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1.
Proc Natl Acad Sci U S A ; 121(28): e2322066121, 2024 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-38968125

RESUMO

The Wnt/Wingless signaling pathway plays critical roles in metazoan development and energy metabolism, but its role in regulating lipid homeostasis remains not fully understood. Here, we report that the activation of canonical Wnt/Wg signaling promotes lipolysis while concurrently inhibiting lipogenesis and fatty acid ß-oxidation in both larval and adult adipocytes, as well as cultured S2R+ cells, in Drosophila. Using RNA-sequencing and CUT&RUN (Cleavage Under Targets & Release Using Nuclease) assays, we identified a set of Wnt target genes responsible for intracellular lipid homeostasis. Notably, active Wnt signaling directly represses the transcription of these genes, resulting in decreased de novo lipogenesis and fatty acid ß-oxidation, but increased lipolysis. These changes lead to elevated free fatty acids and reduced triglyceride (TG) accumulation in adipocytes with active Wnt signaling. Conversely, downregulation of Wnt signaling in the fat body promotes TG accumulation in both larval and adult adipocytes. The attenuation of Wnt signaling also increases the expression of specific lipid metabolism-related genes in larval adipocytes, wing discs, and adult intestines. Taken together, these findings suggest that Wnt signaling-induced transcriptional repression plays an important role in regulating lipid homeostasis by enhancing lipolysis while simultaneously suppressing lipogenesis and fatty acid ß-oxidation.


Assuntos
Proteínas de Drosophila , Via de Sinalização Wnt , Animais , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Adipócitos/metabolismo , Mobilização Lipídica , Drosophila melanogaster/metabolismo , Drosophila melanogaster/genética , Proteína Wnt1/metabolismo , Proteína Wnt1/genética , Lipólise , Lipogênese/genética , Triglicerídeos/metabolismo , Metabolismo dos Lipídeos/genética , Larva/metabolismo , Larva/genética , Transcrição Gênica , Homeostase
2.
Nature ; 569(7758): 718-722, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31118511

RESUMO

Nucleic acids from bacteria or viruses induce potent immune responses in infected cells1-4. The detection of pathogen-derived nucleic acids is a central strategy by which the host senses infection and initiates protective immune responses5,6. Cyclic GMP-AMP synthase (cGAS) is a double-stranded DNA sensor7,8. It catalyses the synthesis of cyclic GMP-AMP (cGAMP)9-12, which stimulates the induction of type I interferons through the STING-TBK1-IRF-3 signalling axis13-15. STING oligomerizes after binding of cGAMP, leading to the recruitment and activation of the TBK1 kinase8,16. The IRF-3 transcription factor is then recruited to the signalling complex and activated by TBK18,17-20. Phosphorylated IRF-3 translocates to the nucleus and initiates the expression of type I interferons21. However, the precise mechanisms that govern activation of STING by cGAMP and subsequent activation of TBK1 by STING remain unclear. Here we show that a conserved PLPLRT/SD motif within the C-terminal tail of STING mediates the recruitment and activation of TBK1. Crystal structures of TBK1 bound to STING reveal that the PLPLRT/SD motif binds to the dimer interface of TBK1. Cell-based studies confirm that the direct interaction between TBK1 and STING is essential for induction of IFNß after cGAMP stimulation. Moreover, we show that full-length STING oligomerizes after it binds cGAMP, and highlight this as an essential step in the activation of STING-mediated signalling. These findings provide a structural basis for the development of STING agonists and antagonists for the treatment of cancer and autoimmune disorders.


Assuntos
Motivos de Aminoácidos , Sequência Conservada , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Cristalografia por Raios X , Ativação Enzimática , Células HEK293 , Humanos , Interferon beta/metabolismo , Proteínas de Membrana/genética , Modelos Moleculares , Mutação , Nucleotídeos Cíclicos/metabolismo , Ligação Proteica , Transdução de Sinais
3.
EMBO Rep ; 22(4): e51298, 2021 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-33594776

RESUMO

Notch signaling and epigenetic factors are known to play critical roles in regulating tissue homeostasis in most multicellular organisms, but how Notch signaling coordinates with epigenetic modulators to control differentiation remains poorly understood. Here, we identify heterochromatin protein 1c (HP1c) as an essential epigenetic regulator of gut homeostasis in Drosophila. Specifically, we observe that HP1c loss-of-function phenotypes resemble those observed after Notch signaling perturbation and that HP1c interacts genetically with components of the Notch pathway. HP1c represses the transcription of Notch target genes by directly interacting with Suppressor of Hairless (Su(H)), the key transcription factor of Notch signaling. Moreover, phenotypes caused by depletion of HP1c in Drosophila can be rescued by expressing human HP1γ, suggesting that HP1γ functions similar to HP1c in Drosophila. Taken together, our findings reveal an essential role of HP1c in normal development and gut homeostasis by suppressing Notch signaling.


