Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 8.943
Filtrar
1.
Methods Mol Biol ; 2854: 19-28, 2025.
Artigo em Inglês | MEDLINE | ID: mdl-39192114

RESUMO

The classic dual luciferase reporter assay has been widely used to rapidly and accurately determine the transcriptional activity of a given promoter induced by certain signal pathways in the cells. In particular, the sensitive characteristics of luciferase highlight its significance in many experiments, such as weak promoter analysis, transfection studies using small amounts of DNA, and detection in cell lines with low transfection efficiency. This chapter presents detailed information and experimental procedures for measuring interferon (IFN)-induced Interferon-Stimulated Response Element (ISRE) promoter activity using the dual luciferase reporter assay.


Assuntos
Genes Reporter , Interferons , Luciferases , Regiões Promotoras Genéticas , Elementos de Resposta , Transdução de Sinais , Humanos , Interferons/metabolismo , Interferons/genética , Luciferases/metabolismo , Luciferases/genética , Transfecção , Animais
2.
Pestic Biochem Physiol ; 204: 106081, 2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-39277394

RESUMO

Xenobiotic response element (XRE) to flavone was the cis- regulatory elements that mediates the induction of the allelochemical-metabolizing CYP321A1 gene from Helicoverpa zea. However, it was unknown whether the XRE-Fla element existed in other species. Recently we have identified and cloned the CYP321A1 gene with promoter region in a related species, Helicoverpa armigera. Sequence similarity of two orthologous CYP321A1 genes was 97.27%, but the promoter sequence similarity was only 56.32%. Sequence alignment showed the XRE-Fla like element owns three mutations in H. armigera compared with H. zea. Progressive 5' deletions and internal mutation indicated that H. armigera XRE-Fla was the essential element of CYP321A1 gene in response to flavone. XRE-Fla mutations and EMSA analysis confirmed that the H. armigera XRE-Fla element binding factor was stronger than H. zea. The findings indicate the XRE element mutations mainly contribute to the differences between the flavone-induced expressions of two CYP321A1 genes, which improve the flexibility and adaptability for allelochemical response of H. armigera.


Assuntos
Sistema Enzimático do Citocromo P-450 , Flavonas , Mariposas , Animais , Mariposas/genética , Mariposas/efeitos dos fármacos , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Flavonas/farmacologia , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Regiões Promotoras Genéticas , Sequência de Bases , Elementos de Resposta , Helicoverpa armigera
3.
PLoS One ; 19(8): e0304183, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39088439

RESUMO

Expression of the androgen receptor is key to the response of cells and tissues to androgenic steroids, such as testosterone or dihydrotestosterone, as well as impacting the benefit of hormone-dependent therapies for endocrine diseases and hormone-dependent cancers. However, the mechanisms controlling androgen receptor expression are not fully understood, limiting our ability to effectively promote or inhibit androgenic signalling therapeutically. An autoregulatory loop has been described in which androgen receptor may repress its own expression in the presence of hormone, although the molecular mechanisms are not fully understood. In this work, we elucidate the mechanisms of autoregulation and demonstrate, for the first time, that a similar repression of the AR gene is facilitated by the progesterone receptor. We show that the progesterone receptor, like the androgen receptor binds to response elements within the AR gene to effect transcriptional repression in response to hormone treatment. Mechanistically, this repression involves hormone-dependent histone deacetylation within the AR 5'UTR region and looping between sequences in intron 2 and the transcription start site (TSS). This novel pathway controlling AR expression in response to hormone stimulation may have important implications for understanding cell or tissue selective receptor signalling.


Assuntos
Regulação da Expressão Gênica , Receptores Androgênicos , Receptores Androgênicos/metabolismo , Receptores Androgênicos/genética , Humanos , Regulação da Expressão Gênica/efeitos dos fármacos , Receptores de Progesterona/metabolismo , Receptores de Progesterona/genética , Regiões 5' não Traduzidas , Elementos de Resposta , Linhagem Celular Tumoral , Acetilação , Transcrição Gênica/efeitos dos fármacos
4.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 40(8): 704-709, 2024 Aug.
Artigo em Chinês | MEDLINE | ID: mdl-39215668

