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1.
Genes Dev ; 33(15-16): 1083-1094, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31296559

RESUMO

The orphan nuclear receptor SHP (small heterodimer partner) is a well-known transcriptional corepressor of bile acid and lipid metabolism in the liver; however, its function in other tissues is poorly understood. Here, we report an unexpected role for SHP in the exocrine pancreas as a modulator of the endoplasmic reticulum (ER) stress response. SHP expression is induced in acinar cells in response to ER stress and regulates the protein stability of the spliced form of X-box-binding protein 1 (XBP1s), a key mediator of ER stress response. Loss of SHP reduces XBP1s protein level and transcriptional activity, which in turn attenuates the ER stress response during the fasting-feeding cycle. Consequently, SHP-deficient mice also are more susceptible to cerulein-induced pancreatitis. Mechanistically, we show that SHP physically interacts with the transactivation domain of XBP1s, thereby inhibiting the polyubiquitination and degradation of XBP1s by the Cullin3-SPOP (speckle-type POZ protein) E3 ligase complex. Together, our data implicate SHP in governing ER homeostasis and identify a novel posttranslational regulatory mechanism for the key ER stress response effector XBP1.


Assuntos
Estresse do Retículo Endoplasmático/genética , Proteólise , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteína 1 de Ligação a X-Box/metabolismo , Células Acinares/metabolismo , Animais , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/genética , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Pâncreas Exócrino/metabolismo , Pancreatite/genética , Processamento de Proteína , Estabilidade Proteica , Receptores Citoplasmáticos e Nucleares/deficiência , Receptores Citoplasmáticos e Nucleares/genética , Ubiquitinação/genética
2.
Nature ; 571(7765): 419-423, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31292545

RESUMO

Single-cell RNA sequencing (scRNA-seq) has highlighted the important role of intercellular heterogeneity in phenotype variability in both health and disease1. However, current scRNA-seq approaches provide only a snapshot of gene expression and convey little information on the true temporal dynamics and stochastic nature of transcription. A further key limitation of scRNA-seq analysis is that the RNA profile of each individual cell can be analysed only once. Here we introduce single-cell, thiol-(SH)-linked alkylation of RNA for metabolic labelling sequencing (scSLAM-seq), which integrates metabolic RNA labelling2, biochemical nucleoside conversion3 and scRNA-seq to record transcriptional activity directly by differentiating between new and old RNA for thousands of genes per single cell. We use scSLAM-seq to study the onset of infection with lytic cytomegalovirus in single mouse fibroblasts. The cell-cycle state and dose of infection deduced from old RNA enable dose-response analysis based on new RNA. scSLAM-seq thereby both visualizes and explains differences in transcriptional activity at the single-cell level. Furthermore, it depicts 'on-off' switches and transcriptional burst kinetics in host gene expression with extensive gene-specific differences that correlate with promoter-intrinsic features (TBP-TATA-box interactions and DNA methylation). Thus, gene-specific, and not cell-specific, features explain the heterogeneity in transcriptomes between individual cells and the transcriptional response to perturbations.


Assuntos
Regulação da Expressão Gênica/genética , Análise de Sequência de RNA/métodos , Análise de Célula Única , Transcrição Genética/genética , Alquilação , Animais , Ciclo Celular , Citomegalovirus/fisiologia , Metilação de DNA , Fibroblastos/metabolismo , Fibroblastos/virologia , Cinética , Camundongos , Regiões Promotoras Genéticas/genética , RNA/análise , RNA/química , Compostos de Sulfidrila/química
3.
Nat Commun ; 10(1): 2766, 2019 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-31235692

RESUMO

A major challenge in biology is that genetically identical cells in the same environment can display gene expression stochasticity (noise), which contributes to bet-hedging, drug tolerance, and cell-fate switching. The magnitude and timescales of stochastic fluctuations can depend on the gene regulatory network. Currently, it is unclear how gene expression noise of specific networks impacts the evolution of drug resistance in mammalian cells. Answering this question requires adjusting network noise independently from mean expression. Here, we develop positive and negative feedback-based synthetic gene circuits to decouple noise from the mean for Puromycin resistance gene expression in Chinese Hamster Ovary cells. In low Puromycin concentrations, the high-noise, positive-feedback network delays long-term adaptation, whereas it facilitates adaptation under high Puromycin concentration. Accordingly, the low-noise, negative-feedback circuit can maintain resistance by acquiring mutations while the positive-feedback circuit remains mutation-free and regains drug sensitivity. These findings may have profound implications for chemotherapeutic inefficiency and cancer relapse.


Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Redes Reguladoras de Genes/genética , Modelos Genéticos , Animais , Antimetabólitos Antineoplásicos/uso terapêutico , Células CHO , Simulação por Computador , Cricetulus , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Retroalimentação Fisiológica , Regulação da Expressão Gênica/genética , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Puromicina/farmacologia , Puromicina/uso terapêutico , Processos Estocásticos
4.
Nat Commun ; 10(1): 2680, 2019 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-31213597

RESUMO

Genetic studies of complex traits in animals have been hindered by the need to generate, maintain, and phenotype large panels of recombinant lines. We developed a new method, C. elegans eXtreme Quantitative Trait Locus (ceX-QTL) mapping, that overcomes this obstacle via bulk selection on millions of unique recombinant individuals. We use ceX-QTL to map a drug resistance locus with high resolution. We also map differences in gene expression in live worms and discovered that mutations in the co-chaperone sti-1 upregulate the transcription of HSP-90. Lastly, we use ceX-QTL to map loci that influence fitness genome-wide confirming previously reported causal variants and uncovering new fitness loci. ceX-QTL is fast, powerful and cost-effective, and will accelerate the study of complex traits in animals.


Assuntos
Caenorhabditis elegans/genética , Mapeamento Cromossômico/métodos , Aptidão Genética/genética , Locos de Características Quantitativas/genética , Característica Quantitativa Herdável , Animais , Mapeamento Cromossômico/economia , Resistência a Medicamentos/genética , Feminino , Regulação da Expressão Gênica/genética , Masculino , Fatores de Tempo
5.
Nat Commun ; 10(1): 2581, 2019 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-31197173

RESUMO

Despite existing reports on differential DNA methylation in type 2 diabetes (T2D) and obesity, our understanding of its functional relevance remains limited. Here we show the effect of differential methylation in the early phases of T2D pathology by a blood-based epigenome-wide association study of 4808 non-diabetic Europeans in the discovery phase and 11,750 individuals in the replication. We identify CpGs in LETM1, RBM20, IRS2, MAN2A2 and the 1q25.3 region associated with fasting insulin, and in FCRL6, SLAMF1, APOBEC3H and the 15q26.1 region with fasting glucose. In silico cross-omics analyses highlight the role of differential methylation in the crosstalk between the adaptive immune system and glucose homeostasis. The differential methylation explains at least 16.9% of the association between obesity and insulin. Our study sheds light on the biological interactions between genetic variants driving differential methylation and gene expression in the early pathogenesis of T2D.


Assuntos
Metilação de DNA/fisiologia , Diabetes Mellitus Tipo 2/genética , Glucose/metabolismo , Insulina/metabolismo , Obesidade/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Simulação por Computador , Ilhas de CpG/genética , Diabetes Mellitus Tipo 2/metabolismo , Epigênese Genética/fisiologia , Epigenômica/métodos , Feminino , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica/genética , Estudo de Associação Genômica Ampla/métodos , Homeostase/genética , Humanos , Masculino , Redes e Vias Metabólicas/genética , Pessoa de Meia-Idade , Obesidade/metabolismo , Polimorfismo de Nucleotídeo Único/fisiologia , Adulto Jovem
6.
Nat Commun ; 10(1): 2673, 2019 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-31209208

RESUMO

Alternative splicing performs a central role in expanding genomic coding capacity and proteomic diversity. However, programming of splicing patterns in engineered biological systems remains underused. Synthetic approaches thus far have predominantly focused on controlling expression of a single protein through alternative splicing. Here, we describe a modular and extensible platform for regulating four programmable exons that undergo a mutually exclusive alternative splicing event to generate multiple functionally-distinct proteins. We present an intron framework that enforces the mutual exclusivity of two internal exons and demonstrate a graded series of consensus sequence elements of varying strengths that set the ratio of two mutually exclusive isoforms. We apply this framework to program the DNA-binding domains of modular transcription factors to differentially control downstream gene activation. This splicing platform advances an approach for generating diverse isoforms and can ultimately be applied to program modular proteins and increase coding capacity of synthetic biological systems.


