , Linhagem Celular , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina/métodos , Regulação Viral da Expressão Gênica/genética , Herpes Simples/metabolismo , Herpes Simples/virologia , Infecções por Herpesviridae/genética , Infecções por Herpesviridae/virologia , Herpesvirus Humano 1/genética , Camundongos , Elementos de Resposta/genética , Células Vero , Proteínas Virais/genética , Ativação Viral/genética , Latência Viral/genética
16.
Front Biosci (Landmark Ed) ; 24: 245-276, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30468654

RESUMO

17beta-estradiol (E2), the main circulating estrogen hormone, is involved in a wide variety of physiological functions ranging from the development to the maintenance of many tissues and organs. The effects of E2 on cells are primarily conveyed by the transcription factors, estrogen receptor (ER) alpha and beta. The regulation of responsive genes by the well-defined ER alpha in response to E2 relies on complex and highly organized processes that dynamically integrate functions of many transcription regulators to induce spatiotemporal alterations in chromatin state and structure. Changes in gene expressions result in cell-specific responses that include proliferation, differentiation and death. Deregulation of E2-ER alpha signaling contributes to the initiation and progression of target tissue malignancies. We aim here to provide a review of recent findings on dynamic transcriptional events mediated by E2-ER alpha with the anticipation that a better understanding of complex regulatory mechanisms underlying ER actions would be a critical basis for the development of effective prognostic tools for and therapeutic interventions against estrogen target tissue malignancies.


Assuntos
Estradiol/farmacologia , Receptor alfa de Estrogênio/metabolismo , Elementos de Resposta/genética , Transcrição Genética/efeitos dos fármacos , Animais , Sítios de Ligação/genética , Estradiol/sangue , Receptor alfa de Estrogênio/química , Humanos , Modelos Moleculares , Conformação de Ácido Nucleico , Domínios Proteicos
17.
Cell Struct Funct ; 44(1): 1-19, 2019 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-30487368

RESUMO

The Golgi stress response is a homeostatic mechanism that augments the functional capacity of the Golgi apparatus when Golgi function becomes insufficient (Golgi stress). Three response pathways of the Golgi stress response have been identified in mammalian cells, the TFE3, HSP47 and CREB3 pathways, which augment the capacity of specific Golgi functions such as N-glycosylation, anti-apoptotic activity and pro-apoptotic activity, respectively. On the contrary, glycosylation of proteoglycans (PGs) is another important function of the Golgi, although the response pathway upregulating expression of glycosylation enzymes for PGs in response to Golgi stress remains unknown. Here, we found that expression of glycosylation enzymes for PGs was induced upon insufficiency of PG glycosylation capacity in the Golgi (PG-Golgi stress), and that transcriptional induction of genes encoding glycosylation enzymes for PGs was independent of the known Golgi stress response pathways and ER stress response. Promoter analyses of genes encoding these glycosylation enzymes revealed the novel enhancer elements PGSE-A and PGSE-B (the consensus sequences are CCGGGGCGGGGCG and TTTTACAATTGGTC, respectively), which regulate their transcriptional induction upon PG-Golgi stress. From these observations, the response pathway we discovered is a novel Golgi stress response pathway, which we have named the PG pathway.Key words: Golgi stress, proteoglycan, ER stress, organelle zone, organelle autoregulation.


Assuntos
Complexo de Golgi/genética , Proteoglicanas/metabolismo , Elementos de Resposta/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Estresse do Retículo Endoplasmático/genética , Proteínas de Choque Térmico HSP47/metabolismo , Células HeLa , Humanos , Transcrição Genética
18.
Free Radic Biol Med ; 130: 278-287, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30391674

RESUMO

The production of nitric oxide (NO) by inducible NO synthase (iNOS) and the regulation of gene expression by hypoxia-inducible factors (HIFs) are important for many aspects of human cell biology. However, little is known about whether iNOS expression is controlled by HIFs in human cells. Stimulation of A549 human lung epithelial cells with cytokines (TNF, IL-1 and IFNγ) increased the nuclear accumulation of HIF-1 in normoxic conditions. Activation of HIF-1 by hypoxia or CoCl2 was not sufficient to induce iNOS expression. However, pharmacological inhibition of HIF-1 reduced the induction of iNOS expression in A549 cells and primary human astrocytes. Moreover, elimination of HIF-1α expression and activity by CRISPR/Cas9 gene editing significantly reduced the induction of human iNOS gene promoter, mRNA and protein expression by cytokine stimulation. Three putative hypoxia response elements (HRE) are present within the human iNOS gene promoter and elimination of an HRE at -4981 bp reduced the induction of human iNOS promoter activity in response to cytokine stimulation. These findings establish an important role for HIF-1α in the induction of human iNOS gene expression in response to cytokine stimulation.


