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1.
Development ; 137(19): 3177-84, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20724452

RESUMO

miR-18 belongs to the Oncomir-1 or miR-17~92 cluster that is intimately associated with the occurrence and progression of different types of cancer. However, the physiological roles of the Oncomir-1 cluster and its individual miRNAs are largely unknown. Here, we describe a novel function for miR-18 in mouse. We show that miR-18 directly targets heat shock factor 2 (HSF2), a transcription factor that influences a wide range of developmental processes including embryogenesis and gametogenesis. Furthermore, we show that miR-18 is highly abundant in testis, displaying distinct cell-type-specific expression during the epithelial cycle that constitutes spermatogenesis. Expression of HSF2 and of miR-18 exhibit an inverse correlation during spermatogenesis, indicating that, in germ cells, HSF2 is downregulated by miR-18. To investigate the in vivo function of miR-18 we developed a novel method, T-GIST, and demonstrate that inhibition of miR-18 in intact seminiferous tubules leads to increased HSF2 protein levels and altered expression of HSF2 target genes. Our results reveal that miR-18 regulates HSF2 activity in spermatogenesis and link miR-18 to HSF2-mediated physiological processes such as male germ cell maturation.


Assuntos
Proteínas de Choque Térmico/metabolismo , MicroRNAs/genética , Espermatogênese , Fatores de Transcrição/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Regulação da Expressão Gênica , Proteínas de Choque Térmico/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Espermatócitos/metabolismo , Fatores de Transcrição/genética
2.
Mol Biol Cell ; 20(5): 1340-7, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19129477

RESUMO

Organisms respond to circumstances threatening the cellular protein homeostasis by activation of heat-shock transcription factors (HSFs), which play important roles in stress resistance, development, and longevity. Of the four HSFs in vertebrates (HSF1-4), HSF1 is activated by stress, whereas HSF2 lacks intrinsic stress responsiveness. The mechanism by which HSF2 is recruited to stress-inducible promoters and how HSF2 is activated is not known. However, changes in the HSF2 expression occur, coinciding with the functions of HSF2 in development. Here, we demonstrate that HSF1 and HSF2 form heterotrimers when bound to satellite III DNA in nuclear stress bodies, subnuclear structures in which HSF1 induces transcription. By depleting HSF2, we show that HSF1-HSF2 heterotrimerization is a mechanism regulating transcription. Upon stress, HSF2 DNA binding is HSF1 dependent. Intriguingly, when the elevated expression of HSF2 during development is mimicked, HSF2 binds to DNA and becomes transcriptionally competent. HSF2 activation leads to activation of also HSF1, revealing a functional interdependency that is mediated through the conserved trimerization domains of these factors. We propose that heterotrimerization of HSF1 and HSF2 integrates transcriptional activation in response to distinct stress and developmental stimuli.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Choque Térmico/metabolismo , Resposta ao Choque Térmico/fisiologia , Fatores de Transcrição/metabolismo , Ativação Transcricional , Animais , Linhagem Celular , DNA Satélite/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Fatores de Transcrição de Choque Térmico , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/fisiologia , Humanos , Masculino , Camundongos , Testículo/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia , Transcrição Gênica
3.
Biochem J ; 382(Pt 1): 239-45, 2004 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-15130092

RESUMO

Retinoic acids and long-chain fatty acids are lipophilic agonists of nuclear receptors such as RXRs (retinoic X receptors) and PPARs (peroxisome-proliferator-activated receptors) respectively. These agonists are also ligands of intracellular lipid-binding proteins, which include FABPs (fatty acid-binding proteins). We reported previously that L (liver-type)-FABP targets fatty acids to the nucleus of hepatocytes and affects PPARalpha activation, which binds together with an RXR subtype to a PPRE (peroxisome-proliferator-responsive element). In the present study, we first determined the optimal combination of murine PPAR/RXR subtypes for binding to known murine FABP-PPREs and to those found by computer search and then tested their in vitro functionality. We show that all PPARs bind to L-FABP-PPRE, PPARalpha, PPARgamma1 and PPARgamma2 to A (adipocyte-type)-FABP-PPRE. All PPAR/RXR heterodimers transactivate L-FABP-PPRE, best are combinations of PPARalpha with RXRalpha or RXRgamma. In contrast, PPARalpha heterodimers do not transactivate A-FABP-PPRE, best combinations are of PPARgamma1 with RXRalpha and RXRgamma, and of PPARgamma2 with all RXR subtypes. We found that the predicted E (epidermal-type)- and H (heart-type)-FABP-PPREs are not activated by any PPAR/RXR combination without or with the PPAR pan-agonist bezafibrate. In the same way, C2C12 myoblasts transfected with promoter fragments of E-FABP and H-FABP genes containing putative PPREs are also not activated through stimulation of PPARs with bezafibrate applied to the cells. These results demonstrate that only PPREs of L- and A-FABP promoters are functional, and that binding of PPAR/RXR heterodimers to a PPRE in vitro does not necessarily predict transactivation.


