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1.
Nat Commun ; 11(1): 575, 2020 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-31996678

RESUMO

mTORC2 phosphorylates AKT in a hydrophobic motif site that is a biomarker of insulin sensitivity. In brown adipocytes, mTORC2 regulates glucose and lipid metabolism, however the mechanism has been unclear because downstream AKT signaling appears unaffected by mTORC2 loss. Here, by applying immunoblotting, targeted phosphoproteomics and metabolite profiling, we identify ATP-citrate lyase (ACLY) as a distinctly mTORC2-sensitive AKT substrate in brown preadipocytes. mTORC2 appears dispensable for most other AKT actions examined, indicating a previously unappreciated selectivity in mTORC2-AKT signaling. Rescue experiments suggest brown preadipocytes require the mTORC2/AKT/ACLY pathway to induce PPAR-gamma and establish the epigenetic landscape during differentiation. Evidence in mature brown adipocytes also suggests mTORC2 acts through ACLY to increase carbohydrate response element binding protein (ChREBP) activity, histone acetylation, and gluco-lipogenic gene expression. Substrate utilization studies additionally implicate mTORC2 in promoting acetyl-CoA synthesis from acetate through acetyl-CoA synthetase 2 (ACSS2). These data suggest that a principal mTORC2 action is controlling nuclear-cytoplasmic acetyl-CoA synthesis.


Assuntos
ATP Citrato (pro-S)-Liase/metabolismo , Adipócitos Marrons/metabolismo , Lipogênese/fisiologia , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Acetato-CoA Ligase/metabolismo , Animais , Proteínas de Transporte , Epigênese Genética , Ácido Graxo Sintases , Edição de Genes , Expressão Gênica , Transportador de Glucose Tipo 4/genética , Transportador de Glucose Tipo 4/metabolismo , Células HEK293 , Histonas/metabolismo , Humanos , Lipogênese/genética , Camundongos , Camundongos Endogâmicos C57BL , PPAR gama/metabolismo , Fosforilação , Proteômica , Elementos de Resposta
2.
J Pharm Biomed Anal ; 177: 112855, 2020 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-31561061

RESUMO

FSH plays a key role in the function of the reproductive system of human beings and is widely used both diagnostically and therapeutically in reproductive medicine. With the growing incidence of infertility, the demand for FSH pharmaceutical products is increasing. For this reason, the quality control process for FSH products is becoming more stringent. An accurate determination of bioactivity is crucial for the safety and efficacy of recombinant human follicle stimulating hormone (rhFSH). Up to now, in-vivo bioassay based on FSH-induced increases in rat ovarian weight has been the only method widely accepted by different pharmacopoeias. However this method has such drawbacks as the complex procedures, long assay period and high variability. Here, we established a reporter gene assay (RGA) based on the CHO-K1-FSHR-CRE-Luc cell line that stably expresses human follicle stimulating hormone receptor (hFSHR), as well as a luciferase reporter under the control of cyclic adenosine monophosphate (cAMP) response elements (CRES). Our study showed that our new assay not only has good dose-dependent responsiveness to rhFSH, but it also performs excellently in terms of specificity, precision, linearity, and simplicity compared with in-vivo rat bioassays. These results implied that this robust reporter gene assay may be a viable supplement to the animal in-vivo bioassay and may be employed in potency determination of rhFSH pharmaceutical products.


Assuntos
Bioensaio/métodos , Hormônio Foliculoestimulante Humano/farmacologia , Genes Reporter/genética , Receptores do FSH/metabolismo , Animais , Células CHO , Linhagem Celular , Cricetulus , AMP Cíclico/genética , Estudos de Viabilidade , Luciferases/química , Luciferases/genética , Receptores do FSH/genética , Proteínas Recombinantes/farmacologia , Elementos de Resposta/genética
3.
Medicine (Baltimore) ; 98(51): e18442, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31861015

RESUMO

Genetic variation and genotype of Hepatitis B virus (HBV) are related to the efficiency of interferon alpha (IFN-α)-based antiviral therapy. However, the correlation of variation in interferon-stimulated response element (ISRE) and HBV genotype response to IFN-α therapy remains elusive.Differences of ISRE between genotype B and C HBV were explored using the HBV sequences retrieved from GenBank, and further investigated by ISRE region cloning and sequencing from 60 clinical samples post-IFN-α therapy. Additionally, ISRE mutants were constructed and their relation to responsiveness of IFN-α was evaluated by real-time PCR and Southern blot analysis.ISRE pattern between genotype B and C were found based on both clinical sample sequencing and full-length sequence alignment. The primary difference is the fourth base within the ISRE region, with T and C for genotype B and C, respectively. HBV with genotype C-type ISRE had a higher replicative capability as compared to HBV with genotype B-type ISRE after IFN-α treatment in huh7 cells. CONCLUSION:: Preference of ISRE between genotype B and C HBV are distinct. Single nucleotide difference (C to T) within the HBV ISRE region may link to the efficacy of IFN-α therapy to genotype B and C HBV. Therefore, this study provides a clue for the determination of IFN-α therapy response to HBV treatment.