RESUMO

Objective To explore the effects of Myxovirus resistance protein A (MxA) on the Janus kinase/Signal transducer and activator of transcription (JAK/STAT) pathway in HepG2 cells. Methods HepG2 cells were transfected with the pcDNA3.1-Flag-MxA construct, and subsequent localization and expression of the MxA protein were detected through immunofluorescence cytochemistry. The presence of MxA protein was further confirmed by using Western blot analysis. Following transfection with MxA small interfering RNA (si-MxA) and subsequent treatment with alpha interferon (IFN-α), real-time fluorescent quantitative PCR was employed to measure the mRNA levels of myxovirus resistance protein A (MxA), protein kinase R (PKR), and oligoadenylate synthase (OAS). Western blot analysis was used to detect the protein expression of MxA, PKR, OAS, signal transducer and activator of transcription 1 (STAT1), phosphorylated STAT1 (pSTAT1), STAT2, phosphorylated STAT2 (p-STAT2) and interferon regulatory factor 9 (IRF9). Additionally, pcDNA3.1-Flag-MxA and pISRE-TA-luc were co-transfected into HepG2 and HepG2.2.15 cells, respectively, to assess the activity of the interferon-stimulated response element (ISRE) by using a luciferase activity assay. Results MxA protein was expressed in both the cytoplasm and nucleus of HepG2 cells, with higher expression levels in the cytoplasm than in the nucleus. Knocking down MxA expression in HepG2 cells did not affect the expression of STAT1, p-STAT1, STAT2, p-STAT2, and IRF9 proteins induced by IFN-α, but significantly reduced the expression of antiviral proteins PKR and OAS. Overexpression of MxA in HepG2 cells enhanced ISRE activity and increased the expression of PKR and OAS proteins, but this effect was inhibited in HepG2.2.15 cells. Conclusion MxA induces the expression of antiviral proteins by enhancing the activity of the JAK/STAT signaling pathway ISRE.


Assuntos
2',5'-Oligoadenilato Sintetase , Proteínas de Resistência a Myxovirus , Fator de Transcrição STAT1 , eIF-2 Quinase , Humanos , Células Hep G2 , Proteínas de Resistência a Myxovirus/genética , Proteínas de Resistência a Myxovirus/metabolismo , 2',5'-Oligoadenilato Sintetase/genética , 2',5'-Oligoadenilato Sintetase/metabolismo , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismo , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/metabolismo , Interferon-alfa/farmacologia , Interferon-alfa/genética , Interferon-alfa/metabolismo , Elementos de Resposta/genética , Transdução de Sinais , Fator de Transcrição STAT2/genética , Fator de Transcrição STAT2/metabolismo , Interferons/genética , Interferons/metabolismo , Fator Gênico 3 Estimulado por Interferon, Subunidade gama/genética , Fator Gênico 3 Estimulado por Interferon, Subunidade gama/metabolismo , Regulação da Expressão Gênica
5.
Sci Rep ; 14(1): 11451, 2024 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-38769443

RESUMO

The SMALL ACIDIC PROTEIN (SMAP) gene is evolutionarily indispensable for organisms. There are two copies of the SMAP gene in the Arabidopsis thaliana genome, namely, SMAP1 and SMAP2. The function of SMAP2 is similar to that of SMAP1, and both can mediate 2,4-D responses in the root of Arabidopsis. This study cloned the AtSMAP2 genetic promoter sequence. Two promoter fragments of different lengths were designed according to the distribution of their cis-acting elements, and the corresponding ß- glucuronidase (GUS) expression vector was constructed. The expression activity of promoters of two lengths, 1993 bp and 997 bp, was studied by the genetic transformation in Arabidopsis. The prediction results of cis-acting elements in the promoter show that there are many hormone response elements in 997 bp, such as three abscisic acid response elements ABRE, gibberellin response elements P-box and GARE-motif and auxin response element AuxRR-core. Through GUS histochemical staining and qRT‒PCR analysis, it was found that the higher promoter activity of PAtSMAP2-997, compared to PAtSMAP2-1993, drove the expression of GUS genes at higher levels in Arabidopsis, especially in the root system. The results provide an important basis for subsequent studies on the regulation of AtSMAP2 gene expression and biological functions.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Clonagem Molecular , Regulação da Expressão Gênica de Plantas , Regiões Promotoras Genéticas , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Plantas Geneticamente Modificadas/genética , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Elementos de Resposta
6.
J Pharm Biomed Anal ; 245: 116185, 2024 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-38723556