Assuntos
Processamento Alternativo/genética , Regulação da Expressão Gênica/genética , Engenharia Genética/métodos , RNA/genética , Fatores de Transcrição/genética , Motivos de Aminoácidos/genética , Animais , Linhagem Celular , Biologia Computacional , Sequência Consenso/genética , Éxons/genética , Biblioteca Gênica , Genes Reporter/genética , Humanos , Íntrons/genética , Mutagênese Sítio-Dirigida/métodos , Domínios Proteicos/genética , Isoformas de Proteínas/genética , RNA/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Genética
7.
Mol Biol (Mosk) ; 53(3): 430-435, 2019.
Artigo em Russo | MEDLINE | ID: mdl-31184608

RESUMO

The Oct-1 (POU2F1) transcription factor is one of the most important regulatory proteins in humans and other mammals. An increase in Oct-1 aids the resistance to oxidative and cytotoxic stresses and radiation exposure. A high level of Oct-1 is found in many human tumors and correlates with low survival. Oct-1 interacts with its binding sites as a monomer, a homodimer, or a multimer. The nucleotide sequence of the Oct-1 binding site determines the character of interaction and the conformation of Oct-1 on target DNA, thus influencing the binding of Oct-1 co-repressors and co-activators. Nucleotide substitutions were introduced in all positions of the PORE and MORE sequences and tested for effect on the Oct-1 capability of forming monomeric and dimeric DNA-protein complexes. The position and nature of nucleotide substitutions were found to affect the type of Oct-1 binding to DNA. Several substitutions suppressed the formation of dimers, while others stimulated the process. Certain nucleotide substitutions completely prevented the binding of both monomers and dimers. The Oct-1 concentration in the cell is another factor that affects the character of DNA-protein interactions. Based on the results, the nature and affinity of interaction with Oct-1 is possible to predict from the nucleotide sequence for PORE and MORE sites of the human genome.


Assuntos
Regulação da Expressão Gênica/genética , Fator 1 de Transcrição de Octâmero/química , Fator 1 de Transcrição de Octâmero/metabolismo , Sítios de Ligação , Humanos , Multimerização Proteica
8.
Zhejiang Da Xue Xue Bao Yi Xue Ban ; 48(1): 65-74, 2019 May 25.
Artigo em Chinês | MEDLINE | ID: mdl-31102360

RESUMO

OBJECTIVE: To explore the expression, localization and regulatory effect on mitochondrial calcium signaling of Rictor in embryonic stem cell-derived cardiomyocytes (ESC-CMs). METHODS: Classical embryonic stem cell cardiomyogenesis model was used for differentiation of mouse embryonic stem cells into cardiomyocytes. The location of Rictor in ESC-CMs was investigated by immunofluorescence and Western blot. The expression of Rictor in mouse embryonic stem cells was interfered with lentiviral technology, then the superposition of mitochondria and endoplasmic reticulum (ER) in ESC-CMs was detected with immunofluorescence method; the cellular ultrastructure of ESC-CMs was observed by transmission electron microscope; the mitochondrial calcium transients of ESC-CMs was detected by living cell workstation;immunoprecipitation was used to detect the interaction between 1,5,5-trisphosphate receptor (IP3 receptor, IP3R), glucose-regulated protein 75 (Grp75) and voltage-dependent anion channel 1 (VDAC1) in mitochondrial outer membrane; the expression of mitochondrial fusion protein (mitonusin-2, Mfn2) was detected by Western blot. RESULTS: Rictor was mainly localized in the endoplasmic reticulum and mitochondrial-endoplasmic reticulum membrane (MAM) in ESC-CMs. Immunofluorescence results showed that Rictor was highly overlapped with ER and mitochondria in ESC-CMs. After mitochondrial and ER were labeled with Mito-Tracker Red and ER-Tracker Green, it was demonstrated that the mitochondria of the myocardial cells in the Rictor group were scattered, and the superimposition rate of mitochondria and ER was lower than that of the negative control group (P<0.01). The MAM structures were decreased in ESC-CMs after knockdown of Rictor. The results of the living cell workstation showed that the amplitude of mitochondrial calcium transients by ATP stimulation in ESC-CMs was decreased after knockdown of Rictor (P<0.01). The results of co-immunoprecipitation showed that the interaction between IP3R, Grp75 and VDAC1 in the MAM structure of the cardiomyocytes in the Rictor group was significantly attenuated (P<0.01); the results of Western blot showed that the expression of Mfn2 protein was significantly decreased (P<0.01). CONCLUSIONS: Using lentiviral technology to interfere Rictor expression in mouse embryonic stem cells, the release of calcium from the endoplasmic reticulum to mitochondria in ESC-CMs decreases, which may be affected by reducing the interaction of IP3R, Grp75, VDAC1 and decreasing the expression of Mfn2, leading to the damage of MAM structure.