Assuntos
Proteínas de Drosophila , Animais , Proteínas Cromossômicas não Histona/genética , Drosophila/genética , Proteínas de Drosophila/genética , Heterocromatina , Homeostase , Humanos , Receptores Notch/genética
4.
PLoS Genet ; 16(5): e1008832, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32463833

RESUMO

Dysregulation of CDK8 (Cyclin-Dependent Kinase 8) and its regulatory partner CycC (Cyclin C), two subunits of the conserved Mediator (MED) complex, have been linked to diverse human diseases such as cancer. Thus, it is essential to understand the regulatory network modulating the CDK8-CycC complex in both normal development and tumorigenesis. To identify upstream regulators or downstream effectors of CDK8, we performed a dominant modifier genetic screen in Drosophila based on the defects in vein patterning caused by specific depletion or overexpression of CDK8 or CycC in developing wing imaginal discs. We identified 26 genomic loci whose haploinsufficiency can modify these CDK8- or CycC-specific phenotypes. Further analysis of two overlapping deficiency lines and mutant alleles led us to identify genetic interactions between the CDK8-CycC pair and the components of the Decapentaplegic (Dpp, the Drosophila homolog of TGFß, or Transforming Growth Factor-ß) signaling pathway. We observed that CDK8-CycC positively regulates transcription activated by Mad (Mothers against dpp), the primary transcription factor downstream of the Dpp/TGFß signaling pathway. CDK8 can directly interact with Mad in vitro through the linker region between the DNA-binding MH1 (Mad homology 1) domain and the carboxy terminal MH2 (Mad homology 2) transactivation domain. Besides CDK8 and CycC, further analyses of other subunits of the MED complex have revealed six additional subunits that are required for Mad-dependent transcription in the wing discs: Med12, Med13, Med15, Med23, Med24, and Med31. Furthermore, our analyses confirmed the positive roles of CDK9 and Yorkie in regulating Mad-dependent gene expression in vivo. These results suggest that CDK8 and CycC, together with a few other subunits of the MED complex, may coordinate with other transcription cofactors in regulating Mad-dependent transcription during wing development in Drosophila.


Assuntos
Ciclina C/genética , Quinase 8 Dependente de Ciclina/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Fatores de Transcrição/metabolismo , Animais , Ciclina C/metabolismo , Quinase 8 Dependente de Ciclina/metabolismo , Drosophila , Regulação da Expressão Gênica no Desenvolvimento , Haploinsuficiência , Discos Imaginais/crescimento & desenvolvimento , Discos Imaginais/metabolismo , Transdução de Sinais , Transcrição Gênica
5.
Proc Natl Acad Sci U S A ; 115(18): 4719-4724, 2018 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-29666231

RESUMO

CRISPR/Cas9-based transcriptional activation (CRISPRa) has recently emerged as a powerful and scalable technique for systematic overexpression genetic analysis in Drosophila melanogaster We present flySAM, a potent tool for in vivo CRISPRa, which offers major improvements over existing strategies in terms of effectiveness, scalability, and ease of use. flySAM outperforms existing in vivo CRISPRa strategies and approximates phenotypes obtained using traditional Gal4-UAS overexpression. Moreover, because flySAM typically requires only a single sgRNA, it dramatically improves scalability. We use flySAM to demonstrate multiplexed CRISPRa, which has not been previously shown in vivo. In addition, we have simplified the experimental use of flySAM by creating a single vector encoding both the UAS:Cas9-activator and the sgRNA, allowing for inducible CRISPRa in a single genetic cross. flySAM will replace previous CRISPRa strategies as the basis of our growing genome-wide transgenic overexpression resource, TRiP-OE.


Assuntos
Animais Geneticamente Modificados , Sistemas CRISPR-Cas , Proteínas de Drosophila , Regulação da Expressão Gênica/genética , Fatores de Transcrição , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/metabolismo , Proteínas de Drosophila/biossíntese , Proteínas de Drosophila/genética , Drosophila melanogaster , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética
6.
Proc Natl Acad Sci U S A ; 114(36): E7469-E7478, 2017 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-28827348

RESUMO

Deregulated Wnt signaling and altered lipid metabolism have been linked to obesity, diabetes, and various cancers, highlighting the importance of identifying inhibitors that can modulate Wnt signaling and aberrant lipid metabolism. We have established a Drosophila model with hyperactivated Wnt signaling caused by partial loss of axin, a key component of the Wnt cascade. The Axin mutant larvae are transparent and have severe adipocyte defects caused by up-regulation of ß-catenin transcriptional activities. We demonstrate pharmacologic mitigation of these phenotypes in Axin mutants by identifying bortezomib and additional peptide boronic acids. We show that the suppressive effect of peptide boronic acids on hyperactive Wnt signaling is dependent on α-catenin; the rescue effect is completely abolished with the depletion of α-catenin in adipocytes. These results indicate that rather than targeting the canonical Wnt signaling pathway directly, pharmacologic modulation of ß-catenin activity through α-catenin is a potentially attractive approach to attenuating Wnt signaling in vivo.