RESUMO

Human epidermal growth factor receptor 2 (HER2) is a key player in the pathogenesis and progression of breast cancer and is currently a primary target for breast cancer immunotherapy. Bioactivity determination is necessary to guarantee the safety and efficacy of therapeutic antibodies targeting HER2. Nevertheless, currently available bioassays for measuring the bioactivity of anti-HER2 mAbs are either not representative or have high variability. Here, we established a reliable reporter gene assay (RGA) based on T47D-SRE-Luc cell line that expresses endogenous HER2 and luciferase controlled by serum response element (SRE) to measure the bioactivity of anti-HER2 antibodies. Neuregulin-1 (NRG-1) can lead to the heterodimerization of HER2 on the cell membrane and induce the expression of downstream SRE-controlled luciferase, while pertuzumab can dose-dependently reverse the reaction, resulting in a good dose-response curve reflecting the activity of the antibody. After optimizing the relevant assay parameters, the established RGA was fully validated based on ICH-Q2 (R1), which demonstrated that the method had excellent specificity, accuracy, precision, linearity, and stability. In summary, this robust and innovative bioactivity determination assay can be applied in the development and screening, release control, biosimilar assessment and stability studies of anti-HER2 mAbs.


Assuntos
Anticorpos Monoclonais Humanizados , Bioensaio , Genes Reporter , Luciferases , Neuregulina-1 , Receptor ErbB-2 , Receptor ErbB-2/genética , Receptor ErbB-2/imunologia , Receptor ErbB-2/antagonistas & inibidores , Humanos , Linhagem Celular Tumoral , Anticorpos Monoclonais Humanizados/farmacologia , Bioensaio/métodos , Luciferases/genética , Neuregulina-1/genética , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/imunologia , Neoplasias da Mama/genética , Feminino , Antineoplásicos Imunológicos/farmacologia , Reprodutibilidade dos Testes , Elementos de Resposta
7.
Nucleic Acids Res ; 52(14): 8515-8533, 2024 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-38783381

RESUMO

MicroRNAs (miRNAs) are critical post-transcriptional regulators in many biological processes. They act by guiding RNA-induced silencing complexes to miRNA response elements (MREs) in target mRNAs, inducing translational inhibition and/or mRNA degradation. Functional MREs are expected to predominantly occur in the 3' untranslated region and involve perfect base-pairing of the miRNA seed. Here, we generate a high-resolution map of miR-181a/b-1 (miR-181) MREs to define the targeting rules of miR-181 in developing murine T cells. By combining a multi-omics approach with computational high-resolution analyses, we uncover novel miR-181 targets and demonstrate that miR-181 acts predominantly through RNA destabilization. Importantly, we discover an alternative seed match and identify a distinct set of targets with repeat elements in the coding sequence which are targeted by miR-181 and mediate translational inhibition. In conclusion, deep profiling of MREs in primary cells is critical to expand physiologically relevant targetomes and establish context-dependent miRNA targeting rules.


Assuntos
MicroRNAs , Elementos de Resposta , MicroRNAs/genética , MicroRNAs/metabolismo , Animais , Camundongos , Estabilidade de RNA/genética , Regiões 3' não Traduzidas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Linfócitos T/metabolismo , Fases de Leitura Aberta/genética , Camundongos Endogâmicos C57BL
8.
Biosci Biotechnol Biochem ; 88(8): 941-947, 2024 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-38782732

RESUMO

Triokinase/FMN cyclase (Tkfc) is involved in fructose metabolism and is responsible for the phosphorylation of glyceraldehyde to glyceraldehyde-3-phosphate. In this study, we showed that refeeding induced hepatic expression of Tkfc in mice. Luciferase reporter gene assays using the Tkfc promoter revealed the existence of 2 hepatocyte nuclear factor 4α (HNF4α)-responsive elements (HNF4RE1 and HNF4RE2) and 1 carbohydrate-responsive element-binding protein (ChREBP)-responsive element (ChoRE1). Deletion and mutation of HNF4RE1 and HNF4RE2 or ChoRE1 abolished HNF4α and ChREBP responsiveness, respectively. HNF4α and ChREBP synergistically stimulated Tkfc promoter activity. ChoRE1 mutation attenuated but maintained HNF4α responsiveness, whereas HNF4RE1 and HNF4RE2 mutations abolished ChREBP responsiveness. Moreover, Tkfc promoter activity stimulation by ChREBP was attenuated upon HNF4α knockdown. Furthermore, Tkfc expression was decreased in the livers of ChREBP-/- and liver-specific HNF4-/- (Hnf4αΔHep) mice. Altogether, our data indicate that Tkfc is a target gene of ChREBP and HNF4α, and Tkfc promoter activity stimulation by ChREBP requires HNF4α.


Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Fator 4 Nuclear de Hepatócito , Fígado , Regiões Promotoras Genéticas , Animais , Humanos , Masculino , Camundongos , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Regulação da Expressão Gênica , Fator 4 Nuclear de Hepatócito/genética , Fator 4 Nuclear de Hepatócito/metabolismo , Fígado/metabolismo , Camundongos Knockout , Elementos de Resposta , Ativação Transcricional , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo
9.
Int J Mol Sci ; 25(9)2024 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-38732071

RESUMO

Iron regulatory proteins (IRP1 and IRP2) are the master regulators of mammalian iron homeostasis. They bind to the iron-responsive elements (IREs) of the transcripts of iron-related genes to regulate their expression, thereby maintaining cellular iron availability. The primary method to measure the IRE-binding activity of IRPs is the electrophoresis mobility shift assay (EMSA). This method is particularly useful for evaluating IRP1 activity, since IRP1 is a bifunctional enzyme and its protein levels remain similar during conversion between the IRE-binding protein and cytosolic aconitase forms. Here, we exploited a method of using a biotinylated-IRE probe to separate IRE-binding IRPs followed by immunoblotting to analyze the IRE-binding activity. This method allows for the successful measurement of IRP activity in cultured cells and mouse tissues under various iron conditions. By separating IRE-binding IRPs from the rest of the lysates, this method increases the specificity of IRP antibodies and verifies whether a band represents an IRP, thereby revealing some previously unrecognized information about IRPs. With this method, we showed that the S711-phosphorylated IRP1 was found only in the IRE-binding form in PMA-treated Hep3B cells. Second, we found a truncated IRE-binding IRP2 isoform that is generated by proteolytic cleavage on sites in the 73aa insert region of the IRP2 protein. Third, we found that higher levels of SDS, compared to 1-2% SDS in regular loading buffer, could dramatically increase the band intensity of IRPs in immunoblots, especially in HL-60 cells. Fourth, we found that the addition of SDS or LDS to cell lysates activated protein degradation at 37 °C or room temperature, especially in HL-60 cell lysates. As this method is more practical, sensitive, and cost-effective, we believe that its application will enhance future research on iron regulation and metabolism.


Assuntos
Proteína 1 Reguladora do Ferro , Ferro , Humanos , Animais , Ferro/metabolismo , Proteína 1 Reguladora do Ferro/metabolismo , Proteína 1 Reguladora do Ferro/genética , Camundongos , Proteína 2 Reguladora do Ferro/metabolismo , Proteína 2 Reguladora do Ferro/genética , Biotinilação , Elementos de Resposta , Fosforilação , Proteínas Reguladoras de Ferro/metabolismo , Proteínas Reguladoras de Ferro/genética , Ligação Proteica , Linhagem Celular Tumoral
10.
Methods Mol Biol ; 2807: 175-194, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38743229

RESUMO

Retroviruses must overcome cellular restrictions to the nucleocytoplasmic export of viral mRNAs that retain introns in order to complete their replication cycle. HIV accomplishes this using a system comprised of a trans-acting viral protein, Rev, and a cis-acting RNA secondary structure in the viral genome, the Rev-Response Element (RRE). HIV primary isolates differ with respect to the sequence and functional activity of the Rev-RRE system. Here, we describe a high throughput assay system for analyzing Rev-RRE functional activity using packageable viral vectors.


Assuntos
RNA Viral , Elementos de Resposta , Produtos do Gene rev do Vírus da Imunodeficiência Humana , Humanos , Produtos do Gene rev do Vírus da Imunodeficiência Humana/genética , Produtos do Gene rev do Vírus da Imunodeficiência Humana/metabolismo , Elementos de Resposta/genética , RNA Viral/genética , HIV-1/genética , HIV-1/fisiologia , Regulação Viral da Expressão Gênica , Replicação Viral/genética , Vetores Genéticos/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
11.
Genome Biol ; 25(1): 128, 2024 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-38773499

RESUMO

BACKGROUND: Cellular iron homeostasis is regulated by iron regulatory proteins (IRP1 and IRP2) that sense iron levels (and other metabolic cues) and modulate mRNA translation or stability via interaction with iron regulatory elements (IREs). IRP2 is viewed as the primary regulator in the liver, yet our previous datasets showing diurnal rhythms for certain IRE-containing mRNAs suggest a nuanced temporal control mechanism. The purpose of this study is to gain insights into the daily regulatory dynamics across IRE-bearing mRNAs, specific IRP involvement, and underlying systemic and cellular rhythmicity cues in mouse liver. RESULTS: We uncover high-amplitude diurnal oscillations in the regulation of key IRE-containing transcripts in the liver, compatible with maximal IRP activity at the onset of the dark phase. Although IRP2 protein levels also exhibit some diurnal variations and peak at the light-dark transition, ribosome profiling in IRP2-deficient mice reveals that maximal repression of target mRNAs at this timepoint still occurs. We further find that diurnal regulation of IRE-containing mRNAs can continue in the absence of a functional circadian clock as long as feeding is rhythmic. CONCLUSIONS: Our findings suggest temporally controlled redundancy in IRP activities, with IRP2 mediating regulation of IRE-containing transcripts in the light phase and redundancy, conceivably with IRP1, at dark onset. Moreover, we highlight the significance of feeding-associated signals in driving rhythmicity. Our work highlights the dynamic nature and regulatory complexity in a metabolic pathway that had previously been considered well-understood.