Assuntos
Sinalização do Cálcio , Células-Tronco Embrionárias Murinas , Miócitos Cardíacos , Proteína Companheira de mTOR Insensível à Rapamicina , Animais , Sinalização do Cálcio/genética , Regulação da Expressão Gênica/genética , Técnicas de Silenciamento de Genes , Camundongos , Mitocôndrias/fisiologia , Miócitos Cardíacos/fisiologia , Transporte Proteico , Proteína Companheira de mTOR Insensível à Rapamicina/genética , Proteína Companheira de mTOR Insensível à Rapamicina/metabolismo
9.
Nat Neurosci ; 22(6): 863-874, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31110321

RESUMO

An expanded GGGGCC hexanucleotide of more than 30 repeats (termed (G4C2)30+) within C9orf72 is the most prominent mutation in familial frontotemporal degeneration (FTD) and amyotrophic lateral sclerosis (ALS) (termed C9+). Through an unbiased large-scale screen of (G4C2)49-expressing Drosophila we identify the CDC73/PAF1 complex (PAF1C), a transcriptional regulator of RNA polymerase II, as a suppressor of G4C2-associated toxicity when knocked-down. Depletion of PAF1C reduces RNA and GR dipeptide production from (G4C2)30+ transgenes. Notably, in Drosophila, the PAF1C components Paf1 and Leo1 appear to be selective for the transcription of long, toxic repeat expansions, but not shorter, nontoxic expansions. In yeast, PAF1C components regulate the expression of both sense and antisense repeats. PAF1C is upregulated following (G4C2)30+ expression in flies and mice. In humans, PAF1 is also upregulated in C9+-derived cells, and its heterodimer partner, LEO1, binds C9+ repeat chromatin. In C9+ FTD, PAF1 and LEO1 are upregulated and their expression positively correlates with the expression of repeat-containing C9orf72 transcripts. These data indicate that PAF1C activity is an important factor for transcription of the long, toxic repeat in C9+ FTD.


Assuntos
Proteína C9orf72/genética , Expansão das Repetições de DNA/genética , Demência Frontotemporal/genética , Regulação da Expressão Gênica/genética , Proteínas Nucleares/genética , Animais , Drosophila melanogaster , Humanos , Camundongos , Fatores de Transcrição/genética
10.
Nat Cell Biol ; 21(5): 651-661, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31036937

RESUMO

A single genome gives rise to diverse tissues through complex epigenomic mechanisms, including N6-methyladenosine (m6A), a widespread RNA modification that is implicated in many biological processes. Here, to explore the global landscape of m6A in human tissues, we generated 21 whole-transcriptome m6A methylomes across major fetal tissues using m6A sequencing. These data reveal dynamic m6A methylation, identify large numbers of tissue differential m6A modifications and indicate that m6A is positively correlated with gene expression homeostasis. We also report m6A methylomes of long intergenic non-coding RNA (lincRNA), finding that enhancer lincRNAs are enriched for m6A. Tissue m6A regions are often enriched for single nucleotide polymorphisms that are associated with the expression of quantitative traits and complex traits including common diseases, which may potentially affect m6A modifications. Finally, we find that m6A modifications preferentially occupy genes with CpG-rich promoters, features of which regulate RNA transcript m6A. Our data indicate that m6A is widely regulated by human genetic variation and promoters, suggesting a broad involvement of m6A in human development and disease.