Assuntos
Hipóxia Celular/genética , Citocinas/farmacologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Óxido Nítrico Sintase Tipo II/genética , Células A549 , Astrócitos/metabolismo , Sistemas CRISPR-Cas/genética , Citocinas/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Interferon gama/efeitos dos fármacos , Interferon gama/genética , Interferon gama/farmacologia , Interleucina-1/farmacologia , Óxido Nítrico/biossíntese , Óxido Nítrico/metabolismo , Regiões Promotoras Genéticas/genética , Elementos de Resposta/genética , Fator de Necrose Tumoral alfa/farmacologia
19.
Biol Trace Elem Res ; 188(2): 373-383, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29931577

RESUMO

Chronic fluorosis is a systemic condition which principally manifests as defects in the skeleton and teeth. Skeletal fluorosis is characterized by aberrant proliferation and activation of osteoblasts, however, the underlying mechanisms of osteoblast activation induced by fluoride are not fully understood. Therefore, we investigated the pathogenic mechanism of human primary osteoblast proliferation and activation in relation to histone acetylation of the promoter p16, a well-known cell cycle regulation-related gene. The results showed that sodium fluoride (NaF) induced deacetylation and decreased expression of the p16 gene via inhibition of specificity protein 1 (Sp1) binding to its response element, which accounts for NaF increasing cell viability and promoting proliferation in human primary osteoblasts. These results reveal the regulatory mechanism of histone acetylation of the p16 gene on osteoblast activation in skeletal fluorosis.


Assuntos
Proliferação de Células/efeitos dos fármacos , Genes p16 , Histonas/metabolismo , Osteoblastos/efeitos dos fármacos , Fluoreto de Sódio/toxicidade , Fator de Transcrição Sp1/metabolismo , Acetilação , Adulto , Proliferação de Células/genética , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Feminino , Intoxicação por Flúor/metabolismo , Intoxicação por Flúor/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Osteoblastos/metabolismo , Cultura Primária de Células , Regiões Promotoras Genéticas/genética , Ligação Proteica , Elementos de Resposta/genética , Adulto Jovem
20.
Dev Biol ; 448(2): 88-100, 2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30583796

RESUMO

The ascidian neural plate consists of a defined number of identifiable cells organized in a grid of rows and columns, representing a useful model to investigate the molecular mechanisms controlling neural patterning in chordates. Distinct anterior brain lineages are specified via unique combinatorial inputs of signalling pathways with Nodal and Delta-Notch signals patterning along the medial-lateral axis and FGF/MEK/ERK signals patterning along the anterior-posterior axis of the neural plate. The Ciona Gsx gene is specifically expressed in the a9.33 cells in the row III/column 2 position of anterior brain lineages, characterised by a combinatorial input of Nodal-OFF, Notch-ON and FGF-ON. Here, we identify the minimal cis-regulatory element (CRE) of 376 bp, which can recapitulate the early activation of Gsx. We show that this minimal CRE responds in the same way as the endogenous Gsx gene to manipulation of FGF- and Notch-signalling pathways and to overexpression of Snail, a mediator of Nodal signals, and Six3/6, which is required to demarcate the anterior boundary of Gsx expression at the late neurula stage. We reveal that sequences proximal to the transcription start site include a temporal regulatory element required for the precise transcriptional onset of gene expression. We conclude that sufficient spatial and temporal information for Gsx expression is integrated in 376 bp of non-coding cis-regulatory sequences.


Assuntos
Ciona/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Placa Neural/metabolismo , Transcrição Genética , Animais , Sequência de Bases , Proteínas de Homeodomínio/metabolismo , Receptores Notch/metabolismo , Elementos de Resposta/genética , Deleção de Sequência , Transdução de Sinais/genética , Fatores de Transcrição da Família Snail/metabolismo , Fatores de Tempo
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