RESUMEN
In any biological system with memory, the state of the system depends on its history. Epigenetic memory maintains gene expression states through cell generations without a change in DNA sequence and in the absence of initiating signals. It is immensely powerful in biological systems - it adds long-term stability to gene expression states and increases the robustness of gene regulatory networks. The Polycomb group (PcG) and Trithorax group (TrxG) proteins can confer long-term, mitotically heritable memory by sustaining silent and active gene expression states, respectively. Several recent studies have advanced our understanding of the molecular mechanisms of this epigenetic memory during DNA replication and mitosis.
Asunto(s)
Ensamble y Desensamble de Cromatina , Proteínas Cromosómicas no Histona/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Epigénesis Genética , Larva/genética , Proteínas del Grupo Polycomb/genética , Animales , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Replicación del ADN , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/metabolismo , Expresión Génica , Genes Homeobox , Larva/crecimiento & desarrollo , Larva/metabolismo , Mitosis/genética , Proteínas del Grupo Polycomb/metabolismoRESUMEN
The Drosophila Trithorax group (TrxG) protein ASH1 remains associated with mitotic chromatin through mechanisms that are poorly understood. ASH1 dimethylates histone H3 at lysine 36 via its SET domain. Here, we identify domains of the TrxG protein ASH1 that are required for mitotic chromatin attachment in living Drosophila. Quantitative live imaging demonstrates that ASH1 requires AT hooks and the BAH domain but not the SET domain for full chromatin binding in metaphase, and that none of these domains are essential for interphase binding. Genetic experiments show that disruptions of the AT hooks and the BAH domain together, but not deletion of the SET domain alone, are lethal. Transcriptional profiling demonstrates that intact ASH1 AT hooks and the BAH domain are required to maintain expression levels of a specific set of genes, including several involved in cell identity and survival. This study identifies in vivo roles for specific ASH1 domains in mitotic binding, gene regulation, and survival that are distinct from its functions as a histone methyltransferase.
Asunto(s)
Cromatina , Proteínas de Unión al ADN , Proteínas de Drosophila , Drosophila/citología , Factores de Transcripción , Secuencias AT-Hook , Animales , Cromatina/metabolismo , Proteínas de Unión al ADN/metabolismo , Drosophila/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Dominios PR-SET , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Epigenetic memory mediated by Polycomb group (PcG) proteins must be maintained during cell division, but must also be flexible to allow cell fate transitions. Here we quantify dynamic chromatin-binding properties of PH::GFP and PC::GFP in living Drosophila in two cell types that undergo defined differentiation and mitosis events. Quantitative fluorescence recovery after photobleaching (FRAP) analysis demonstrates that PcG binding has a higher plasticity in stem cells than in more determined cells and identifies a fraction of PcG proteins that binds mitotic chromatin with up to 300-fold longer residence times than in interphase. Mathematical modeling examines which parameters best distinguish stem cells from differentiated cells. We identify phosphorylation of histone H3 at Ser 28 as a potential mechanism governing the extent and rate of mitotic PC dissociation in different lineages. We propose that regulation of the kinetic properties of PcG-chromatin binding is an essential factor in the choice between stability and flexibility in the establishment of cell identities.
Asunto(s)
Diferenciación Celular , Cromatina/metabolismo , Mitosis , Proteínas Represoras/metabolismo , Células Madre/citología , Animales , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citología , Recuperación de Fluorescencia tras Fotoblanqueo , Proteínas Fluorescentes Verdes/metabolismo , Histonas/metabolismo , Modelos Biológicos , Fosforilación , Proteínas del Grupo Polycomb , Serina/metabolismo , Células Madre/metabolismoRESUMEN
The Polycomb (PcG) and Trithorax (TrxG) group proteins work antagonistically on several hundred developmentally important target genes, giving stable mitotic memory, but also allowing flexibility of gene expression states. How this is achieved in quantitative terms is poorly understood. Here, we present a quantitative kinetic analysis in living Drosophila of the PcG proteins Enhancer of Zeste, (E(Z)), Pleiohomeotic (PHO) and Polycomb (PC) and the TrxG protein absent, small or homeotic discs 1 (ASH1). Fluorescence recovery after photobleaching and fluorescence correlation spectroscopy reveal highly dynamic chromatin binding behaviour for all proteins, with exchange occurring within seconds. We show that although the PcG proteins substantially dissociate from mitotic chromatin, ASH1 remains robustly associated with chromatin throughout mitosis. Finally, we show that chromatin binding by ASH1 and PC switches from an antagonistic relationship in interphase, to a cooperative one during mitosis. These results provide quantitative insights into PcG and TrxG chromatin-binding dynamics and have implications for our understanding of the molecular nature of epigenetic memory.
Asunto(s)
Cromatina/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/metabolismo , Mitosis/genética , Proteínas Nucleares/metabolismo , Complejo Represivo Polycomb 2/metabolismo , Proteínas del Grupo Polycomb/metabolismo , Factores de Transcripción/metabolismo , Animales , Animales Modificados Genéticamente , Proteínas de Unión al ADN/genética , Drosophila/embriología , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Embrión no Mamífero/metabolismo , Proteínas Fluorescentes Verdes/genética , Células-Madre Neurales/metabolismo , Proteínas Nucleares/genética , Complejo Represivo Polycomb 2/genética , Proteínas del Grupo Polycomb/genética , Proteínas Recombinantes de Fusión/análisis , Factores de Transcripción/genéticaRESUMEN
How fast? How strong? How many? So what? Why do numbers matter in biology? Chromatin binding proteins are forever in motion, exchanging rapidly between bound and free pools. How do regulatory systems whose components are in constant flux ensure stability and flexibility? This review explores the application of quantitative and mathematical approaches to mechanisms of epigenetic regulation. We discuss methods for measuring kinetic parameters and protein quantities in living cells, and explore the insights that have been gained by quantifying and modelling dynamics of chromatin binding proteins.
Asunto(s)
Algoritmos , Cromatina/genética , Simulación por Computador , Epigénesis Genética , Modelos Genéticos , Animales , Cromatina/metabolismo , Proteínas de Unión al ADN/metabolismo , Humanos , Cinética , Unión Proteica , Procesos EstocásticosRESUMEN
Non-coding RNAs regulate dosage compensation in mammals by controlling transcriptional silencing of one of the two X chromosomes in females. The two major transcripts involved in this process are Xist and its antisense counterpart Tsix. Expression of Xist and Tsix from the X inactivation center is mutually exclusive. Xist expression triggers chromosome wide silencing of the X chromosome from which it is transcribed. Tsix is a repressor of Xist and is specifically expressed from the other X chromosome, maintaining its activity. Here, we review non-coding RNAs that have been implicated in X chromosome inactivation. Focusing on the best studied transcripts Xist and Tsix we portray a current perspective on chromosome wide gene regulation by non-coding RNAs.