Assuntos
Adipócitos/efeitos dos fármacos , Ácidos Borônicos/farmacologia , Peptídeos/farmacologia , Proteínas Wnt/metabolismo , Via de Sinalização Wnt/efeitos dos fármacos , Animais , Proteína Axina/metabolismo , Drosophila/efeitos dos fármacos , Drosophila/metabolismo , Transcrição Gênica/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , beta Catenina/metabolismo
7.
Genes Dev ; 26(4): 356-68, 2012 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-22345517

RESUMO

E2F transcription factors are important regulators of cell proliferation and are frequently dysregulated in human malignancies. To identify novel regulators of E2F function, we used Drosophila as a model system to screen for mutations that modify phenotypes caused by reduced levels of dE2F1. This screen identified components of the Pumilio translational repressor complex (Pumilio, Nanos, and Brain tumor) as suppressors of dE2F1-RNAi phenotypes. Subsequent experiments provided evidence that Pumilio complexes repress dE2F1 levels and that this mechanism of post-transcriptional regulation is conserved in human cells. The human Pumilio homologs Pum 1 and Pum 2 repress the translation of E2F3 by binding to the E2F3 3' untranslated region (UTR) and also enhance the activity of multiple E2F3 targeting microRNAs (miRNAs). E2F3 is an oncogene with strong proliferative potential and is regularly dysregulated or overexpressed in cancer. Interestingly, Pumilio/miRNA-mediated regulation of E2F3 is circumvented in cancer cells in several different ways. Bladder carcinomas selectively down-regulate miRNAs that cooperate with Pumilio to target E2F3, and multiple tumor cell lines shorten the 3' end of the E2F3 mRNA, removing the Pumilio regulatory elements. These studies suggest that Pumilio-miRNA repression of E2F3 translation provides an important level of E2F regulation that is frequently abrogated in cancer cells.


Assuntos
Proteínas de Drosophila/metabolismo , Fatores de Transcrição E2F/genética , Regulação Neoplásica da Expressão Gênica , MicroRNAs/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição/metabolismo , Regiões 3' não Traduzidas/genética , Animais , Linhagem Celular Tumoral , Drosophila , Proteínas de Drosophila/genética , Fatores de Transcrição E2F/metabolismo , Células HeLa , Humanos , MicroRNAs/genética , Processamento Pós-Transcricional do RNA , Proteínas de Ligação a RNA/genética , Fatores de Transcrição/genética
8.
Int J Mol Sci ; 21(20)2020 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-33053834

RESUMO

Cyclin-dependent kinase 8 (CDK8) and its regulatory partner Cyclin C (CycC) play conserved roles in modulating RNA polymerase II (Pol II)-dependent gene expression. To understand the structure and function relations of CDK8, we analyzed the structures of human and Drosophila CDK8 proteins using molecular dynamics simulations, combined with functional analyses in Drosophila. Specifically, we evaluated the structural differences between hCDK8 and dCDK8 to predict the effects of the LXXLL motif mutation (AQKAA), the P154L mutations, and drug binding on local structures of the CDK8 proteins. First, we have observed that both the LXXLL motif and the kinase activity of CDK8 are required for the normal larval-to-pupal transition in Drosophila. Second, our molecular dynamic analyses have revealed that hCDK8 has higher hydrogen bond occupation of His149-Asp151 and Asp151-Asn156 than dCDK8. Third, the substructure of Asp282, Phe283, Arg285, Thr287 and Cys291 can distinguish human and Drosophila CDK8 structures. In addition, there are two hydrogen bonds in the LXXLL motif: a lower occupation between L312 and L315, and a relatively higher occupation between L312 and L316. Human CDK8 has higher hydrogen bond occupation between L312 and L316 than dCDK8. Moreover, L312, L315 and L316 in the LXXLL motif of CDK8 have the specific pattern of hydrogen bonds and geometries, which could be crucial for the binding to nuclear receptors. Furthermore, the P154L mutation dramatically decreases the hydrogen bond between L312 and L315 in hCDK8, but not in dCDK8. The mutations of P154L and AQKAA modestly alter the local structures around residues 154. Finally, we identified the inhibitor-induced conformational changes of hCDK8, and our results suggest a structural difference in the drug-binding site between hCDK8 and dCDK8. Taken together, these results provide the structural insights into the roles of the LXXLL motif and the kinase activity of CDK8 in vivo.