Assuntos
Vírus da Hepatite B/efeitos dos fármacos , Hepatite B/tratamento farmacológico , Interferon-alfa/farmacologia , Elementos de Resposta , Adulto , Feminino , Genótipo , Vírus da Hepatite B/genética , Humanos , Interferon-alfa/uso terapêutico , Masculino , Pessoa de Meia-Idade , Mutação , Adulto Jovem
4.
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
5.
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
6.
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
7.
PLoS Genet ; 15(9): e1008400, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31553720

RESUMO

Auxin is a major developmental regulator in plants and the acquisition of a transcriptional response to auxin likely contributed to developmental innovations at the time of water-to-land transition. Auxin Response Factors (ARFs) Transcription Factors (TFs) that mediate auxin-dependent transcriptional changes are divided into A, B and C evolutive classes in land plants. The origin and nature of the first ARF proteins in algae is still debated. Here, we identify the most 'ancient' ARF homologue to date in the early divergent charophyte algae Chlorokybus atmophyticus, CaARF. Structural modelling combined with biochemical studies showed that CaARF already shares many features with modern ARFs: it is capable of oligomerization, interacts with the TOPLESS co-repressor and specifically binds Auxin Response Elements as dimer. In addition, CaARF possesses a DNA-binding specificity that differs from class A and B ARFs and that was maintained in class C ARF along plants evolution. Phylogenetic evidence together with CaARF biochemical properties indicate that the different classes of ARFs likely arose from an ancestral proto-ARF protein with class C-like features. The foundation of auxin signalling would have thus happened from a pre-existing hormone-independent transcriptional regulation together with the emergence of a functional hormone perception complex.


Assuntos
Carofíceas/genética , Ácidos Indolacéticos/metabolismo , Proteínas de Plantas/genética , Receptores de Superfície Celular/genética , Proteínas de Ligação a DNA/genética , Evolução Molecular , Regulação da Expressão Gênica de Plantas/genética , Família Multigênica/genética , Filogenia , Reguladores de Crescimento de Planta/genética , Elementos de Resposta/genética , Fatores de Transcrição/genética
8.
Int J Mol Sci ; 20(18)2019 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-31500275

RESUMO

Mitochondria are multifunctional cellular organelles that are major producers of reactive oxygen species (ROS) in eukaryotes; to maintain the redox balance, they are supplemented with different ROS scavengers, including mitochondrial peroxiredoxins (Prdxs). Mitochondrial Prdxs have physiological and pathological significance and are associated with the initiation and progression of various cancer types. In this review, we have focused on signaling involving ROS and mitochondrial Prdxs that is associated with cancer development and progression. An upregulated expression of Prdx3 and Prdx5 has been reported in different cancer types, such as breast, ovarian, endometrial, and lung cancers, as well as in Hodgkin's lymphoma and hepatocellular carcinoma. The expression of Prdx3 and Prdx5 in different types of malignancies involves their association with different factors, such as transcription factors, micro RNAs, tumor suppressors, response elements, and oncogenic genes. The microenvironment of mitochondrial Prdxs plays an important role in cancer development, as cancerous cells are equipped with a high level of antioxidants to overcome excessive ROS production. However, an increased production of Prdx3 and Prdx5 is associated with the development of chemoresistance in certain types of cancers and it leads to further complications in cancer treatment. Understanding the interplay between mitochondrial Prdxs and ROS in carcinogenesis can be useful in the development of anticancer drugs with better proficiency and decreased resistance. However, more targeted studies are required for exploring the tumor microenvironment in association with mitochondrial Prdxs to improve the existing cancer therapies and drug development.


