RESUMEN
Methylation of histone H3 at lysine 9 (H3K9) is a hallmark of heterochromatin that plays crucial roles in gene silencing, genome stability, and chromosome segregation. In Schizosaccharomyces pombe, Clr4 mediates both di- and tri-methylation of H3K9. Although H3K9 methylation has been intensely studied in mitotic cells, its role during sexual differentiation remains unclear. Here, we map H3K9 methylation genome-wide during meiosis and show that constitutive heterochromatin temporarily loses H3K9me2 and becomes H3K9me3 when cells commit to meiosis. Cells lacking the ability to tri-methylate H3K9 exhibit meiotic chromosome segregation defects. Finally, the H3K9 methylation switch is accompanied by differential phosphorylation of Clr4 by the cyclin-dependent kinase Cdk1. Our results suggest that a conserved master regulator of the cell cycle controls the specificity of an H3K9 methyltransferase to prevent ectopic H3K9 methylation and to ensure faithful gametogenesis.
Asunto(s)
Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Metilación , Histonas/genética , Histonas/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Fosforilación , Heterocromatina/metabolismo , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Metiltransferasas/genética , Metiltransferasas/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Gametogénesis/genéticaRESUMEN
Faithful propagation of functionally distinct chromatin states is crucial for maintaining cellular identity, and its breakdown can lead to diseases such as cancer. Whereas mechanisms that sustain repressed states have been intensely studied, regulatory circuits that protect active chromatin from inactivating signals are not well understood. Here we report a positive feedback loop that preserves the transcription-competent state of RNA polymerase II-transcribed genes. We found that Pdp3 recruits the histone acetyltransferase Mst2 to H3K36me3-marked chromatin. Thereby, Mst2 binds to all transcriptionally active regions genome-wide. Besides acetylating histone H3K14, Mst2 also acetylates Brl1, a component of the histone H2B ubiquitin ligase complex. Brl1 acetylation increases histone H2B ubiquitination, which positively feeds back on transcription and prevents ectopic heterochromatin assembly. Our work uncovers a molecular pathway that secures epigenome integrity and highlights the importance of opposing feedback loops for the partitioning of chromatin into transcriptionally active and inactive states.
Asunto(s)
Ensamble y Desensamble de Cromatina , Eucromatina/enzimología , Silenciador del Gen , Histona Acetiltransferasas/metabolismo , Histonas/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Nucleares/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/enzimología , Acetilación , Eucromatina/genética , Retroalimentación Fisiológica , Regulación Fúngica de la Expresión Génica , Heterocromatina/enzimología , Heterocromatina/genética , Histona Acetiltransferasas/genética , Proteínas de la Membrana/genética , Mutación , Proteínas Nucleares/genética , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Transcripción Genética , Activación Transcripcional , UbiquitinaciónRESUMEN
Small non-protein coding RNAs are involved in pathways that control the genome at the level of chromatin. In Schizosaccharomyces pombe, small interfering RNAs (siRNAs) are required for the faithful propagation of heterochromatin that is found at peri-centromeric repeats. In contrast to repetitive DNA, protein-coding genes are refractory to siRNA-mediated heterochromatin formation, unless siRNAs are expressed in mutant cells. Here we report the identification of 20 novel mutant alleles that enable de novo formation of heterochromatin at a euchromatic protein-coding gene by using trans-acting siRNAs as triggers. For example, a single amino acid substitution in the pre-mRNA cleavage factor Yth1 enables siRNAs to trigger silent chromatin formation with unparalleled efficiency. Our results are consistent with a kinetic nascent transcript processing model for the inhibition of small-RNA-directed de novo formation of heterochromatin and lay a foundation for further mechanistic dissection of cellular activities that counteract epigenetic gene silencing.
Asunto(s)
Regulación Fúngica de la Expresión Génica , Silenciador del Gen , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Schizosaccharomyces/genética , Factores de Escisión y Poliadenilación de ARNm/genética , Alelos , Sustitución de Aminoácidos , Centrómero/química , Centrómero/metabolismo , Ensamble y Desensamble de Cromatina , Perfilación de la Expresión Génica , Heterocromatina/química , Heterocromatina/metabolismo , Cinética , Modelos Genéticos , Anotación de Secuencia Molecular , Mutación , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Schizosaccharomyces/metabolismo , Factores de Escisión y Poliadenilación de ARNm/metabolismoRESUMEN
Site-directed RNA editing allows for the manipulation of RNA and protein function by reprogramming genetic information at the RNA level. For this we assemble artificial RNA-guided editases and demonstrate their transcript repair activity in cells and in developing embryos of the annelid Platynereis dumerilii. A hallmark of our assembly strategy is the covalent attachment of guideRNA and editing enzyme by applying the SNAP-tag technology, a process that we demonstrate here to be readily triggered by light in vitro, in mammalian cell culture, and also in P. dumerilii. Lacking both sophisticated chemistry and extensive genetic engineering, this technology provides a convenient route for the light-dependent switching of protein isoforms. The presented strategy may also serve as a blue-print for the engineering of addressable machineries that apply tailored nucleic acid analogues to manipulate RNA or DNA site-specifically in living organisms.