Assuntos
Motivos de Aminoácidos , Sítios de Ligação , Quinase 8 Dependente de Ciclina/química , Proteínas de Drosophila/química , Modelos Moleculares , Domínios e Motivos de Interação entre Proteínas , Inibidores de Proteínas Quinases/química , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Quinase 8 Dependente de Ciclina/antagonistas & inibidores , Proteínas de Drosophila/antagonistas & inibidores , Humanos , Ligação de Hidrogênio , Ligantes , Conformação Molecular , Mutação , Ligação Proteica , Inibidores de Proteínas Quinases/farmacologia , Especificidade da Espécie , Relação Estrutura-Atividade
9.
Dev Biol ; 444(2): 62-70, 2018 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-30352217

RESUMO

The complex interplay between genetic and environmental factors, such as diet and lifestyle, defines the initiation and progression of multifactorial diseases, including cancer, cardiovascular and metabolic diseases, and neurological disorders. Given that most of the studies have been performed in controlled experimental settings to ensure the consistency and reproducibility, the impacts of environmental factors, such as dietary perturbation, on the development of animals with different genotypes and the pathogenesis of these diseases remain poorly understood. By analyzing the cdk8 and cyclin C (cycC) mutant larvae in Drosophila, we have previously reported that the CDK8-CycC complex coordinately regulates lipogenesis by repressing dSREBP (sterol regulatory element-binding protein)-activated transcription and developmental timing by activating EcR (ecdysone receptor)-dependent gene expression. Here we report that dietary nutrients, particularly proteins and carbohydrates, modulate the developmental timing through the CDK8/CycC/EcR pathway. We observed that cdk8 and cycC mutants are sensitive to the levels of dietary proteins and seven amino acids (arginine, glutamine, isoleucine, leucine, methionine, threonine, and valine). Those mutants are also sensitive to dietary carbohydrates, and they are more sensitive to monosaccharides than disaccharides. These results suggest that CDK8-CycC mediates the dietary effects on lipid metabolism and developmental timing in Drosophila larvae.


Assuntos
Quinase 8 Dependente de Ciclina/fisiologia , Proteínas de Drosophila/fisiologia , Larva/metabolismo , Necessidades Nutricionais/fisiologia , Animais , Ciclina C/metabolismo , Ciclina C/fisiologia , Quinase 8 Dependente de Ciclina/metabolismo , Dieta , Proteínas Alimentares/metabolismo , Drosophila/embriologia , Drosophila/genética , Proteínas de Drosophila/metabolismo , Expressão Gênica , Reprodutibilidade dos Testes
10.
Mol Cell ; 42(5): 689-99, 2011 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-21596603

RESUMO

Epigenetic regulation of gene expression by histone-modifying corepressor complexes is central to normal animal development. The NAD(+)-dependent deacetylase and gene repressor SIRT1 removes histone H4K16 acetylation marks and facilitates heterochromatin formation. However, the mechanistic contribution of SIRT1 to epigenetic regulation at euchromatic loci and whether it acts in concert with other chromatin-modifying activities to control developmental gene expression programs remain unclear. We describe here a SIRT1 corepressor complex containing the histone H3K4 demethylase LSD1/KDM1A and several other LSD1-associated proteins. SIRT1 and LSD1 interact directly and play conserved and concerted roles in H4K16 deacetylation and H3K4 demethylation to repress genes regulated by the Notch signaling pathway. Mutations in Drosophila SIRT1 and LSD1 orthologs result in similar developmental phenotypes and genetically interact with the Notch pathway in Drosophila. These findings offer new insights into conserved mechanisms of epigenetic gene repression and regulation of development by SIRT1 in metazoans.


Assuntos
Proteínas de Drosophila/fisiologia , Drosophila melanogaster/genética , Oxirredutases N-Desmetilantes/fisiologia , Receptores Notch/genética , Sirtuína 1/fisiologia , Animais , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Epigênese Genética , Regulação da Expressão Gênica no Desenvolvimento , Histonas/metabolismo , Imunoprecipitação , Mutação , Oxirredutases N-Desmetilantes/genética , Oxirredutases N-Desmetilantes/metabolismo , Fenótipo , Receptores Notch/metabolismo , Sirtuína 1/genética , Sirtuína 1/metabolismo
11.
Adv Exp Med Biol ; 1167: 129-155, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31520353