Assuntos
Neoplasias/metabolismo , Peroxirredoxina III/metabolismo , Peroxirredoxinas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica , Humanos , Mitocôndrias/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/genética , Elementos de Resposta , Transdução de Sinais , Microambiente Tumoral , Regulação para Cima
9.
Gene ; 721: 144113, 2019 Dec 30.
Artigo em Inglês | MEDLINE | ID: mdl-31505214

RESUMO

Vaspin, initially identified in visceral adipose tissue, is an adipokine, and administration of recombinant vaspin leads to lowering of the endoplasmic reticulum stress which is elevated in obesity or enhancement of insulin sensitivity. CCAAT/enhancer binding protein (C/EBP), as a basic leucine zipper transcription factor, plays a critical role in adipocyte development and glucose and lipid metabolisms in liver. The present study aimed to investigate the effect of C/EBPα on vaspin gene expression. The expression of hepatic vaspin was markedly decreased in liver-specific C/EBPα knockout mice. A reporter assay indicated that two C/EBP-responsive elements (CEBPREs) are necessary for C/EBPα-dependent induction of vaspin promoter activities. Furthermore, electrophoretic mobility shift assay showed that C/EBPα in mouse liver is capable of directly binding the two CEBPREs. These results suggest that C/EBPα positively regulates hepatic vaspin expression through two functional CEBPREs. Thus, vaspin is a novel C/EBPα target gene in the liver.


Assuntos
Adipocinas/biossíntese , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Regulação da Expressão Gênica/fisiologia , Fígado/metabolismo , Elementos de Resposta/fisiologia , Serpinas/biossíntese , Adipocinas/genética , Animais , Camundongos , Camundongos Knockout , Serpinas/genética
10.
Clin Biochem ; 73: 70-76, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31386834

RESUMO

BACKGROUND: Chromosomal region 9p21.3 is most robustly associated with coronary artery disease (CAD) in western European populations. However, heterogeneity in CAD phenotypes leads to uncertainty whether 9p21.3 is associated with stable and/or acute clinical presentations of CAD. 9p21.3 is rich in regulatory elements, but the underlying mechanisms of its actions in CAD remain unclear. We investigate the association of 9p21.3 two haplotype blocks lead variants (rs10757278 and rs518394) with first-ever non-fatal myocardial infarction (MI) in CAD patients and their association with CDKN2B mRNA expression in peripheral blood mononuclear cells 6 months after the event. METHODS: We included CAD patients with sustained first MI (n = 523) and controls (n = 583). Gene expression was assessed in 72 patients 6 months after MI and 43 healthy controls. TaqMan® technology was used for the gene expression and genotyping analysis. RESULTS: CDKN2B mRNA was significantly lower in MI patients compared with the controls (p = 0.002) and in patients carrying the rs10757278 G risk allele versus AA homozygotes (p = 0.012) 6 months after the event. While we confirmed the association of rs10757278 with CDKN2B expression in MI patients, we failed to find an association between the investigated variants and MI or disease burden. CONCLUSIONS: We suggest a dysregulation of gene expression in the 9p21.3 region six months after acute MI, which is affected by a genetic variant in patients. The rs10757278 rare allele is one factor that might lead to prolonged risk for proatherogenic complications.


Assuntos
Cromossomos Humanos Par 9/genética , Doença da Artéria Coronariana , Inibidor de Quinase Dependente de Ciclina p15/biossíntese , Regulação da Expressão Gênica , Haplótipos , Infarto do Miocárdio , Elementos de Resposta , Adulto , Idoso , Alelos , Cromossomos Humanos Par 9/metabolismo , Doença da Artéria Coronariana/enzimologia , Doença da Artéria Coronariana/genética , Inibidor de Quinase Dependente de Ciclina p15/genética , Feminino , Seguimentos , Homozigoto , Humanos , Masculino , Pessoa de Meia-Idade , Infarto do Miocárdio/enzimologia , Infarto do Miocárdio/genética
11.
Mol Cell ; 75(6): 1178-1187.e4, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31402096

RESUMO

In complex genetic loci, individual enhancers interact most often with specific basal promoters. Here we investigate the activation of the Bicoid target gene hunchback (hb), which contains two basal promoters (P1 and P2). Early in embryogenesis, P1 is silent, while P2 is strongly activated. In vivo deletion of P2 does not cause activation of P1, suggesting that P2 contains intrinsic sequence motifs required for activation. We show that a two-motif code (a Zelda binding site plus TATA) is required and sufficient for P2 activation. Zelda sites are present in the promoters of many embryonically expressed genes, but the combination of Zelda plus TATA does not seem to be a general code for early activation or Bicoid-specific activation per se. Because Zelda sites are also found in Bicoid-dependent enhancers, we propose that simultaneous binding to both enhancers and promoters independently synchronizes chromatin accessibility and facilitates correct enhancer-promoter interactions.


