RESUMEN
The growth suppressive function of the retinoblastoma (pRB) tumor suppressor family is largely attributed to its ability to negatively regulate the family of E2F transcriptional factors and, as a result, to repress E2F-dependent transcription. Deregulation of the pRB pathway is thought to be an obligatory event in most types of cancers. The large number of mammalian E2F proteins is one of the major obstacles that complicate their genetic analysis. In Drosophila, the E2F family consists of only two members. They are classified as an activator (dE2F1) and a repressor (dE2F2). It has been previously shown that proliferation of de2f1 mutant cells is severely reduced due to unchecked activity of the repressor dE2F2 in these cells. We report here a mosaic screen utilizing the de2f1 mutant phenotype to identify suppressors that overcome the dE2F2/RBF-dependent proliferation block. We have isolated l(3)mbt and B52, which are known to be required for dE2F2 function, as well as genes that were not previously linked to the E2F/pRB pathway such as Doa, gfzf, and CG31133. Inactivation of gfzf, Doa, or CG31133 does not relieve repression by dE2F2. We have shown that gfzf and CG31133 potentiate E2F-dependent activation and synergize with inactivation of RBF, suggesting that they may act in parallel to dE2F. Thus, our results demonstrate the efficacy of the described screening strategy for studying regulation of the dE2F/RBF pathway in vivo.
Asunto(s)
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Pruebas Genéticas/métodos , Supresión Genética , Factores de Transcripción/genética , Algoritmos , Animales , Animales Modificados Genéticamente , Proliferación Celular , Células Cultivadas , Femenino , Prueba de Complementación Genética/métodos , Masculino , Modelos Genéticos , Mosaicismo , Proteínas Mutantes/genética , Proteínas Mutantes/fisiología , Fenotipo , Proteína de Retinoblastoma/genética , Supresión Genética/genéticaRESUMEN
The endoplasmic reticulum (ER) is the cell organelle where secretory and membrane proteins are synthesized and folded. Correctly folded proteins exit the ER and are transported to the Golgi and other destinations within the cell, but proteins that fail to fold properly-misfolded proteins-are retained in the ER and their accumulation may constitute a form of stress to the cell-ER stress. Several signaling pathways, collectively known as unfolded protein response (UPR), have evolved to detect the accumulation of misfolded proteins in the ER and activate a cellular response that attempts to maintain homeostasis and a normal flux of proteins in the ER. In certain severe situations of ER stress, however, the protective mechanisms activated by the UPR are not sufficient to restore normal ER function and cells die by apoptosis. Most research on the UPR used yeast or mammalian model systems and only recently Drosophila has emerged as a system to study the molecular and cellular mechanisms of the UPR. Here, we review recent advances in Drosophila UPR research, in the broad context of mammalian and yeast literature.
Asunto(s)
Apoptosis , Retículo Endoplásmico/metabolismo , Estrés Fisiológico , Animales , Humanos , Chaperonas Moleculares/metabolismo , Pliegue de Proteína , Transducción de SeñalRESUMEN
In Drosophila melanogaster, the loss of activator de2f1 leads to a severe reduction in cell proliferation and repression of E2F targets. To date, the only known way to rescue the proliferation block in de2f1 mutants was through the inactivation of dE2F2. This suggests that dE2F2 provides a major contribution to the de2f1 mutant phenotype. Here, we report that in mosaic animals, in addition to de2f2, the loss of a DEAD box protein Belle (Bel) also rescues proliferation of de2f1 mutant cells. Surprisingly, the rescue occurs in a dE2F2-independent manner since the loss of Bel does not relieve dE2F2-mediated repression. In the eye disc, bel mutant cells fail to undergo a G1 arrest in the morphogenetic furrow, delay photoreceptor recruitment and differentiation, and show a reduction of the transcription factor Ci155. The down-regulation of Ci155 is important since it is sufficient to partially rescue proliferation of de2f1 mutant cells. Thus, mutation of bel relieves the dE2F2-mediated cell cycle arrest in de2f1 mutant cells through a novel Ci155-dependent mechanism without functional inactivation of the dE2F2 repressor.