RESUMO

Multiple large-scale epidemiological studies have identified obesity as an important risk factor for a variety of human cancers, particularly cancers of the uterus, gallbladder, kidney, liver, colon, and ovary, but there is much uncertainty regarding how obesity increases the cancer risks. Given that obesity has been consistently identified as a major risk factor for uterine tumors, the most common malignancies of the female reproductive system, we use uterine tumors as a pathological context to survey the relevant literature and propose a novel hypothesis: chronic downregulation of the cyclin-dependent kinase 8 (CDK8) module, composed of CDK8 (or its paralog CDK19), Cyclin C, MED12 (or MED12L), and MED13 (or MED13L), by elevated insulin or insulin-like growth factor signaling in obese women may increase the chances to dysregulate the activities of transcription factors regulated by the CDK8 module, thereby increasing the risk of uterine tumors. Although we focus on endometrial cancer and uterine leiomyomas (or fibroids), two major forms of uterine tumors, our model may offer additional insights into how obesity increases the risk of other types of cancers and diseases. To illustrate the power of model organisms for studying human diseases, here we place more emphasis on the findings obtained from Drosophila melanogaster.


Assuntos
Drosophila melanogaster , Obesidade/complicações , Neoplasias Uterinas/patologia , Animais , Quinase 8 Dependente de Ciclina/genética , Modelos Animais de Doenças , Proteínas de Drosophila/genética , Feminino , Humanos , Complexo Mediador/genética , Fatores de Risco
12.
Proc Natl Acad Sci U S A ; 113(24): E3403-12, 2016 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-27302953

RESUMO

Type I IFNs are key cytokines mediating innate antiviral immunity. cGMP-AMP synthase, ritinoic acid-inducible protein 1 (RIG-I)-like receptors, and Toll-like receptors recognize microbial double-stranded (ds)DNA, dsRNA, and LPS to induce the expression of type I IFNs. These signaling pathways converge at the recruitment and activation of the transcription factor IRF-3 (IFN regulatory factor 3). The adaptor proteins STING (stimulator of IFN genes), MAVS (mitochondrial antiviral signaling), and TRIF (TIR domain-containing adaptor inducing IFN-ß) mediate the recruitment of IRF-3 through a conserved pLxIS motif. Here we show that the pLxIS motif of phosphorylated STING, MAVS, and TRIF binds to IRF-3 in a similar manner, whereas residues upstream of the motif confer specificity. The structure of the IRF-3 phosphomimetic mutant S386/396E bound to the cAMP response element binding protein (CREB)-binding protein reveals that the pLxIS motif also mediates IRF-3 dimerization and activation. Moreover, rotavirus NSP1 (nonstructural protein 1) employs a pLxIS motif to target IRF-3 for degradation, but phosphorylation of NSP1 is not required for its activity. These results suggest a concerted mechanism for the recruitment and activation of IRF-3 that can be subverted by viral proteins to evade innate immune responses.


Assuntos
Fator Regulador 3 de Interferon/química , Rotavirus/química , Proteínas não Estruturais Virais/química , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Proteínas Adaptadoras de Transporte Vesicular/química , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/imunologia , Motivos de Aminoácidos , Proteína de Ligação a CREB/química , Proteína de Ligação a CREB/genética , Proteína de Ligação a CREB/imunologia , Humanos , Evasão da Resposta Imune , Imunidade Inata , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/imunologia , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Domínios Proteicos , Rotavirus/genética , Rotavirus/imunologia , Infecções por Rotavirus/genética , Infecções por Rotavirus/imunologia , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/imunologia
13.
PLoS Biol ; 13(7): e1002207, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-26222308

RESUMO

The steroid hormone ecdysone and its receptor (EcR) play critical roles in orchestrating developmental transitions in arthropods. However, the mechanism by which EcR integrates nutritional and developmental cues to correctly activate transcription remains poorly understood. Here, we show that EcR-dependent transcription, and thus, developmental timing in Drosophila, is regulated by CDK8 and its regulatory partner Cyclin C (CycC), and the level of CDK8 is affected by nutrient availability. We observed that cdk8 and cycC mutants resemble EcR mutants and EcR-target genes are systematically down-regulated in both mutants. Indeed, the ability of the EcR-Ultraspiracle (USP) heterodimer to bind to polytene chromosomes and the promoters of EcR target genes is also diminished. Mass spectrometry analysis of proteins that co-immunoprecipitate with EcR and USP identified multiple Mediator subunits, including CDK8 and CycC. Consistently, CDK8-CycC interacts with EcR-USP in vivo; in particular, CDK8 and Med14 can directly interact with the AF1 domain of EcR. These results suggest that CDK8-CycC may serve as transcriptional cofactors for EcR-dependent transcription. During the larval-pupal transition, the levels of CDK8 protein positively correlate with EcR and USP levels, but inversely correlate with the activity of sterol regulatory element binding protein (SREBP), the master regulator of intracellular lipid homeostasis. Likewise, starvation of early third instar larvae precociously increases the levels of CDK8, EcR and USP, yet down-regulates SREBP activity. Conversely, refeeding the starved larvae strongly reduces CDK8 levels but increases SREBP activity. Importantly, these changes correlate with the timing for the larval-pupal transition. Taken together, these results suggest that CDK8-CycC links nutrient intake to developmental transitions (EcR activity) and fat metabolism (SREBP activity) during the larval-pupal transition.


