RESUMO
Specification of cell identity and the proper functioning of a mature cell depend on precise regulation of gene expression. Both binary ON/OFF regulation of transcription, as well as more fine-tuned control of transcription levels in the ON state, are required to define cell types. The Drosophila melanogaster Hox gene, Ultrabithorax (Ubx), exhibits both of these modes of control during development. While ON/OFF regulation is needed to specify the fate of the developing wing (Ubx OFF) and haltere (Ubx ON), the levels of Ubx within the haltere differ between compartments along the proximal-distal axis. Here, we identify and molecularly dissect the novel contribution of a previously identified Ubx cis-regulatory module (CRM), anterobithorax (abx), to a negative auto-regulatory loop that decreases Ubx expression in the proximal compartment of the haltere as compared to the distal compartment. We find that Ubx, in complex with the known Hox cofactors, Homothorax (Hth) and Extradenticle (Exd), acts through low-affinity Ubx-Exd binding sites to reduce the levels of Ubx transcription in the proximal compartment. Importantly, we also reveal that Ubx-Exd-binding site mutations sufficient to result in de-repression of abx activity in a transgenic context are not sufficient to de-repress Ubx expression when mutated at the endogenous locus, suggesting the presence of multiple mechanisms through which Ubx-mediated repression occurs. Our results underscore the complementary nature of CRM analysis through transgenic reporter assays and genome modification of the endogenous locus; but, they also highlight the increasing need to understand gene regulation within the native context to capture the potential input of multiple genomic elements on gene control.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/metabolismo , Homeostase/genética , Fatores de Transcrição/metabolismo , Asas de Animais/embriologia , Animais , Animais Geneticamente Modificados , Sítios de Ligação/fisiologia , Proteínas de Drosophila/genética , Drosophila melanogaster/embriologia , Genes de Insetos/fisiologia , Proteínas de Homeodomínio/genética , Larva/crescimento & desenvolvimento , Mutação , Elementos Reguladores de Transcrição/fisiologia , Fatores de Transcrição/genéticaRESUMO
Budding yeast cells suffering a single unrepaired double-strand break (DSB) trigger the Mec1 (ATR)-dependent DNA damage response that causes them to arrest before anaphase for 12-15 h. Here we find that hyperactivation of the cytoplasm-to-vacuole (CVT) autophagy pathway causes the permanent G2/M arrest of cells with a single DSB that is reflected in the nuclear exclusion of both Esp1 and Pds1. Transient relocalization of Pds1 is also seen in wild-type cells lacking vacuolar protease activity after induction of a DSB. Arrest persists even as the DNA damage-dependent phosphorylation of Rad53 diminishes. Permanent arrest can be overcome by blocking autophagy, by deleting the vacuolar protease Prb1, or by driving Esp1 into the nucleus with a SV40 nuclear localization signal. Autophagy in response to DNA damage can be induced in three different ways: by deleting the Golgi-associated retrograde protein complex (GARP), by adding rapamycin, or by overexpression of a dominant ATG13-8SA mutation.
Assuntos
Anáfase/fisiologia , Autofagia/fisiologia , Pontos de Checagem do Ciclo Celular/fisiologia , Quebras de DNA de Cadeia Dupla , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transporte Ativo do Núcleo Celular/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Autofagia/efeitos dos fármacos , Proteínas Relacionadas à Autofagia , Western Blotting , Proteínas de Ciclo Celular/metabolismo , Endopeptidases/metabolismo , Proteínas de Fluorescência Verde , Proteínas Nucleares/metabolismo , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética , Saccharomycetales , Securina , Separase , Sirolimo/farmacologiaRESUMO
For gene products that must be present in cells at defined concentrations, expression levels must be tightly controlled to ensure robustness against environmental, genetic, and developmental noise. By studying the regulation of the concentration-sensitive Drosophila melanogaster Hox gene Ultrabithorax (Ubx), we found that Ubx enhancer activities respond to both increases in Ubx levels and genetic background. Large, transient increases in Ubx levels are capable of silencing all enhancer input into Ubx transcription, resulting in the complete silencing of this gene. Small increases in Ubx levels, brought about by duplications of the Ubx locus, cause sporadic silencing of subsets of Ubx enhancers. Ubx enhancer silencing can also be induced by outcrossing laboratory stocks to D. melanogaster strains established from wild flies from around the world. These results suggest that enhancer activities are not rigidly determined, but instead are sensitive to genetic background. Together, these findings suggest that enhancer silencing may be used to maintain gene product levels within the correct range in response to natural genetic variation.