RESUMO
Heat shock factor 1 (HSF1) is an important transcription factor in cellular stress responses, cancer, aging, and developmental processes including gametogenesis. Disruption of Hsf1, together with another HSF family member, Hsf2, causes male sterility and complete lack of mature sperm in mice, but the specific role of HSF1 in spermatogenesis has remained unclear. Here, we show that HSF1 is transiently expressed in meiotic spermatocytes and haploid round spermatids in mouse testis. The Hsf1(-/-) male mice displayed regions of seminiferous tubules containing only spermatogonia and increased morphological abnormalities in sperm heads. In search for HSF1 target genes, we identified 742 putative promoters in mouse testis. Among them, the sex chromosomal multicopy genes that are expressed in postmeiotic cells were occupied by HSF1. Given that the sex chromatin mostly is repressed during and after meiosis, it is remarkable that HSF1 directly regulates the transcription of sex-linked multicopy genes during postmeiotic repression. In addition, our results show that HSF1 localizes to the sex body prior to the meiotic divisions and to the sex chromocenter after completed meiosis. To the best of our knowledge, HSF1 is the first known transcription factor found at the repressed sex chromatin during meiosis.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Meiose/fisiologia , Túbulos Seminíferos/metabolismo , Cromatina Sexual/metabolismo , Espermatogênese/fisiologia , Espermatozoides/metabolismo , Fatores de Transcrição/metabolismo , Animais , Proteínas de Ligação a DNA/genética , Feminino , Regulação da Expressão Gênica/fisiologia , Fatores de Transcrição de Choque Térmico , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Infertilidade Masculina/genética , Infertilidade Masculina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Família Multigênica/fisiologia , Cromatina Sexual/genética , Fatores de Transcrição/genéticaRESUMO
Heat-shock factor 1 (HSF1) protects cells and organisms against various types of stress, either by triggering a complex response that promotes cell survival or by triggering cell death when stress-induced alterations cannot be rescued. Although this dual role of HSF1 was observed in spermatogenesis exposed to heat shock or proteotoxic stress, HSF1 was also reported to contribute to cell resistance against genotoxic stress, such as that caused by doxorubicin, an anticancer drug in common clinical use. To better understand the stress/cell-dependent functions of HSF1, we used wild-type and Hsf1(tm1Ijb)/Hsf1(tm1Ijb) males to determine the role of HSF1 in the genotoxic stress response elicited in spermatogenic cells. Within 2 days after a single intraperitoneal injection of doxorubicin (DOXO; 5 mg/kg), proliferation of Hsf1+/+ but not Hsf1-/- spermatogenic cells was significantly reduced, whereas cell death was increased in mitotic germ cells and metaphase I spermatocytes. By 21 days, meiotic cells were depleted in all treated Hsf1+/+ testes but not in Hsf1-/- ones. Nevertheless, after 3 mo, spermatogenesis showed better signs of recovery in Hsf1+/+ than in Hsf1-/- males. Taken together, these data indicate that acute response to genotoxic stress in the testis involves HSF1-dependent mechanisms that induce apoptotic cell death in a TRP53-independent manner, but also intervene on a longer term to restore seminiferous tubules.
Assuntos
Antibióticos Antineoplásicos/toxicidade , Proteínas de Ligação a DNA/fisiologia , Doxorrubicina/toxicidade , Mutagênese/efeitos dos fármacos , Mutagênicos/toxicidade , Testículo/efeitos dos fármacos , Testículo/fisiologia , Fatores de Transcrição/fisiologia , Animais , Antimetabólitos , Western Blotting , Bromodesoxiuridina , Proteínas de Ligação a DNA/genética , Fatores de Transcrição de Choque Térmico , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Masculino , Meiose/efeitos dos fármacos , Meiose/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Antígeno Nuclear de Célula em Proliferação/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Contagem de Espermatozoides , Cabeça do Espermatozoide/efeitos dos fármacos , Cabeça do Espermatozoide/fisiologia , Espermatogênese/efeitos dos fármacos , Fatores de Transcrição/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/fisiologiaRESUMO
Transcription of the Escherichia coli osmB gene is induced by several stress conditions. osmB is expressed from two promoters, osmBp1 and osmBp2. The downstream promoter, osmBp2, is induced after osmotic shock or upon entry into stationary phase in a sigma(S)-dependent manner. The upstream promoter, osmBp1, is independent of sigma(S) and is activated by RcsB, the response regulator of the His-Asp phosphorelay signal transduction system RcsCDB. RcsB is responsible for the induction of osmBp1 following treatment with chlorpromazine. Activation of osmBp1 by RcsB requires a sequence upstream of its -35 element similar to the RcsB binding site consensus, suggesting a direct regulatory role. osmB appears as another example of a multistress-responsive gene whose transcription involves both a sigma(S)-dependent promoter and a second one independent of sigma(S) but controlled by stress-specific transcription factors.
Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Bactérias/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli/fisiologia , Regulação Bacteriana da Expressão Gênica , Lipoproteínas/genética , Proteínas Periplásmicas/genética , Regiões Promotoras Genéticas , Fator sigma/metabolismo , Fatores de Transcrição/metabolismo , Adaptação Fisiológica , Ácido Aspártico/metabolismo , Proteínas da Membrana Bacteriana Externa/biossíntese , Sequência de Bases , DNA Bacteriano , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/fisiologia , Histidina/metabolismo , Lipoproteínas/biossíntese , Dados de Sequência Molecular , Proteínas Periplásmicas/biossíntese , Transdução de SinaisRESUMO
The relationship between the survival of Escherichia coli during long-term starvation in rich medium and the supercoiling of a reporter plasmid (pBR322) has been studied. In aerated continuously shaken cultures, E. coli lost the ability to form colonies earlier in rich NaCl-free Luria-Bertani medium than in NaCl-containing medium, and the negative supercoiling of plasmid pBR322 declined more rapidly in the absence of NaCl. Addition of NaCl at the 24th hour restored both viability and negative supercoiling in proportion to the concentration of added NaCl. Addition of ofloxacin, a quinolone inhibitor of gyrase, abolished rescue by added NaCl in proportion to the ofloxacin added. This observation raises the possibility that cells had the ability to recover plasmid supercoiling even if nutrients were not available and could survive during long-term starvation in a manner linked, at least in part, to the topological state of DNA and gyrase activity.
Assuntos
DNA Super-Helicoidal/metabolismo , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/genética , Plasmídeos/genética , Cloreto de Sódio/metabolismo , Meios de Cultura , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , DNA Super-Helicoidal/genética , Regulação Bacteriana da Expressão Gênica , Fatores de TempoRESUMO
Transcription of the Escherichia coli osmC gene is induced by several stress conditions. osmC is expressed from two overlapping promoters, osmCp1 and osmCp2. The proximal promoter, osmCp2, is transcribed at the entry into the stationary phase by the sigma(s) sigma factor. The distal promoter, osmCp1, is activated by NhaR and RcsB. NhaR is a positive regulator of the LysR family and is known to be an activator of the nhaA gene encoding an Na(+)/H(+) antiporter. RcsB is the response regulator of the RcsCDB His-Asp phosphorelay signal transduction system. Genetic data indicated that activation of osmCp1 by both NhaR and RcsB requires the same short sequences upstream of the -35 region of the promoter. Accordingly, DNase I footprint analysis indicated that both activators protect an overlapping region close to the -35 box of the promoter and suggested that the regulatory effect is direct. Despite the overlap of the binding sites, each activator acts independent of the other and is specific for a particular stress. NhaR can stimulate osmCp1 in response to an osmotic signal even in the absence of RcsB. RcsB is responsible for the induction of osmCp1 by alteration of the cell envelope, even in the absence of NhaR. osmCp1 as an example of multiple-stress-responsive promoter is discussed in light of a comparison of the NhaR and RcsB target regions in the Enterobacteriaceae.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/fisiologia , Regulação Bacteriana da Expressão Gênica , Regiões Promotoras Genéticas/genética , Fatores de Transcrição/metabolismo , Sequência de Bases , Sítios de Ligação , Pegada de DNA , Desoxirribonucleases/metabolismo , Enterobacteriaceae/química , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Resposta ao Choque Térmico , Dados de Sequência Molecular , Alinhamento de Sequência , Transativadores/metabolismoRESUMO
The RcsCB His-Asp phosphorelay system regulates the expression of several genes of Escherichia coli, but the molecular nature of the inducing signal is still unknown. We show here that treatment of an exponentially growing culture of E. coli with the cationic amphipathic compound chlorpromazine (CPZ) stimulates expression of a set of genes positively regulated by the RcsCB system. This induction is abolished in rcsB or rcsC mutant strains. In addition, treatment with CPZ inhibits growth. The wild-type strain is able to recover from this inhibition and resume growth after a period of adaptation. In contrast, strains deficient in the RcsCB His-Asp phosphorelay system are hypersensitive to CPZ. These results suggest that cells must express specific RcsCB-regulated genes in order to cope with the CPZ-induced stress. This is the first report of the essential role of the RcsCB system in a stress situation. These results also strengthen the notion that alterations of the cell envelope induce a signal recognized by the RcsC sensor.