Assuntos
Ritmo Circadiano , Proteína 1 Reguladora do Ferro , Proteína 2 Reguladora do Ferro , Ferro , Fígado , RNA Mensageiro , Animais , Proteína 1 Reguladora do Ferro/metabolismo , Proteína 1 Reguladora do Ferro/genética , Proteína 2 Reguladora do Ferro/metabolismo , Proteína 2 Reguladora do Ferro/genética , Ritmo Circadiano/genética , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Camundongos , Fígado/metabolismo , Ferro/metabolismo , Regulação da Expressão Gênica , Elementos de Resposta , Camundongos Endogâmicos C57BL , Masculino , Comportamento Alimentar
12.
J Virol ; 98(6): e0042324, 2024 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-38771044

RESUMO

Bovine alphaherpesvirus 1 (BoHV-1) infection causes respiratory tract disorders and immune suppression and may induce bacterial pneumonia. BoHV-1 establishes lifelong latency in sensory neurons after acute infection. Reactivation from latency consistently occurs following stress or intravenous injection of the synthetic corticosteroid dexamethasone (DEX), which mimics stress. The immediate early transcription unit 1 (IEtu1) promoter drives expression of infected cell protein 0 (bICP0) and bICP4, two viral transcriptional regulators necessary for productive infection and reactivation from latency. The IEtu1 promoter contains two glucocorticoid receptor (GR) responsive elements (GREs) that are transactivated by activated GR. GC-rich motifs, including consensus binding sites for specificity protein 1 (Sp1), are in the IEtu1 promoter sequences. E2F family members bind a consensus sequence (TTTCCCGC) and certain specificity protein 1 (Sp1) sites. Consequently, we hypothesized that certain E2F family members activate IEtu1 promoter activity. DEX treatment of latently infected calves increased the number of E2F2+ TG neurons. GR and E2F2, but not E2F1, E2F3a, or E2F3b, cooperatively transactivate a 436-bp cis-regulatory module in the IEtu1 promoter that contains both GREs. A luciferase reporter construct containing a 222-bp fragment downstream of the GREs was transactivated by E2F2 unless two adjacent Sp1 binding sites were mutated. Chromatin immunoprecipitation studies revealed that E2F2 occupied IEtu1 promoter sequences when the BoHV-1 genome was transfected into mouse neuroblastoma (Neuro-2A) or monkey kidney (CV-1) cells. In summary, these findings revealed that GR and E2F2 cooperatively transactivate IEtu1 promoter activity, which is predicted to influence the early stages of BoHV-1 reactivation from latency. IMPORTANCE: Bovine alpha-herpesvirus 1 (BoHV-1) acute infection in cattle leads to establishment of latency in sensory neurons in the trigeminal ganglia (TG). A synthetic corticosteroid dexamethasone consistently initiates BoHV-1 reactivation in latently infected calves. The BoHV-1 immediate early transcription unit 1 (IEtu1) promoter regulates expression of infected cell protein 0 (bICP0) and bICP4, two viral transcriptional regulators. Hence, the IEtu1 promoter must be activated for the reactivation to occur. The number of TG neurons expressing E2F2, a transcription factor and cell cycle regulator, increased during early stages of reactivation from latency. The glucocorticoid receptor (GR) and E2F2, but not E2F1, E2F3a, or E2F3b, cooperatively transactivated a 436-bp cis-regulatory module (CRM) in the IEtu1 promoter that contains two GR responsive elements. Chromatin immunoprecipitation studies revealed that E2F2 occupies IEtu1 promoter sequences in cultured cells. GR and E2F2 mediate cooperative transactivation of IEtu1 promoter activity, which is predicted to stimulate viral replication following stressful stimuli.