Asunto(s)
Proteínas de Drosophila/deficiencia , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citología , Drosophila melanogaster/metabolismo , Transactivadores/deficiencia , Transactivadores/metabolismo , Factores de Transcripción/deficiencia , Factores de Transcripción/metabolismo , Animales , Diferenciación Celular , Proliferación Celular , Células Clonales , Regulación hacia Abajo , Proteínas de Drosophila/genética , Factor de Transcripción E2F2/deficiencia , Factor de Transcripción E2F2/metabolismo , Ojo/citología , Ojo/embriología , Fase G1 , Mosaicismo , Mutación/genética , Proteínas Represoras/metabolismoRESUMEN
E2F and retinoblastoma tumor suppressor protein pRB are important regulators of cell proliferation; however, the regulation of these proteins in vivo is not well understood. In Drosophila there are two E2F genes, an activator, de2f1, and a repressor, de2f2. The loss of de2f1 gives rise to the G(1)/S block accompanied by the repression of E2F-dependent transcription. These defects can be suppressed by mutation of de2f2. In this work, we show that the de2f1 mutant phenotype is rescued by the loss of the pre-mRNA splicing factor SR protein B52. Mutations in B52 restore S phase in clones of de2f1 mutant cells and phenocopy the loss of the de2f2 function. B52 acts upstream of de2f2 and plays a specific role in regulation of de2f2 pre-mRNA splicing. In B52-deficient cells, the level of dE2F2 protein is severely reduced and the expression of dE2F2-dependent genes is deregulated. Reexpression of the intronless copy of dE2F2 in B52-deficient cells restores the dE2F2-mediated repression. These results uncover a previously unrecognized role of the splicing factor in maintaining the G(1)/S block in vivo by specific regulation of the dE2F2 repressor function.
Asunto(s)
Ciclo Celular/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/fisiología , Drosophila/crecimiento & desarrollo , Factor de Transcripción E2F2/genética , Ojo/crecimiento & desarrollo , Proteínas Nucleares/fisiología , Fosfoproteínas/fisiología , Empalme del ARN/genética , Animales , Regulación hacia Abajo , Drosophila/genética , Proteínas de Drosophila/metabolismo , Factor de Transcripción E2F2/metabolismo , Ojo/anatomía & histología , Regulación del Desarrollo de la Expresión Génica , Mutación , Proteínas Nucleares/genética , Fenotipo , Fosfoproteínas/genética , Factores de Empalme de ARN , ARN Mensajero/metabolismo , Fase S/genética , Factores de Transcripción/genética , Transcripción GenéticaRESUMEN
In polytene chromosomes of D. melanogaster the heterochromatic pericentric regions are underreplicated (underrepresented). In this report, we analyze the effects of eu-heterochromatic rearrangements involving a cluster of the X-linked heterochromatic (Xh) Stellate repeats on the representation of these sequences in salivary gland polytene chromosomes. The discontinuous heterochromatic Stellate cluster contains specific restriction fragments that were mapped along the distal region of Xh. We found that transposition of a fragment of the Stellate cluster into euchromatin resulted in its replication in polytene chromosomes. Interestingly, only the Stellate repeats that remain within the pericentric Xh and are close to a new eu-heterochromatic boundary were replicated, strongly suggesting the existence of a spreading effect exerted by the adjacent euchromatin. Internal rearrangements of the distal Xh did not affect Stellate polytenization. We also demonstrated trans effects exerted by heterochromatic blocks on the replication of the rearranged heterochromatin; replication of transposed Stellate sequences was suppressed by a deletion of Xh and restored by addition of Y heterochromatin. This phenomenon is discussed in light of a possible role of heterochromatic proteins in the process of heterochromatin underrepresentation in polytene chromosomes.