Assuntos
Proteínas de Ligação a DNA/biossíntese , Proteínas de Drosophila/biossíntese , Proteínas de Drosophila/metabolismo , Proteínas de Homeodomínio/metabolismo , Motivos de Nucleotídeos , Elementos de Resposta , Transativadores/metabolismo , Fatores de Transcrição/biossíntese , Animais , Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Drosophila melanogaster , Proteínas de Homeodomínio/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Transativadores/genética , Fatores de Transcrição/genética
12.
Gen Comp Endocrinol ; 284: 113263, 2019 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-31454497

RESUMO

Corticotropin-releasing hormone (CRH) is known to act as a potent thyrotropin-releasing factor in non-mammalian species such as chicken and bullfrog. This interaction is mediated by type 2 CRH receptors (CRHR2) expressed by the thyrotropes in the pituitary gland. However, the response elements (REs) and their corresponding transcription factors (TFs) that control CRHR2 expression in thyrotropes are not known. Since thyrotrope-specific expression of the ß-subunit of thyrotropin is synergistically stimulated by the co-expression of POU1F1 and GATA2, we hypothesised that in non-mammalian vertebrates like chicken, CRHR2 expression is controlled by the same TFs and that their REs are present in the chicken CRHR2 gene promoter. In situ hybridisation and immunohistochemistry suggest that chicken thyrotropes, like those of mammals, express the mRNAs for the TFs GATA2, POU1F1 and PITX1, but not NR5A1. Using luciferase reporter assays, we show that both GATA2 and PITX1 can activate the promoter of CRHR2, but PITX1 requires a functional GATA2 RE to be present. POU1F1 alone did not affect promoter activity, but synergistically increased the effect of GATA2. Promoter deletion analysis and mutagenesis showed that essential GATA2 and PITX1 REs are located between 116 and 198 bp upstream of the start codon. These REs are highly conserved in non-mammalian species. Additionally, NR5A1 (steroidogenic factor 1) suppressed both GATA2- and PITX1-induced promoter activity and may therefore play a role in restricting CRHR2 expression in gonadotropes. We conclude that the expression of CRHR2 in chicken thyrotropes is stimulated by GATA2 with interactions with POU1F1 and PITX1, in the absence of NR5A1.


Assuntos
Galinhas/genética , Regulação da Expressão Gênica , Hipófise/metabolismo , Receptores de Hormônio Liberador da Corticotropina/genética , Fatores de Transcrição/metabolismo , Animais , Células COS , Sequência Conservada , Evolução Molecular , Luciferases/metabolismo , Mutação/genética , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Elementos de Resposta/genética , Fatores de Transcrição/genética
13.
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
14.
Nat Genet ; 51(9): 1380-1388, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31427791

RESUMO

Chromatin architecture has been implicated in cell type-specific gene regulatory programs, yet how chromatin remodels during development remains to be fully elucidated. Here, by interrogating chromatin reorganization during human pluripotent stem cell (hPSC) differentiation, we discover a role for the primate-specific endogenous retrotransposon human endogenous retrovirus subfamily H (HERV-H) in creating topologically associating domains (TADs) in hPSCs. Deleting these HERV-H elements eliminates their corresponding TAD boundaries and reduces the transcription of upstream genes, while de novo insertion of HERV-H elements can introduce new TAD boundaries. The ability of HERV-H to create TAD boundaries depends on high transcription, as transcriptional repression of HERV-H elements prevents the formation of boundaries. This ability is not limited to hPSCs, as these actively transcribed HERV-H elements and their corresponding TAD boundaries also appear in pluripotent stem cells from other hominids but not in more distantly related species lacking HERV-H elements. Overall, our results provide direct evidence for retrotransposons in actively shaping cell type- and species-specific chromatin architecture.


Assuntos
Cromatina/genética , Retrovirus Endógenos/genética , Regulação da Expressão Gênica , Células-Tronco Pluripotentes/citologia , Elementos de Resposta , Retroelementos/genética , Transcrição Genética , Animais , Diferenciação Celular , Humanos , Células-Tronco Pluripotentes/fisiologia , Primatas , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
15.
Chem Commun (Camb) ; 55(58): 8402-8405, 2019 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-31257385

RESUMO

A proof-of-principle for the application of hemi-indigo derivatives as RNA binders with photocontrollable fluorescence is presented. The photoswitch binds to the human immunodeficiency virus type 1 (HIV-1) RNA with a significant light-up effect. The fluorescence of the RNA-bound ligand can be reversibly switched ON and OFF by light without destroying the ligand-RNA associates.