Assuntos
Proteínas de Transporte/fisiologia , Genes/fisiologia , Proliferadores de Peroxissomos/metabolismo , Elementos de Resposta/fisiologia , Animais , Composição de Bases/fisiologia , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Proteínas de Transporte/genética , Linhagem Celular Tumoral , DNA/genética , DNA/metabolismo , Dimerização , Proteínas de Ligação a Ácido Graxo , Regulação da Expressão Gênica/fisiologia , Genes Reporter/genética , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Camundongos , Mioblastos/química , Mioblastos/metabolismo , Receptores Ativados por Proliferador de Peroxissomo/metabolismo , Receptores Ativados por Proliferador de Peroxissomo/fisiologia , Regiões Promotoras Genéticas/fisiologia , Receptores X de Retinoides/metabolismo , Receptores X de Retinoides/fisiologia , Ativação Transcricional/genética , Ativação Transcricional/fisiologia
4.
J Cell Biol ; 164(1): 15-7, 2004 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-14709538

RESUMO

Nuclear stress granules are subnuclear compartments that form in response to heat shock and other stress stimuli. Although many components of nuclear stress granules have been identified, including HSF1 and pre-mRNA processing factors, their function remains a mystery. A paper in this issue describes the stress-induced transcriptional activation of one of the nuclear stress granule target sites, a heterochromatic region that has been considered silent (Jolly et al., 2004). These intriguing findings will certainly give the research of these structures a new twist.


Assuntos
Estruturas do Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Precursores de RNA/metabolismo , Estresse Fisiológico/metabolismo , Ativação Transcricional/genética , Animais , Estruturas do Núcleo Celular/genética , Estruturas do Núcleo Celular/ultraestrutura , Cromossomos Humanos Par 9/genética , DNA Satélite/genética , Proteínas de Ligação a DNA/genética , Fatores de Transcrição de Choque Térmico , Heterocromatina/genética , Humanos , Precursores de RNA/genética , Estresse Fisiológico/genética , Fatores de Transcrição
5.
J Cell Sci ; 116(Pt 17): 3557-70, 2003 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-12865437

RESUMO

The heat-shock response is characterized by the activation of heat-shock transcription factor 1 (HSF1), followed by increased expression of heat-shock proteins (Hsps). The stress-induced subnuclear compartmentalization of HSF1 into nuclear stress granules has been suggested to be an important control step in the regulation of stress response and cellular homeostasis in human cells. In this study, we demonstrate that the less-well characterized HSF2 interacts physically with HSF1 and is a novel stress-responsive component of the stress granules. Based on analysis of our deletion mutants, HSF2 influences to the localization of HSF1 in stress granules. Moreover, our results indicate that the stress granules are dynamic structures and suggest that they might be regulated in an Hsp70-dependent manner. The reversible localization of Hsp70 in the nucleoli strictly coincides with the presence of HSF1 in stress granules and is dramatically suppressed in thermotolerant cells. We propose that the regulated subcellular distribution of Hsp70 is an important regulatory mechanism of HSF1-mediated heat shock response.


Assuntos
Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico/metabolismo , Resposta ao Choque Térmico/fisiologia , Fatores de Transcrição/metabolismo , Nucléolo Celular/metabolismo , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Células HeLa , Fatores de Transcrição de Choque Térmico , Humanos , Células K562 , Ligação Proteica , Estrutura Terciária de Proteína/fisiologia
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