Assuntos
Ciclina C/metabolismo , Quinase 8 Dependente de Ciclina/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/crescimento & desenvolvimento , Drosophila/metabolismo , Receptores de Esteroides/metabolismo , Animais , Animais Geneticamente Modificados , Ciclina C/genética , Quinase 8 Dependente de Ciclina/genética , Proteínas de Ligação a DNA/metabolismo , Drosophila/genética , Proteínas de Drosophila/genética , Ecdisteroides/biossíntese , Feminino , Privação de Alimentos , Regulação da Expressão Gênica , Larva/crescimento & desenvolvimento , Larva/metabolismo , Mutação , Proteínas de Ligação a Elemento Regulador de Esterol/metabolismo , Fatores de Transcrição/metabolismo
14.
Genes Dev ; 24(13): 1403-17, 2010 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-20595232

RESUMO

The sterol regulatory element-binding protein (SREBP) transcription factor family is a critical regulator of lipid and sterol homeostasis in eukaryotes. In mammals, SREBPs are highly active in the fed state to promote the expression of lipogenic and cholesterogenic genes and facilitate fat storage. During fasting, SREBP-dependent lipid/cholesterol synthesis is rapidly diminished in the mouse liver; however, the mechanism has remained incompletely understood. Moreover, the evolutionary conservation of fasting regulation of SREBP-dependent programs of gene expression and control of lipid homeostasis has been unclear. We demonstrate here a conserved role for orthologs of the NAD(+)-dependent deacetylase SIRT1 in metazoans in down-regulation of SREBP orthologs during fasting, resulting in inhibition of lipid synthesis and fat storage. Our data reveal that SIRT1 can directly deacetylate SREBP, and modulation of SIRT1 activity results in changes in SREBP ubiquitination, protein stability, and target gene expression. In addition, chemical activators of SIRT1 inhibit SREBP target gene expression in vitro and in vivo, correlating with decreased hepatic lipid and cholesterol levels and attenuated liver steatosis in diet-induced and genetically obese mice. We conclude that SIRT1 orthologs play a critical role in controlling SREBP-dependent gene regulation governing lipid/cholesterol homeostasis in metazoans in response to fasting cues. These findings may have important biomedical implications for the treatment of metabolic disorders associated with aberrant lipid/cholesterol homeostasis, including metabolic syndrome and atherosclerosis.


Assuntos
Regulação para Baixo , Jejum/fisiologia , Sirtuína 1/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 2/metabolismo , Acetilação , Animais , Benzamidas/farmacologia , Caenorhabditis elegans , Linhagem Celular , Colesterol/biossíntese , Regulação para Baixo/efeitos dos fármacos , Células HeLa , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Lipídeos/biossíntese , Camundongos , Naftóis/farmacologia , Niacinamida/farmacologia , Estabilidade Proteica/efeitos dos fármacos , Sirtuínas/antagonistas & inibidores
15.
PLoS Genet ; 10(5): e1004357, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24809668

RESUMO

Inactivation of the Rb tumor suppressor can lead to increased cell proliferation or cell death depending on specific cellular context. Therefore, identification of the interacting pathways that modulate the effect of Rb loss will provide novel insights into the roles of Rb in cancer development and promote new therapeutic strategies. Here, we identify a novel synthetic lethal interaction between Rb inactivation and deregulated Wg/Wnt signaling through unbiased genetic screens. We show that a weak allele of axin, which deregulates Wg signaling and increases cell proliferation without obvious effects on cell fate specification, significantly alters metabolic gene expression, causes hypersensitivity to metabolic stress induced by fasting, and induces synergistic apoptosis with mutation of fly Rb ortholog, rbf. Furthermore, hyperactivation of Wg signaling by other components of the Wg pathway also induces synergistic apoptosis with rbf. We show that hyperactivated Wg signaling significantly increases TORC1 activity and induces excessive energy stress with rbf mutation. Inhibition of TORC1 activity significantly suppressed synergistic cell death induced by hyperactivated Wg signaling and rbf inactivation, which is correlated with decreased energy stress and decreased induction of apoptotic regulator expression. Finally the synthetic lethality between Rb and deregulated Wnt signaling is conserved in mammalian cells and that inactivation of Rb and APC induces synergistic cell death through a similar mechanism. These results suggest that elevated TORC1 activity and metabolic stress underpin the evolutionarily conserved synthetic lethal interaction between hyperactivated Wnt signaling and inactivated Rb tumor suppressor.