Assuntos
Corantes Fluorescentes/metabolismo , HIV-1/genética , Indóis/metabolismo , RNA/metabolismo , Fluorescência , Corantes Fluorescentes/química , Corantes Fluorescentes/efeitos da radiação , Indóis/química , Indóis/efeitos da radiação , Ligantes , Luz , Estudo de Prova de Conceito , RNA/genética , Elementos de Resposta , Estereoisomerismo
16.
Nucleic Acids Res ; 47(15): 7781-7797, 2019 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-31340029

RESUMO

Polycomb Response Elements (PREs) are cis-regulatory DNA elements that maintain gene transcription states through DNA replication and mitosis. PREs have little sequence similarity, but are enriched in a number of sequence motifs. Previous methods for modelling Drosophila melanogaster PRE sequences (PREdictor and EpiPredictor) have used a set of 7 motifs and a training set of 12 PREs and 16-23 non-PREs. Advances in experimental methods for mapping chromatin binding factors and modifications has led to the publication of several genome-wide sets of Polycomb targets. In addition to the seven motifs previously used, PREs are enriched in the GTGT motif, recently associated with the sequence-specific DNA binding protein Combgap. We investigated whether models trained on genome-wide Polycomb sites generalize to independent PREs when trained with control sequences generated by naive PRE models and including the GTGT motif. We also developed a new PRE predictor: SVM-MOCCA. Training PRE predictors with genome-wide experimental data improves generalization to independent data, and SVM-MOCCA predicts the majority of PREs in three independent experimental sets. We present 2908 candidate PREs enriched in sequence and chromatin signatures. 2412 of these are also enriched in H3K4me1, a mark of Trithorax activated chromatin, suggesting that PREs/TREs have a common sequence code.


Assuntos
Algoritmos , DNA/genética , Drosophila melanogaster/genética , Genoma de Inseto , Proteínas do Grupo Polycomb/genética , Elementos de Resposta , Animais , Sítios de Ligação , Cromatina/química , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , DNA/química , DNA/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Embrião não Mamífero , Ontologia Genética , Histonas/genética , Histonas/metabolismo , Larva/genética , Larva/metabolismo , Anotação de Sequência Molecular , Motivos de Nucleotídeos , Proteínas do Grupo Polycomb/metabolismo , Ligação Proteica , Software , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
17.
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
18.
J Exp Clin Cancer Res ; 38(1): 330, 2019 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-31351496

RESUMO

BACKGROUND: RelA/p65 a crucial member of NF-κB signaling pathway plays diverse role in mediating oncogenesis. Limited knowledge prevails on the mechanistic insights of RelA gene regulation. RNA helicase p68 apart from being a vital player of RNA metabolism acts as a transcriptional coactivator of several oncogenic transcription factors including ß-catenin and is highly implicated in cancer progression. In this study, we aim to discern the molecular mechanism of how an RNA helicase, p68 deploys a major oncogenic signaling pathway, Wnt/ ß-catenin to regulate the expression of RelA, an indispensable component of NF-κB signaling pathway towards driving colon carcinogenesis. METHODS: Immunoblotting and quantitative RT-PCR was performed for determining the protein and mRNA expressions of the concerned genes respectively. Luciferase assay was employed for studying the promoter activity of RelA. Chromatin immunoprecipitation was used to evaluate the occupancy of transcription factors on the RelA promoter. Immunohistochemical analysis was conducted using FFPE sections derived from normal human colon and colon cancer patient samples. Finally, a syngeneic colorectal allograft mouse model was used to assess physiological significance of the in vitro findings. RESULTS: p68, ß-catenin and RelA proteins were found to bear strong positive correlation in normal and colon carcinoma patient samples. Both p68 and ß-catenin increased RelA mRNA and protein expression. p68, ß-catenin and Wnt3a elevated RelA promoter activity. Conversely, p68 and ß-catenin knockdown diminished RelA promoter activity and led to reduced RelA mRNA and protein expression. p68 was perceived to occupy RelA promoter with ß-catenin at the TCF4/LEF (TBE) sites thereby potentiating RelA transcription. p68 and ß-catenin alliance positively modulated the expression of signature NF-κB target genes. Enhanced NF-κB target gene expression by p68 was corroborated by findings in clinical samples. Tumors generated in mice colorectal allograft model, stably expressing p68 further reinforced our in vitro findings. CONCLUSIONS: We report for the first time a novel mechanism of alliance between p68 and ß-catenin in regulating the expression of RelA and stimulating the NF-κB signaling axis towards driving colon carcinogenesis. This study unravels novel modes of p68-mediated colon carcinogenesis, marking it a potential target for therapy.