Assuntos
Adenosina/análogos & derivados , Elementos Facilitadores Genéticos , Desenvolvimento Fetal/genética , Feto , Adenosina/genética , Epigenômica , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/genética , Humanos , Metilação , Polimorfismo de Nucleotídeo Único/genética , Regiões Promotoras Genéticas , RNA Longo não Codificante/genética , Transcriptoma/genética
11.
Nat Cell Biol ; 21(5): 535, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31048769
12.
Biol Res ; 52(1): 27, 2019 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-31054579

RESUMO

BACKGROUND: To assess the expression of T-box transcription factor 4 (TBX4) during the anorectal development in normal and ethylenethiourea (ETU)-induced anorectal malformations (ARM) rat embryos. METHODS: Anorectal malformations was induced by ETU on the 10th gestational day (E10) in rat embryos. Spatio-temporal expression of TBX4 was evaluated in normal (n = 490) and ETU-induced ARM rat embryos (n = 455) from E13 to E16 by immunohistochemical staining, Western blot analysis and real-time RT-PCR. RESULTS: In the normal embryos, immunohistochemical staining revealed that TBX4 expression was detected in the epithelium of hindgut and urorectal septum (URS) on E13. TBX4-immunopositive cells were increased significantly in the epithelium of hindgut and URS, the future anal orifice part of cloacal membrane on E14. On E15, abundant stained cells were observed in the rectum, URS and dorsal cloacal membrane and the expression of positive cells reached its peak. On E16, only sporadic positive cells were distributed in the epithelium of the distal rectum. In the ARM embryos, the hindgut/rectum, URS and dorsal cloacal membrane were faint for TBX4 immunohistochemical staining. In the normal group, TBX4 protein and mRNA expression showed time-dependent changes in the hindgut/rectum from E13 to E16 on Western blot and real-time RT-PCR. On E13 and E15, the expression level of TBX4 mRNA in the ARM group was significantly lower than that in the normal group (P < 0.05). On E15, the expression level of TBX4 protein in the ARM group was significantly lower than that in the normal group (P < 0.05). CONCLUSIONS: The expression of TBX4 was downregulated in ETU-induced ARM embryos, which may play important roles in the pathogenesis of anorectal development.


Assuntos
Malformações Anorretais/genética , Etilenotioureia/farmacologia , Regulação da Expressão Gênica/genética , Proteínas com Domínio T/genética , Animais , Malformações Anorretais/induzido quimicamente , Western Blotting , Feminino , Imuno-Histoquímica , Gravidez , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase em Tempo Real , Proteínas com Domínio T/metabolismo
13.
Parasitol Res ; 118(6): 1919-1926, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31069534

RESUMO

In our previous study, proteomics analyses of host cells infected with Eimeria tenella sporozoites coupled with isobaric tags for relative and absolute quantitation, identified several host proteins related to Eimeria invasion. In this study, A 458-bp Gallus gallus fatty acid-binding protein 4 (FABP4) gene was cloned and subcloned to pET-28c(+) vector to construct the prokaryotic recombinant expression plasmid pET-28c(+)-FABP4. The 18.5 kDa recombinant FABP4 protein (rFABP4) was expressed and identified by western blotting. Expression of FABP4 in E. tenella sporozoite-infected DF-1 cells was downregulated significantly than in non-infected cells detected by western blotting and immunohistochemistry. The antibody inhibition assay showed that antibodies against FABP4 at 50, 100, 200, 300, and 400 µg/mL had no significant effect on sporozoite invasion. BMS-309403 and transforming growth factor-ß3 (TGF-ß3) was used to inhibit and improve the expression of FABP4 in DF-1 cells, respectively, and their effect on the sporozoite invasion of cells was detected by flow cytometry. Sporozoite invasion rate in the BMS-309403-treated group was not significantly affected; however, the invasion rate in the TGF-ß3-treated group declined significantly. These results show that host FABP4 plays a negative role in Eimeria invasion. However, further studies are needed to elucidate the exact mechanism of how FABP4 negatively regulates Eimeria invasion.