Assuntos
Genes do Retinoblastoma , Complexos Multiproteicos/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Proteínas Wnt/metabolismo , Animais , Apoptose , Sequência de Bases , Primers do DNA , Drosophila , Genes Letais , Alvo Mecanístico do Complexo 1 de Rapamicina , Reação em Cadeia da Polimerase
16.
Proc Natl Acad Sci U S A ; 110(47): 19012-7, 2013 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-24191015

RESUMO

The ability to engineer genomes in a specific, systematic, and cost-effective way is critical for functional genomic studies. Recent advances using the CRISPR-associated single-guide RNA system (Cas9/sgRNA) illustrate the potential of this simple system for genome engineering in a number of organisms. Here we report an effective and inexpensive method for genome DNA editing in Drosophila melanogaster whereby plasmid DNAs encoding short sgRNAs under the control of the U6b promoter are injected into transgenic flies in which Cas9 is specifically expressed in the germ line via the nanos promoter. We evaluate the off-targets associated with the method and establish a Web-based resource, along with a searchable, genome-wide database of predicted sgRNAs appropriate for genome engineering in flies. Finally, we discuss the advantages of our method in comparison with other recently published approaches.


Assuntos
Sistemas CRISPR-Cas/genética , Drosophila melanogaster/genética , Engenharia Genética/métodos , Genômica/métodos , Células Germinativas , Animais , Animais Geneticamente Modificados , Bases de Dados Genéticas , Proteínas de Drosophila/genética , Mutagênese/genética , Regiões Promotoras Genéticas/genética , Proteínas de Ligação a RNA/genética
17.
J Biol Chem ; 289(43): 29937-47, 2014 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-25190802

RESUMO

Dysregulation of lipid homeostasis is a common feature of several major human diseases, including type 2 diabetes and cardiovascular disease. However, because of the complex nature of lipid metabolism, the regulatory mechanisms remain poorly defined at the molecular level. As the key transcriptional activators of lipogenic genes, such as fatty acid synthase (FAS), sterol regulatory element-binding proteins (SREBPs) play a pivotal role in stimulating lipid biosynthesis. Several studies have shown that SREBPs are regulated by the NAD(+)-dependent histone deacetylase SIRT1, which forms a complex with the lysine-specific histone demethylase LSD1. Here, we show that LSD1 plays a role in regulating SREBP1-mediated gene expression. Multiple lines of evidence suggest that LSD1 is required for SREBP1-dependent activation of the FAS promoter in mammalian cells. LSD1 knockdown decreases SREBP-1a at the transcription level. Although LSD1 affects nuclear SREBP-1 abundance indirectly through SIRT1, it is also required for SREBP1 binding to the FAS promoter. As a result, LSD1 knockdown decreases triglyceride levels in hepatocytes. Taken together, these results show that LSD1 plays a role in regulating lipogenic gene expression, suggesting LSD1 as a potential target for treating dysregulation of lipid metabolism.


Assuntos
Regulação da Expressão Gênica , Histona Desmetilases/metabolismo , Lipogênese/genética , Animais , Núcleo Celular/metabolismo , Ácido Graxo Sintases/genética , Ácido Graxo Sintases/metabolismo , Células HEK293 , Células Hep G2 , Hepatócitos/metabolismo , Humanos , Masculino , Camundongos , Regiões Promotoras Genéticas , Ligação Proteica , Sirtuína 1/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Transcrição Gênica
18.
J Biol Chem ; 289(23): 16374-88, 2014 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-24770415

RESUMO

The circadian clockworks gate macrophage inflammatory responses. Given the association between clock dysregulation and metabolic disorders, we conducted experiments to determine the extent to which over-nutrition modulates macrophage clock function and whether macrophage circadian dysregulation is a key factor linking over-nutrition to macrophage proinflammatory activation, adipose tissue inflammation, and systemic insulin resistance. Our results demonstrate that 1) macrophages from high fat diet-fed mice are marked by dysregulation of the molecular clockworks in conjunction with increased proinflammatory activation, 2) global disruption of the clock genes Period1 (Per1) and Per2 recapitulates this amplified macrophage proinflammatory activation, 3) adoptive transfer of Per1/2-disrupted bone marrow cells into wild-type mice potentiates high fat diet-induced adipose and liver tissue inflammation and systemic insulin resistance, and 4) Per1/2-disrupted macrophages similarly exacerbate inflammatory responses and decrease insulin sensitivity in co-cultured adipocytes in vitro. Furthermore, PPARγ levels are decreased in Per1/2-disrupted macrophages and PPARγ2 overexpression ameliorates Per1/2 disruption-associated macrophage proinflammatory activation, suggesting that this transcription factor may link the molecular clockworks to signaling pathways regulating macrophage polarization. Thus, macrophage circadian clock dysregulation is a key process in the physiological cascade by which diet-induced obesity triggers macrophage proinflammatory activation, adipose tissue inflammation, and insulin resistance.