Assuntos
Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , RNA Helicases DEAD-box/metabolismo , Regulação Neoplásica da Expressão Gênica , Fator de Transcrição RelA/genética , beta Catenina/metabolismo , Animais , Biomarcadores , Linhagem Celular Tumoral , Modelos Animais de Doenças , Xenoenxertos , Humanos , Modelos Biológicos , NF-kappa B/metabolismo , Regiões Promotoras Genéticas , Elementos de Resposta , Transdução de Sinais , Fator de Transcrição RelA/metabolismo
19.
Int J Mol Sci ; 20(15)2019 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-31357595

RESUMO

The p53 canonical consensus sequence is a 10-bp repeat of PuPuPuC(A/T)(A/T)GPyPyPy, separated by a spacer with up to 13 bases. C(A/T)(A/T)G is the core sequence and purine (Pu) and pyrimidine (Py) bases comprise the flanking sequence. However, in the p53 noncanonical sequences, there are many variations, such as length of consensus sequence, variance of core sequence or flanking sequence, and variance in number of bases making up the spacer or AT gap composition. In comparison to p53, the p53 family members p63 and p73 have been found to have more tolerance to bind and activate several of these noncanonical sequences. The p53 protein forms monomers, dimers, and tetramers, and its nonspecific binding domain is well-defined; however, those for p63 or p73 are still not fully understood. Study of p63 and p73 structure to determine the monomers, dimers or tetramers to bind and regulate noncanonical sequence is a new challenge which is crucial to obtaining a complete picture of structure and function in order to understand how p63 and p73 regulate genes differently from p53. In this review, we will summarize the rules of p53 family non-canonical sequences, especially focusing on the structure of p53 family members in the regulation of specific target genes. In addition, we will compare different software programs for prediction of p53 family responsive elements containing parameters with canonical or non-canonical sequences.


Assuntos
Elementos de Resposta , Ativação Transcricional , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Sequência Consenso , Regulação da Expressão Gênica , Variação Genética , Humanos , Família Multigênica , Ligação Proteica , Multimerização Proteica , Relação Estrutura-Atividade , Proteína Supressora de Tumor p53/genética
20.
Int J Mol Sci ; 20(12)2019 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-31212732

RESUMO

Light influences a wide range of physiological processes from prokaryotes to mammals. Neurospora crassa represents an important model system used for studying this signal pathway. At molecular levels, the WHITE COLLAR Complex (WCC), a heterodimer formed by WC-1 (the blue light photo-sensor) and WC-2 (the transcriptional activator), is the critical positive regulator of light-dependent gene expression. GATN (N indicates any other nucleotide) repeats are consensus sequences within the promoters of light-dependent genes recognized by the WCC. The distal GATN is also known as C-box since it is involved in the circadian clock. However, we know very little about the role of the proximal GATN, and the molecular mechanism that controls the transcription of light-induced genes during the dark/light transition it is still unclear. Here we showed a first indication that mutagenesis of the proximal GATA sequence within the target promoter of the albino-3 gene or deletion of the WC-1 zinc finger domain led to a rise in expression of light-dependent genes already in the dark, effectively decoupling light stimuli and transcriptional activation. This is the first observation of cis-/trans-acting repressive machinery, which is not consistent with the light-dependent regulatory mechanism observed in the eukaryotic world so far.


Assuntos
Sítios de Ligação , Escuridão , Fatores de Transcrição GATA/metabolismo , Regulação da Expressão Gênica/efeitos da radiação , Luz , Elementos de Resposta , Fatores de Transcrição/metabolismo , Sequência de Bases , Cromatina/genética , Cromatina/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Fatores de Transcrição GATA/química , Mutação , Neurospora/genética , Neurospora/metabolismo , Neurospora/efeitos da radiação , Motivos de Nucleotídeos , Fenótipo , Regiões Promotoras Genéticas , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Ativação Transcricional , Dedos de Zinco/genética
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