Assuntos
Ciclo Celular , Fator de Transcrição E2F2 , Regulação Viral da Expressão Gênica , Herpesvirus Bovino 1 , Proteínas Imediatamente Precoces , Regiões Promotoras Genéticas , Receptores de Glucocorticoides , Ativação Transcricional , Animais , Bovinos , Camundongos , Sítios de Ligação , Linhagem Celular , Dexametasona/farmacologia , Fator de Transcrição E2F2/metabolismo , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Regulação Viral da Expressão Gênica/genética , Infecções por Herpesviridae/virologia , Infecções por Herpesviridae/metabolismo , Infecções por Herpesviridae/veterinária , Infecções por Herpesviridae/genética , Herpesvirus Bovino 1/genética , Herpesvirus Bovino 1/fisiologia , Proteínas Imediatamente Precoces/genética , Neurônios/virologia , Receptores de Glucocorticoides/metabolismo , Elementos de Resposta/genética , Fator de Transcrição Sp1/metabolismo , Transativadores/metabolismo , Gânglio Trigeminal/citologia , Gânglio Trigeminal/virologia , Ativação Viral , Latência Viral
13.
Adv Healthc Mater ; 13(22): e2401087, 2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-38696899

RESUMO

Hypoxia, a ubiquitous hallmark in cancer, underscores the significance of targeting HIF-1α, the principal transcriptional factor of hypoxic responses, for effective cancer therapy. Herein, DNA yokes, a novel class of DNA nanomaterials harboring specific HIF-1α binding sequences (hypoxia response elements, HREs), are introduced as nanopharmaceuticals for cancer treatment. Comprising a basal tetrahedral DNA nanostructure and four HRE-bearing overhanging chains, DNA yokes exhibit exceptional stability and prolonged intracellular retention. The investigation reveals their capacity to bind HIF-1α, thereby disrupting its interaction with the downstream genomic DNAs and impeding transcriptional activity. Moreover, DNA yokes facilitate HIF-1α degradation via the ubiquitination pathway, thereby sequestering it from downstream targets and ultimately promoting its degradation. In addition, DNA yokes attenuate cancer cell proliferation, migration, and invasion under hypoxic conditions, while also displaying preferential accumulation within tumors, thereby inhibiting tumor growth and metastasis in vivo. This study pioneers a novel approach to cancer therapy through the development of DNA-based drugs characterized by high stability and low toxicity to normal cells, positioning DNA yokes as promising candidates for cancer treatment.


Assuntos
DNA , Subunidade alfa do Fator 1 Induzível por Hipóxia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Humanos , DNA/química , DNA/metabolismo , Animais , Linhagem Celular Tumoral , Camundongos , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/patologia , Proliferação de Células/efeitos dos fármacos , Camundongos Nus , Nanoestruturas/química , Antineoplásicos/farmacologia , Antineoplásicos/química , Camundongos Endogâmicos BALB C , Movimento Celular/efeitos dos fármacos , Elementos de Resposta
14.
Int J Mol Sci ; 25(8)2024 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-38673804

RESUMO

Throughout its lifecycle, Entamoeba histolytica encounters a variety of stressful conditions. This parasite possesses Heat Shock Response Elements (HSEs) which are crucial for regulating the expression of various genes, aiding in its adaptation and survival. These HSEs are regulated by Heat Shock Transcription Factors (EhHSTFs). Our research has identified seven such factors in the parasite, designated as EhHSTF1 through to EhHSTF7. Significantly, under heat shock conditions and in the presence of the antiamoebic compound emetine, EhHSTF5, EhHSTF6, and EhHSTF7 show overexpression, highlighting their essential role in gene response to these stressors. Currently, only EhHSTF7 has been confirmed to recognize the HSE as a promoter of the EhPgp5 gene (HSE_EhPgp5), leaving the binding potential of the other EhHSTFs to HSEs yet to be explored. Consequently, our study aimed to examine, both in vitro and in silico, the oligomerization, and binding capabilities of the recombinant EhHSTF5 protein (rEhHSTF5) to HSE_EhPgp5. The in vitro results indicate that the oligomerization of rEhHSTF5 is concentration-dependent, with its dimeric conformation showing a higher affinity for HSE_EhPgp5 than its monomeric state. In silico analysis suggests that the alpha 3 α-helix (α3-helix) of the DNA-binding domain (DBD5) of EhHSTF5 is crucial in binding to the major groove of HSE, primarily through hydrogen bonding and salt-bridge interactions. In summary, our results highlight the importance of oligomerization in enhancing the affinity of rEhHSTF5 for HSE_EhPgp5 and demonstrate its ability to specifically recognize structural motifs within HSE_EhPgp5. These insights significantly contribute to our understanding of one of the potential molecular mechanisms employed by this parasite to efficiently respond to various stressors, thereby enabling successful adaptation and survival within its host environment.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP , Entamoeba histolytica , Regiões Promotoras Genéticas , Proteínas de Protozoários , Sítios de Ligação , Simulação por Computador , Entamoeba histolytica/genética , Entamoeba histolytica/metabolismo , Resposta ao Choque Térmico/genética , Ligação Proteica , Multimerização Proteica , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/química , Elementos de Resposta , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/química , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo
15.
Sci Adv ; 10(17): eadn1837, 2024 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-38657072