Assuntos
Galinhas/parasitologia , Coccidiose/veterinária , Eimeria tenella/metabolismo , Proteínas de Ligação a Ácido Graxo/metabolismo , Regulação da Expressão Gênica/genética , Esporozoítos/metabolismo , Animais , Anticorpos/imunologia , Western Blotting , Linhagem Celular , Coccidiose/parasitologia , Regulação para Baixo , Eimeria tenella/genética , Eimeria tenella/imunologia , Proteínas de Ligação a Ácido Graxo/genética , Proteínas de Ligação a Ácido Graxo/imunologia , Coelhos/parasitologia , Fator de Crescimento Transformador beta3/farmacologia
14.
Biomed Res Int ; 2019: 1425281, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31058184

RESUMO

Leishmania major, a protozoan parasite that diverged early from the main eukaryotic lineage, exhibits unusual mechanisms of gene expression. Little is known in this organism about the transcription factors involved in the synthesis of tRNA, 5S rRNA, and snRNAs, transcribed by RNA Polymerase III (Pol III). Here we identify and characterize the TFIIIB subunit Bdp1 in L. major (LmBdp1). Bdp1 plays key roles in Pol III transcription initiation in other organisms, as it participates in Pol III recruitment and promoter opening. In silico analysis showed that LmBdp1 contains the typical extended SANT domain as well as other Bdp1 conserved regions. Nevertheless, LmBdp1 also displays distinctive features, including the presence of only one aromatic residue in the N-linker region. We were not able to produce null mutants of LmBdp1 by homologous recombination, as the obtained double replacement cell line contained an extra copy of LmBdp1, indicating that LmBdp1 is essential for the viability of L. major promastigotes. Notably, the mutant cell line showed reduced levels of the LmBdp1 protein, and its growth was significantly decreased in relation to wild-type cells. Nuclear run-on assays demonstrated that Pol III transcription was affected in the mutant cell line, and ChIP experiments showed that LmBdp1 binds to 5S rRNA, tRNA, and snRNA genes. Thus, our results indicate that LmBdp1 is an essential protein required for Pol III transcription in L. major.


Assuntos
Leishmania major/genética , RNA Polimerase III/genética , Fator de Transcrição TFIIIB/genética , Transcrição Genética , Simulação por Computador , Sequência Conservada/genética , Regulação da Expressão Gênica/genética , Recombinação Homóloga/genética , Proteínas Mutantes/genética , Regiões Promotoras Genéticas , Domínios Proteicos/genética , Subunidades Proteicas/genética , RNA Ribossômico 5S/biossíntese , RNA Nuclear Pequeno/biossíntese , RNA de Transferência/biossíntese
15.
Biomed Res Int ; 2019: 8973076, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31058195

RESUMO

Ovaries represent one of the primary steroidogenic organs, producing estrogen and progesterone under the regulation of gonadotropins during the estrous cycle. Gonadotropins fluctuate the expression of various steroidogenesis-related genes, such as those encoding steroidogenic enzymes, cholesterol deliverer, and electronic transporter. Steroidogenic factor-1 (SF-1)/adrenal 4-binding protein (Ad4BP)/NR5A1 and liver receptor homolog-1 (LRH-1) play important roles in these phenomena via transcriptional regulation. With the aid of cAMP, SF-1/Ad4BP and LRH-1 can induce the differentiation of stem cells into steroidogenic cells. This model is a useful tool for studying the molecular mechanisms of steroidogenesis. In this article, we will provide insight into the transcriptional regulation of steroidogenesis-related genes in ovaries that are revealed from stem cell-derived steroidogenic cells. Using the cells derived from the model, novel SF-1/Ad4BP- and LRH-1-regulated genes were identified by combined DNA microarray and promoter tiling array analyses. The interaction of SF-1/Ad4BP and LRH-1 with transcriptional regulators in the regulation of ovarian steroidogenesis was also revealed.


