RESUMO
A flexible method to image unmodified transcripts and transcription in vivo would be a valuable tool to understand the regulation and dynamics of transcription. Here, we present a novel approach to follow native transcription, with fluorescence microscopy, in live C. elegans. By using the fluorescently tagged Argonaute protein NRDE-3, programmed by exposure to defined dsRNA to bind to nascent transcripts of the gene of interest, we demonstrate transcript labelling of multiple genes, at the transcription site and in the cytoplasm. This flexible approach does not require genetic manipulation, and can be easily scaled up by relying on whole-genome dsRNA libraries. We apply this method to image the transcriptional dynamics of the heat-shock inducible gene hsp-4 (a member of the hsp70 family), as well as two transcription factors: ttx-3 (a LHX2/9 orthologue) in embryos, and hlh-1 (a MyoD orthologue) in larvae, respectively involved in neuronal and muscle development.
Assuntos
Proteínas Argonautas/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Homeodomínio/genética , Proteínas Musculares/genética , Neuropeptídeos/genética , Proteínas Nucleares/genética , Proteínas de Ligação a RNA/genética , Fatores de Transcrição/genética , Animais , Caenorhabditis elegans/genética , Citoplasma/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Choque Térmico HSP70/genética , Resposta ao Choque Térmico/genética , Desenvolvimento Muscular/genética , Neurônios/metabolismo , Transcrição Gênica/genéticaRESUMO
Tight cell-cycle regulation of the histone H4-K20 methyltransferase PR-Set7 is essential for the maintenance of genome integrity. In mammals, this mainly involves the interaction of PR-Set7 with the replication factor PCNA, which triggers the degradation of the enzyme by the CRL4CDT2 E3 ubiquitin ligase. PR-Set7 is also targeted by the SCFß-TRCP ligase, but the role of this additional regulatory pathway remains unclear. Here, we show that Drosophila PR-Set7 undergoes a cell-cycle proteolytic regulation, independently of its interaction with PCNA. Instead, Slimb, the ortholog of ß-TRCP, is specifically required for the degradation of the nuclear pool of PR-Set7 prior to S phase. Consequently, inactivation of Slimb leads to nuclear accumulation of PR-Set7, which triggers aberrant chromatin compaction and G1/S arrest. Strikingly, these phenotypes result from non-enzymatic PR-Set7 functions that prevent proper histone H4 acetylation independently of H4K20 methylation. Altogether, these results identify the Slimb-mediated PR-Set7 proteolysis as a new critical regulatory mechanism required for proper interphase chromatin organization at G1/S transition.
Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Pontos de Checagem da Fase G1 do Ciclo Celular/genética , Histona-Lisina N-Metiltransferase/genética , Mutação , Animais , Animais Geneticamente Modificados , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Cromatina/genética , Cromatina/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Interfase/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ligação Proteica , Processamento de Proteína Pós-Traducional , Proteólise , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
In metazoans, the pausing of RNA polymerase II at the promoter (paused Pol II) has emerged as a widespread and conserved mechanism in the regulation of gene transcription. While critical in recruiting Pol II to the promoter, the role transcription factors play in transitioning paused Pol II into productive Pol II is, however, little known. By studying how Drosophila Hox transcription factors control transcription, we uncovered a molecular mechanism that increases productive transcription. We found that the Hox proteins AbdA and Ubx target gene promoters previously bound by the transcription pausing factor M1BP, containing paused Pol II and enriched with promoter-proximal Polycomb Group (PcG) proteins, yet lacking the classical H3K27me3 PcG signature. We found that AbdA binding to M1BP-regulated genes results in reduction in PcG binding, the release of paused Pol II, increases in promoter H3K4me3 histone marks and increased gene transcription. Linking transcription factors, PcG proteins and paused Pol II states, these data identify a two-step mechanism of Hox-driven transcription, with M1BP binding leading to Pol II recruitment followed by AbdA targeting, which results in a change in the chromatin landscape and enhanced transcription.
Assuntos
Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/fisiologia , Regulação da Expressão Gênica , Proteínas de Homeodomínio/fisiologia , Proteínas Nucleares/fisiologia , Fatores de Transcrição/metabolismo , Fatores de Transcrição/fisiologia , Transcrição Gênica/genética , Animais , Animais Geneticamente Modificados , Células Cultivadas , Proteínas de Drosophila/genética , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Embrião não Mamífero , Feminino , Proteínas de Homeodomínio/metabolismo , Masculino , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , RNA Polimerase II/metabolismoRESUMO
The emergence following gene duplication of a large repertoire of Hox paralogue proteins underlies the importance taken by Hox proteins in controlling animal body plans in development and evolution. Sequence divergence of paralogous proteins accounts for functional specialization, promoting axial morphological diversification in bilaterian animals. Yet functionally specialized paralogous Hox proteins also continue performing ancient common functions. In this study, we investigate how highly divergent Hox proteins perform an identical function. This was achieved by comparing in Drosophila the mode of limb suppression by the central (Ultrabithorax and AbdominalA) and posterior class (AbdominalB) Hox proteins. Results highlight that Hox-mediated limb suppression relies on distinct modes of DNA binding and a distinct use of TALE cofactors. Control of common functions by divergent Hox proteins, at least in the case studied, relies on evolving novel molecular properties. Thus, changes in protein sequences not only provide the driving force for functional specialization of Hox paralogue proteins, but also provide means to perform common ancient functions in distinct ways.