RESUMO

Polycomb group (PcG) proteins mediate epigenetic silencing of important developmental genes by modifying histones and compacting chromatin through two major protein complexes, PRC1 and PRC2. These complexes are recruited to DNA by CpG islands (CGIs) in mammals and Polycomb response elements (PREs) in Drosophila. When PcG target genes are turned OFF, PcG proteins bind to PREs or CGIs, and PREs serve as anchors that loop together and stabilize gene silencing. Here, we address which PcG proteins bind to PREs and whether PREs mediate looping when their targets are in the ON transcriptional state. While the binding of most PcG proteins decreases at PREs in the ON state, one PRC1 component, Ph, remains bound. Further, PREs can loop to each other and with presumptive enhancers in the ON state and, like CGIs, may act as tethering elements between promoters and enhancers. Overall, our data suggest that PREs are important looping elements for developmental loci in both the ON and OFF states.


Assuntos
Proteínas de Drosophila , Proteínas do Grupo Polycomb , Ligação Proteica , Elementos de Resposta , Transcrição Gênica , Animais , Proteínas do Grupo Polycomb/metabolismo , Proteínas do Grupo Polycomb/genética , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Ilhas de CpG , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Cromatina/metabolismo , Cromatina/genética , Regiões Promotoras Genéticas
16.
Cell Rep ; 43(4): 113972, 2024 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-38517892

RESUMO

Hypoxia-inducible factor 1 (HIF-1) is a transcriptional activator that mediates cellular adaptation to decreased oxygen availability. HIF-1 recruits chromatin-modifying enzymes leading to changes in histone acetylation, citrullination, and methylation at target genes. Here, we demonstrate that hypoxia-inducible gene expression in estrogen receptor (ER)-positive MCF7 and ER-negative SUM159 human breast cancer cells requires the histone H2A/H2B chaperone facilitates chromatin transcription (FACT) and the H2B ubiquitin ligase RING finger protein 20/40 (RNF20/40). Knockdown of FACT or RNF20/40 expression leads to decreased transcription initiation and elongation at HIF-1 target genes. Mechanistically, FACT and RNF20/40 are recruited to hypoxia response elements (HREs) by HIF-1 and stabilize binding of HIF-1 (and each other) at HREs. Hypoxia induces the monoubiquitination of histone H2B at lysine 120 at HIF-1 target genes in an HIF-1-dependent manner. Together, these findings delineate a cooperative molecular mechanism by which FACT and RNF20/40 stabilize multiprotein complex formation at HREs and mediate histone ubiquitination to facilitate HIF-1 transcriptional activity.


Assuntos
Proteínas de Ligação a DNA , Fator 1 Induzível por Hipóxia , Ubiquitina-Proteína Ligases , Humanos , Hipóxia Celular , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Histonas/metabolismo , Fator 1 Induzível por Hipóxia/metabolismo , Células MCF-7 , Ligação Proteica , Elementos de Resposta , Fatores de Transcrição/metabolismo , Ativação Transcricional , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitinação
17.
Int J Mol Sci ; 25(3)2024 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-38338965

RESUMO

A birthweight centile (BWC) below the 25th is associated with an elevated risk of adverse perinatal outcomes, particularly among males. This male vulnerability may stem from alterations in placenta-specific androgen signalling, a signalling axis that involves the androgen receptor (AR)-mediated regulation of target genes containing androgen response elements (AREs). In this study, we examined global and ARE-specific transcriptomic signatures in term male placentae (≥37 weeks of gestation) across BWC subcategories (<10th, 10th-30th, >30th) using RNA-seq and gene set enrichment analysis. ARE-containing transcripts in placentae with BWCs below the 10th percentile were upregulated compared to those in the 10th-30th and >30th percentiles, which coincided with the enrichment of gene sets related to hypoxia and the suppression of gene sets associated with mitochondrial function. In the absence of ARE-containing transcripts in silico, <10th and 10th-30th BWC subcategory placentae upregulated gene sets involved in vasculature development, immune function, and cell adhesion when compared to those in the >30th BWC subcategory. Collectively, our in silico findings suggest that changes in the expression of ARE-containing transcripts in male placentae may contribute to impaired placental vasculature and therefore result in reduced fetal growth outcomes.