Assuntos
Ovário/crescimento & desenvolvimento , Receptores Citoplasmáticos e Nucleares/genética , Fator Esteroidogênico 1/genética , Transcrição Genética , Diferenciação Celular/genética , Feminino , Regulação da Expressão Gênica/genética , Humanos , Ovário/metabolismo , Regiões Promotoras Genéticas , Células-Tronco/citologia , Células-Tronco/metabolismo , Fatores de Transcrição/genética
16.
Nat Cell Biol ; 21(5): 614-626, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31036939

RESUMO

Cell growth is controlled by a lysosomal signalling complex containing Rag small GTPases and mammalian target of rapamycin complex 1 (mTORC1) kinase. Here, we carried out a microscopy-based genome-wide human short interfering RNA screen and discovered a lysosome-localized G protein-coupled receptor (GPCR)-like protein, GPR137B, that interacts with Rag GTPases, increases Rag localization and activity, and thereby regulates mTORC1 translocation and activity. High GPR137B expression can recruit and activate mTORC1 in the absence of amino acids. Furthermore, GPR137B also regulates the dissociation of activated Rag from lysosomes, suggesting that GPR137B controls a cycle of Rag activation and dissociation from lysosomes. GPR137B-knockout cells exhibited defective autophagy and an expanded lysosome compartment, similar to Rag-knockout cells. Like zebrafish RagA mutants, GPR137B-mutant zebrafish had upregulated TFEB target gene expression and an expanded lysosome compartment in microglia. Thus, GPR137B is a GPCR-like lysosomal regulatory protein that controls dynamic Rag and mTORC1 localization and activity as well as lysosome morphology.


Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Genoma Humano/genética , Proteínas Monoméricas de Ligação ao GTP/genética , Receptores Acoplados a Proteínas-G/genética , Animais , Autofagia/genética , Regulação da Expressão Gênica/genética , Humanos , Lisossomos/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Microglia/metabolismo , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , RNA Interferente Pequeno/genética , Receptores Acoplados a Proteínas-G/antagonistas & inibidores , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento
17.
Int J Mol Sci ; 20(9)2019 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-31071917

RESUMO

The tooth has an unusual sensory system that converts external stimuli predominantly into pain, yet its sensory afferents in teeth demonstrate cytochemical properties of non-nociceptive neurons. This review summarizes the recent knowledge underlying this paradoxical nociception, with a focus on the ion channels involved in tooth pain. The expression of temperature-sensitive ion channels has been extensively investigated because thermal stimulation often evokes tooth pain. However, temperature-sensitive ion channels cannot explain the sudden intense tooth pain evoked by innocuous temperatures or light air puffs, leading to the hydrodynamic theory emphasizing the microfluidic movement within the dentinal tubules for detection by mechanosensitive ion channels. Several mechanosensitive ion channels expressed in dental sensory systems have been suggested as key players in the hydrodynamic theory, and TRPM7, which is abundant in the odontoblasts, and recently discovered PIEZO receptors are promising candidates. Several ligand-gated ion channels and voltage-gated ion channels expressed in dental primary afferent neurons have been discussed in relation to their potential contribution to tooth pain. In addition, in recent years, there has been growing interest in the potential sensory role of odontoblasts; thus, the expression of ion channels in odontoblasts and their potential relation to tooth pain is also reviewed.


Assuntos
Canais Iônicos/genética , Dor/genética , Proteínas Serina-Treonina Quinases/genética , Canais de Cátion TRPM/genética , Dente/fisiopatologia , Polpa Dentária/crescimento & desenvolvimento , Polpa Dentária/fisiopatologia , Dentina/metabolismo , Regulação da Expressão Gênica/genética , Humanos , Neurônios Aferentes/metabolismo , Neurônios Aferentes/patologia , Odontoblastos/metabolismo , Odontoblastos/patologia , Dor/fisiopatologia , Dente/crescimento & desenvolvimento , Gânglio Trigeminal/fisiopatologia
18.
Biomed Res Int ; 2019: 2497509, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31073522