Assuntos
Proteínas de Bactérias/fisiologia , Clorpromazina/farmacologia , Proteínas de Escherichia coli , Escherichia coli/efeitos dos fármacos , Complexos Multienzimáticos/fisiologia , Fosfoproteínas Fosfatases/fisiologia , Proteínas Quinases/fisiologia , Fatores de Transcrição , Escherichia coli/crescimento & desenvolvimentoRESUMO
Two overlapping promoters, osmC(p1) and osmC(p2), direct the transcription of the osmC gene of Escherichia coli. The proximal promoter, osmC(p2), is induced upon entry into stationary phase under the control of Esigma(s), the RNA polymerase that uses the sigma(s) (RpoS) sigma factor. Transcription from the distal promoter, osmC(p1), is independent of sigma(s). Previous analysis demonstrated that the osmolarity of the growth medium modulates expression of both promoters. The use of an E. coli genomic library showed that the cloned nhaR gene was able to stimulate transcription of an osmC-lac reporter fusion. NhaR is a positive regulator of the LysR family, previously identified as an activator of nhaA, a gene encoding a Na+/H+ antiporter involved in adaptation to Na+ and alkaline pH in E. coli and other enteric bacteria. NhaR was shown to activate only the expression of osmC(p1) and to be necessary for the induction of this promoter by LiCl, NaCl and sucrose. Therefore, activation by NhaR is responsible for the osmotic induction of osmC(p1). In contrast to its action on nhaA, NhaR activation of osmC(p1) is independent of H-NS. Activation of osmC(p1) by NhaR requires a site located just upstream of the atypical -35 region of the promoter.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA , Proteínas de Escherichia coli , Escherichia coli/fisiologia , Regulação Bacteriana da Expressão Gênica , Regiões Promotoras Genéticas , Fatores de Transcrição/metabolismo , Ativação Transcricional , Proteínas de Bactérias/efeitos dos fármacos , Proteínas de Bactérias/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Pressão Osmótica , Fatores de Transcrição/genética , Equilíbrio HidroeletrolíticoRESUMO
The sigmaS subunit of RNA polymerase is a key regulator of Escherichia coli transcription in stress conditions. sigmaS accumulates in cells subjected to stresses such as an osmotic upshift or the entry into stationary phase. We show here that, at elevated osmolarity, sigmaS accumulates long before the beginning of the sigmaS-dependent induction of osmEp, one of its target promoters. A combination of in vivo and in vitro evidence indicates that a high level of DNA negative supercoiling inhibits transcription by EsigmaS. The variations in superhelical densities occurring as a function of growth conditions can modulate transcription of a subset of sigmaS targets and thereby contribute to the temporal disconnection between the accumulation of sigmaS and sigmaS-driven transcription. We propose that, in stress conditions leading to the accumulation of sigmaS without lowering the growth rate, the level of DNA supercoiling acts as a checkpoint that delays the shift from the major (Esigma70) to the general stress (EsigmaS) transcriptional machinery, retarding the induction of a subset of the sigmaS regulon until the conditions become unfavourable enough to cause entry into stationary phase.