Assuntos
Células da Medula Óssea/metabolismo , Dieta Hiperlipídica , Inflamação/metabolismo , Resistência à Insulina , Proteínas Circadianas Period/metabolismo , Adipócitos/metabolismo , Animais , Técnicas de Cocultura , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , PPAR gama/metabolismo
19.
Nature ; 455(7212): 552-6, 2008 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-18794899

RESUMO

The E2F1 transcription factor can promote proliferation or apoptosis when activated, and is a key downstream target of the retinoblastoma tumour suppressor protein (pRB). Here we show that E2F1 is a potent and specific inhibitor of beta-catenin/T-cell factor (TCF)-dependent transcription, and that this function contributes to E2F1-induced apoptosis. E2F1 deregulation suppresses beta-catenin activity in an adenomatous polyposis coli (APC)/glycogen synthase kinase-3 (GSK3)-independent manner, reducing the expression of key beta-catenin targets including c-MYC. This interaction explains why colorectal tumours, which depend on beta-catenin transcription for their abnormal proliferation, keep RB1 intact. Remarkably, E2F1 activity is also repressed by cyclin-dependent kinase-8 (CDK8), a colorectal oncoprotein. Elevated levels of CDK8 protect beta-catenin/TCF-dependent transcription from inhibition by E2F1. Thus, by retaining RB1 and amplifying CDK8, colorectal tumour cells select conditions that collectively suppress E2F1 and enhance the activity of beta-catenin.


Assuntos
Quinases Ciclina-Dependentes/metabolismo , Fator de Transcrição E2F1/antagonistas & inibidores , Fator de Transcrição E2F1/metabolismo , Proteína do Retinoblastoma/metabolismo , Transcrição Gênica , beta Catenina/antagonistas & inibidores , beta Catenina/metabolismo , Proteína da Polipose Adenomatosa do Colo/metabolismo , Apoptose , Linhagem Celular , Quinase 8 Dependente de Ciclina , Regulação da Expressão Gênica , Genes myc/genética , Quinase 3 da Glicogênio Sintase/metabolismo , Humanos , Proteína do Retinoblastoma/genética , Transdução de Sinais , Fatores de Transcrição TCF/metabolismo , Proteínas Wnt/metabolismo
20.
Appl Microbiol Biotechnol ; 98(3): 1357-66, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23749227

RESUMO

The compartmentalized anaerobic reactor (CAR) is a patent novel high-rate reactor, which consists of three compartments. The reactor has a great potential for application due to its many advantages. In this work, the microbial consortium, spatial distribution, and their relationship with performance of CAR were investigated by means of polymerase chain reaction, denaturing gradient gel electrophoresis, and fluorescence in situ hybridization. The results showed that the predominant archaea were Methanobacterium, Methanosaeta, and Methanospirillum, and the predominant bacteria were Firmicutes, Deltaproteobacteria, Spirochaetes, Actinobacteria, and Gammaproteobacteria in the microbial consortium. The methanogenic archaea (MA), the hydrogen-producing acetogenic bacteria (HAB), and the hydrolytic fermentative bacteria (HFB) were found to be predominant in the upper, middle, and bottom compartments, respectively. The results revealed that the granular sludge took on a stratified microbial structure. The HFB, HAB, and MA were located in the outer shell, middle layer, and core, respectively. The microbial populations from the bottom compartment were relatively homogeneous in the granular sludge, and from the middle and upper compartments, they were relatively heterogeneous in the granular sludge. The microbial consortia and their spatial distribution were in accordance with the organic loading rate and chemical components in the three compartments.


Assuntos
Archaea/isolamento & purificação , Bactérias/isolamento & purificação , Reatores Biológicos/microbiologia , Biota , Consórcios Microbianos , Anaerobiose , Archaea/classificação , Archaea/genética , Bactérias/classificação , Bactérias/genética , Eletroforese em Gel de Gradiente Desnaturante , Hibridização in Situ Fluorescente , Reação em Cadeia da Polimerase , Esgotos/microbiologia
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