RESUMO

To analyze gene expression data with sophisticated grouping structures and to extract hidden patterns from such data, feature selection is of critical importance. It is well known that genes do not function in isolation but rather work together within various metabolic, regulatory, and signaling pathways. If the biological knowledge contained within these pathways is taken into account, the resulting method is a pathway-based algorithm. Studies have demonstrated that a pathway-based method usually outperforms its gene-based counterpart in which no biological knowledge is considered. In this article, a pathway-based feature selection is firstly divided into three major categories, namely, pathway-level selection, bilevel selection, and pathway-guided gene selection. With bilevel selection methods being regarded as a special case of pathway-guided gene selection process, we discuss pathway-guided gene selection methods in detail and the importance of penalization in such methods. Last, we point out the potential utilizations of pathway-guided gene selection in one active research avenue, namely, to analyze longitudinal gene expression data. We believe this article provides valuable insights for computational biologists and biostatisticians so that they can make biology more computable.


Assuntos
Regulação da Expressão Gênica/genética , Transdução de Sinais/genética , Transcriptoma/genética , Algoritmos , Biologia Computacional , Bases de Dados Genéticas , Humanos
19.
Artif Cells Nanomed Biotechnol ; 47(1): 1782-1787, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31062612

RESUMO

Hepatic steatosis is one of the most important features of the pathogenesis for non-alcoholic fatty liver disease. Fat deposition in liver cells can influence hepatic lipogenesis along with other metabolic pathways and further lead to the irreversible liver cirrhosis and injury. However, the underlying mechanism of steatosis remains largely unexplored. Our previous study revealed that AQP7 played an important role in liver steatosis. In this study, we determined that the transcriptional level of AQP7 was up-regulated by estrogen receptor alpha (ERα) upon 17ß-estradiol (E2) and oleic acids treated HepG2 cells. Furthermore, we identified long non-coding RNA nuclear enriched abundant transcript 1 (NEAT1) as a potential hallmark which was down-regulated in ERα silencing HepG2 cells by RNA-Seq. Finally, we validated that the 3' terminal nucleotides of NEAT1 were contributed for the interaction with ERα to facilitate AQP7 transcription to suppress liver steatosis. Overall, our study gave evidence that NEAT1 played an important role in the activation of ERα to regulate AQP7-mediated hepatic steatosis.


Assuntos
Aquaporinas/genética , Receptor alfa de Estrogênio/metabolismo , Fígado Gorduroso/genética , Regulação da Expressão Gênica/genética , RNA Longo não Codificante/genética , Fígado Gorduroso/metabolismo , Fígado Gorduroso/patologia , Células Hep G2 , Humanos , RNA Longo não Codificante/metabolismo , Transcrição Genética/genética
20.
Genes Dev ; 33(13-14): 747-762, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31123067

RESUMO

Prolonged cold exposure stimulates the recruitment of beige adipocytes within white adipose tissue. Beige adipocytes depend on mitochondrial oxidative phosphorylation to drive thermogenesis. The transcriptional mechanisms that promote remodeling in adipose tissue during the cold are not well understood. Here we demonstrate that the transcriptional coregulator transducin-like enhancer of split 3 (TLE3) inhibits mitochondrial gene expression in beige adipocytes. Conditional deletion of TLE3 in adipocytes promotes mitochondrial oxidative metabolism and increases energy expenditure, thereby improving glucose control. Using chromatin immunoprecipitation and deep sequencing, we found that TLE3 occupies distal enhancers in proximity to nuclear-encoded mitochondrial genes and that many of these binding sites are also enriched for early B-cell factor (EBF) transcription factors. TLE3 interacts with EBF2 and blocks its ability to promote the thermogenic transcriptional program. Collectively, these studies demonstrate that TLE3 regulates thermogenic gene expression in beige adipocytes through inhibition of EBF2 transcriptional activity. Inhibition of TLE3 may provide a novel therapeutic approach for obesity and diabetes.


Assuntos
Adipócitos Bege/metabolismo , Proteínas Correpressoras/genética , Proteínas Correpressoras/metabolismo , Glucose/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Células Cultivadas , Dieta Hiperlipídica , Metabolismo Energético/genética , Deleção de Genes , Regulação da Expressão Gênica/genética , Estudo de Associação Genômica Ampla , Resistência à Insulina/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/genética , Mitocôndrias/metabolismo , Termogênese/genética
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