Assuntos
Androgênios , Placenta , Gravidez , Masculino , Humanos , Feminino , Androgênios/farmacologia , Desenvolvimento Fetal , Perfilação da Expressão Gênica , Elementos de Resposta
18.
Neuropsychopharmacol Rep ; 44(1): 262-266, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38267013

RESUMO

BACKGROUND: More than 800 000 people die by suicide annually. The heritability of suicide is 30%-50%. We focused on the hypoxia response element (HRE), which promotes the expression of macrophage migration inhibitory factor (MIF) via the hypoxia-inducible factor (HIF) pathway, important in neurogenesis and neuroprotection. We examined a genetic polymorphism of rs17004038, a single-nucleotide polymorphism (SNP), in suicide completers and controls. METHODS: The study population included 1336 suicide completers and 814 unrelated healthy controls. All participants were Japanese. We obtained peripheral blood, extracted DNA, and genotyped the patients for SNP rs17004038 (C > A). RESULTS: No significant differences were observed between the two groups in either the allele or genotype analyses. Subgroup analyses by sex, age (<40 or ≥40), and suicide method (violent or nonviolent suicide) were performed with similar results. CONCLUSION: No association was observed between SNP rs17004038 and suicide completion. Although it is challenging to collect a large number of samples from suicide completers, further MIF-related genetic studies, including those of rs17004038, are necessary with larger sample sizes.


Assuntos
Fatores Inibidores da Migração de Macrófagos , Suicídio , Humanos , Predisposição Genética para Doença , Hipóxia/genética , Japão , Fatores Inibidores da Migração de Macrófagos/genética , Polimorfismo de Nucleotídeo Único , Elementos de Resposta
19.
Nat Commun ; 15(1): 586, 2024 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-38233393

RESUMO

X-chromosomal genetic variants are understudied but can yield valuable insights into sexually dimorphic human traits and diseases. We performed a sex-stratified cross-ancestry X-chromosome-wide association meta-analysis of seven kidney-related traits (n = 908,697), identifying 23 loci genome-wide significantly associated with two of the traits: 7 for uric acid and 16 for estimated glomerular filtration rate (eGFR), including four novel eGFR loci containing the functionally plausible prioritized genes ACSL4, CLDN2, TSPAN6 and the female-specific DRP2. Further, we identified five novel sex-interactions, comprising male-specific effects at FAM9B and AR/EDA2R, and three sex-differential findings with larger genetic effect sizes in males at DCAF12L1 and MST4 and larger effect sizes in females at HPRT1. All prioritized genes in loci showing significant sex-interactions were located next to androgen response elements (ARE). Five ARE genes showed sex-differential expressions. This study contributes new insights into sex-dimorphisms of kidney traits along with new prioritized gene targets for further molecular research.


Assuntos
Androgênios , Estudo de Associação Genômica Ampla , Humanos , Masculino , Feminino , Androgênios/genética , Rim , Cromossomos Humanos X/genética , Elementos de Resposta , Polimorfismo de Nucleotídeo Único , Predisposição Genética para Doença , Tetraspaninas/genética
20.
Evolution ; 78(1): 195-208, 2024 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-37996091

RESUMO

In males of many vertebrate species, sexual selection has led to the evolution of sexually dimorphic traits, which are often developmentally controlled by androgen signaling involving androgen response elements (AREs). Evolutionary changes in the number and genomic locations of AREs can modify patterns of receptor regulation and potentially alter gene expression. Here, we use recently sequenced primate genomes to evaluate the hypothesis that the strength of sexual selection is related to the genome-wide number of AREs in a diversifying lineage. In humans, we find a higher incidence of AREs near male-biased genes and androgen-responsive genes when compared with randomly selected genes from the genome. In a set of primates, we find that gains or losses of AREs proximal to genes are correlated with changes in male expression levels and the degree of sex-biased expression of those genes. In a larger set of primates, we find that an increase in one indicator of sexual selection, canine size sexual dimorphism, is correlated with genome-wide ARE counts. Our results suggest that the responsiveness of the genome to androgens in humans and their close relatives has been shaped by sexual selection that arises from competition among males for mating access to females.


Assuntos
Androgênios , Caracteres Sexuais , Feminino , Humanos , Masculino , Animais , Cães , Primatas/genética , Elementos de Resposta , Proliferação de Células
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA